STUDIES ON THE INFECTION OF YAMS BY SCUTELLONEMA BRADYS (STEINER AND LEHEW) BY SAMUEL OLADEJI ADE3IYAN, B.Sc. (A g r ic . Science) Nott. A thesis in the Department o f AGRICULTURAL BIOLOGY Submitted to the Faculty o f Agricu lture, Forestry and Veterinary Science in p a r t ia l fu lfilm en t o f the requirements fo r the degree o f DOCTOR OF PHILOSOPHY o f the UNIVERSITY OF IBADAN August 1975 1 ABSTRACT A general survey o f phytoparasitic nematodes associated with yam ( Dioscorea spp.) in the Mid-Western State o f N igeria showed that Seutellonema bradys and Meloidogyne spp. were the economically important nematodes o f yam tubers. J3 . bradys was associated with the ’ dry ro t ' o f yam tubers causing storage losses estimated between 80 and 100%. Nematodes o f the genus Meloidogyne were found associated with ga llin g o f tubers o f water yam (D. a la ta ). Studies on the rate o f population build-up o f S. bradys in storage showed that _S. bradys increased 9- fo ld , 8- fo ld and 5- fo ld in the tubers o f D. rotundata/ D. cayenensis and D. a la ta respective ly during 6 months o f storage. These increases in population influenced the severity o f ’ dry r o t ’ disease. Results o f investigations into the depth o f penetration o f S. bradys in 5 d iffe ren t v a r ie t ie s o f D. rotundata showed that there were d ifferences in v a r ie ta l su scep tib ility . The bulk o f the nematode population was found in the periderm to a depth o f between 0 - 1 . 5 cm, but depth o f penetration was greater in the 2 head portions o f each o f the tubers than e ith er the middle or bottom portions. Observations on the a c t iv it ie s o f the nematodes in tuber tissues (h istopathology) suggested that the 'dry ro t ' was mainly due to mechanical damage to the c e lls and the host reaction to in tra ce llu la r feeding by S. bradys. Studies on changes in the carbohydrate constituents o f the yam tuber in fected by S. bradys showed an increase in the percentages o f monosaccharides and disaccharides lik e and sucrose, glucose^ galactose, fruobose with a concomitant decrease in starch, amylose and amylopectin when compared with healthy yam. Q ualita tive and quantitative determination o f amino acid constituents o f nematode-infected tubers o f white yam (D. rotundata) . yellow yam (D. cayenensis) and water yam (D. a la ta ) showed that the re la t iv e numbers o f free amino acids were not m ateria lly changed fo llow ing in fec tion by 5, bradys. but a reduction occurred in the number o f 'e s s en tia l' amino acids in the in fected tubers. Eighteen ninhydrin p os itiv e amino acids were detected in the protein hydrolysate. Except in the case o f white yam and in a oW few cases, increases in protein amino acids were recorded 3 in the in fected tubers o f yellow and water yam. The percentage protein was also increased by in fec tion in a l l species except white yam (D. rotundata) . loss Observations on the rate o f weightA (cumulative percentage weight and mean percentage weight lo ss ) in 3 d iffe ren t species o f Dioscorea stored in a yam barn showed that there was a s ign ifican t d ifferen ce in the rate o f weight loss between nematode-infected and nematode-free tubers o f D. rotundata and D. cavenensis. but no s ign ifican t d ifferen ce was recorded between the in fected and healthy yam tubers o f D. a la ta . Estimation o f the ed ible portions in nematode-infected and nematode-free tubers o f £• rotundata. D. cavenensis and D. a la ta showed a s ign ifican t d ifferen ce in the percentage peeling losses between the in fected and healthy tubers. Chromatographic analysis o f the incubation solution o f JL* bradys showed that 5 amino acids - aspartic acid, phenylalanine, hydroxylnol acetic a c id , leucine and isoleucine were discharged by th is nematode. The absence o f the steroid group o f compounds in the nematode-infected yam tubers revealed by spectrometrie analysis might be d isease-re lated as evidenced by i t s appearance in the 4 healthy tubers. Polygalacturonase and amylase a c t iv it ie s were detected in homogenates o f S, bradvs. Studies on fungi associated with the dry rot disease o f yam tubers showed Aspergillus n iger . Penicillium sclerotigenum. Triohoderma v ir id e . Rhizopus n igricans and Fusarium oxvsporum. Botrvodiplodia theobromae and Fusarium moniliforme as the main species. Studies on the possible interrelationshipsbetween J3. bradvs and 3 fungi A. n ic e r . P . sclerotigenum and F . oxvsporum showed that the presence o f the nematode seemed to increase the degree o f pathogenicity o f Fusarium and Pen icillium species on yams. But the presence o f S. bradvs did not increase the degree o f pathogenicity o f Aspergillus n ig e r . In greenhouse experiments, the in teraction between S. bradvs and A. n iger was found to be disadvantageous to the nematode. The presence o f the fungus seemed to have some e ffe c t on the number o f nematodes that invaded the roots and tubers and subsequently on nematode development. This was thought to be due to an a n ti-b io tic action o f A. n iger on £3. bradvs. A host range study of 30 crop plants and weeds revealed that beniseed ( Sesamum indicum L . ) , cowpea (Yigna unguioulata (L . ) W alp.), were good a ltern a tive hosts o f 5 S. bradys. Small populations o f the nematode also survived endoparasitica lly in the roots o f Bupatorium. avn ed re lla . ro s e lle fa ib 1 scus sab dar i f f a L . ) . kenaf (Hibiscus cannabinus L . ) , melon (Cucurbita pepo L . ) ; jute ( Corchorus o lito r iu s L » ) ; yam bean ( Sphenost.vlis stenooarpa) (Hochst ex A Rich) Harms., soko (Oelosia araentia L . ) and pigeon pea ( Ca.lanus ca.ian (L . ) Druce). Non-hosts included maize and tobacco. Dipping nematode-infected tubers o f D. a la ta and D. cayenensis in hot water at temperatures ranging between 50 and 60°C for l+O minutes completely eliminated the nematode. However, at temperatures above 55°G fo r an exposure time o f 1+0 minutes, the tubers so treated su ffered a physio log ica l damage and rotted very rap id ly . Temperatures between 50 and 55°C had no adverse e f fe c t on percentage emergence, growth, y ie ld and p a la ta b ility o f tubers o f D. a la ta . F ie ld t r ia ls on chemical and cu ltu ra l control o f S. bradys on D. a lata showed that the y ie ld o f yam was increased and the nematode population suppressed by the application o f organic manure at the rate o f 1 . 5 kg/heap 6 or 1 ,886.3 kg/ha. Although the application of nemagon at the rate o f 35 .2 kg/ha. considerably suppressed nematode population, the y ie ld o f yam was s ign ific a n tly reduced. The resu lts showed that there is a good deal o f p o ten tia l for experimentation with various cu ltural methods o f nematode con tro l. Studies on the e f fe c t o f gamma irrad ia tion on S. bradys showed that dosages between 5 and 15 Krad did not elim inate the nematodes completely, but suppressed sprouting and signs o f deterioration in tubers. Dosages between 20 - 30 Krad eliminated about 70 - 80% o f the nematode population. 8 ACKNOl/LEDGEMEI'TTS I wish to thank Drs. R.A. Odihirin and M.0 „ A den iji, who supervised my research for their constructive critic ism s and guidance throughout the period of this study. My profound gratitude goes to Professor T, A jibo la Taylor, Head o f the Department of Agricu ltu ra l B iology, University of Ibadan, for his support and encouragement. I should also thank Dr. John Bridge (O.D.M, Hematologist), Mrs. O.A. Egunjobi, Mr. J.A. MacKenzie, Drs. J . I . Okogun, R .I . Bgwuatu, Matt. F. Ivb ija ro , and Messrs A.O. Imonikhe, F.O. Makinde, G.A. Paulissen and M.O. Awoyomi, a l l o f the University of Ibadan, and Mr. B.M. Nana o f the International In stitu te for Trop ica l Agricu ltu re, Ibadan, for th eir technical assistance and useful suggestions. The assistance o f Messrs A, Omokanjuola, K.A. Yussuf, R.O. Coker, S.D. Owoyomi and A.B. Bashorun is also g ra te fu lly acknowledged. I have to thank ttie University o f Ibadan and the United Kingdom M inistry of Overseas Development (O.D.M.) for the award of Fellowships which have made th is research possib le. I am gra te fu l to the s t a f f o f the Department o f 9 Chemical Pathology, U.C.H., Ibadan, and the S p ille rs o f Cambridge for the analysis o f amino acids in yam tubers, and also to Mr. Akpan A. Udo who typed the scrip ts . F in a lly , I am immensely g ra te fu l to my w ife , Idowu, fo r her patience, understanding, moral and fin an c ia l support throughout the period of this study. 10 CERTIFICATION We c e r t i fy that th is work was carried out hy Mr. S.O. Adesiyan in the Department o f Agricu ltu ra l B iology, University o f Ibadan, Ibadan. S U P E R V I S O R S R.A. Odihirin, B.Sc. (Lond .), M.O. A d en iji, GB.Sc. (Lond.) M.Sc. (Rutgers), Ph.D, (N. C arolina), M.Sc., Ph.D. (N o t t . ) , Lecturer (Nematology), Department o f Senior Lecturer Agricu ltu ra l B io logy, University of (Phytopathology), Ibadan, Ibadan, N igeria , Department o f Agricu ltu ra l B iology, University o f Ibadan, N igeria . August 1975. 11 CONTENTS PAGE ABSTRACTS *. . 1 ACKNOWLEDGEMENT S 8 CERTIFICATION 10 TABLE OF CONTENT S 11 LIST OF PLATES • « • 18 LIST OF FIGURES 22 LIST OF TABLES 24 CHAPTER 1 1. INTRODUCTION 27 OBJECTIVES OF THE RESEARCH . . . ............... 29 2.1 REVIEW OF LITERATURE . . . . . . ............... 32 PART I I . Evidence for the occurrence o f nematodes in yam tuber . . . . . . ............... 32 I I . B r ie f taxonomy o f the plant paras itic nematodes of economic importance............. 34 I I I . Description o f Honlolaimidae attacking yams . . . . . . ............... 35 IV. Description of Heteroderidae attacking yams . . . • * • # • • 38 12 PAGE V . Control of S., brad vs in yams • • • . . . 39 (a ) Chemical control • • • . . . 39 (b ) Hot water treatment • • • • • • . . . 41 (c ) Crop rotation . . • • • • % • . . . 42 (d ) B io log ica l control • • • • •• . . . 43 PART I I Evolution of Dioscor eaceae; Its botanical featu res., . . . . . . . 44 I I . Global yam production . . . . . . . 4 5 I I I . Yam production in N igeria . Acreage planted to yam and y ie ld per hectare , 47 IV. Consumption of yam per head in N igeria . 50 V. Economics o f yam production . . . . 51 V I. Important species grown . . . . . . . 52 V I I . Yam cu ltiva tion . . . . . . . . . . 54 V I I I . Storage practices . . . . . . . . • 55 IX. Magnitude of storage losses . . . • 57 X. N u tritiona l value of yams... . . . . 58 X I. Uses other than as food . . . . . . . 59 13 PAGE CHAPTER 3 3. EXPERIMENTAL WORK AND RESULTS . . . . . . 60 3.1 D istribution of nematode parasites o f yam tubers in Mid-West S tate, N igeria . . . . 60 3.2 Surface s te r i l iz a t io n o f nematode. . . . 72 ( i ) Use of 0.1% streptomycin sulphate ••• 72 ( i i ) Use of 0.1% streptomycin sulphate and 20 ppm o f malachite green so lu tion s ... 72 ( i i i ) Use of 20 ppm malachite green solution on ly«. . . . . . . . . . . . . 73 ( i v ) Use of 0.1% mercuric chloride ••• 73 3.3 Studies on population build-up of S. bradys in storage . . . . . . . . . . . . 77 ( i ) Population build-up o f S. bradys . . . 77 ( i i ) The e f fe c t of temperature and humidity on nematode population densities in stored tub ers . . . . . . . . . . . . 78 3.U Depth of penetration . . . . . . . . . 83 ( i ) V a r ie ta l su scep tib ility . . . . . . 84 ( i i ) D istribution o f nematodes within the peri derm . . . ' . . . . . . . . . 84 ( i i i ) Depth o f penetration in -the top, middle and bottom portions of the yam tuber ••• 85 14 P.AGE 3 . 5 Histopathology studies o f the white yam (Dioscor ea rotundata P o ir ) in fected with Scutellonema bradys . . . ••• ••• 90 ( i ) Sections across dry rot area. . . . 91 ( i i ) Sections across wet ro t area. ••• 92 ( H i ) L/S through yam roots . . . »•• 94 3.6 Changes in carbohydrate constituents induced in Dioscor ea rotundata var efon by Scutellonema bradys . . . . . . . . . 115 ( i ) Preparation of samples . . . . . . 115 ( i i ) Quantitative estimation of starch . . . 116 ( i i i ) Estimation of amylose ••• ••• 117 ( i v ) Sugar estimation . . . . . . . . . 117 3.7 Q ualitative and quantitative changes in the free and protein amino acids in the healthy and nematode-infected tubers in three species of Dioscor ea . . . . . . . . . . . . 123 ( i ) Extraction of free amino acids by paper chromatography . . . . . . . . . 123 ( i i ) Extraction of protein amino acids by column chromatography . . . . . . 124 15 PAGE 3.8 The e f fe c t of nematode in fection on percentage weight loss and ed ib le portions in three species o f Dioscorea . . . . . . 131 ( i ) Weight lo s s . . . . . . . . . . . 131 ( i i ) Estimation o f edible portions in nematode-infected tubers . . . . . . 132 3.9 Substances discharged by the yam nematode S. 138 ( i ) Sources of the nematodes and extraction procedure . . , . . . . . . . . . 138 ( i i ) Preparation of the surface s te r il iz e d nematodes for microchemical te s ts ' ••. 138 ( i i i ) Chromatographic analysis of incubation s olution . . . . . . . . . . . . 139 ( i v ) Preparation o f nematode homogenate for spectrometric analysis . . . « « . 140 (v ) Enzymes of S. bradys . . . . 141 ( v i ) Assay systems for hydrolytic en2ymes. • 141 ( v i i ) Relationship between nematode homogenate in ml and percentage drop in v isco s ity 142 ( v i i i ) Assay systems for amylase and invertase 143 16 PAGE 3.10 Fungi associated with Hie dry rot disease o f yam tubers . . . 158 ( i ) D irect p lating method . . . . . . 158 ( i i ) Keeping yam pieces in humidity chambers before p lating . . . . . . . . . 158 3.11 The in terrela tionsh ips of Scutellonems. bradys and fungi associated with wet rot o f yams. . . . . . . . . . . . . . . . 162 ( i ) Greenhouse experiment . . . . . . 162 ( i i ) Storage experim ent.. . . . . . . 164 3.12 Comparison of possib le nematicidal e ffe c ts o f A. n ig e r . P. sclerotigenum and F. oxysporum 186 3.13 Host range studies of the yam nematode - Scutellonema bradys . . . . . . . . . 192 3.1 U Control of S. bradys by hot water treatment 196 (a ) E ffe c t o f hot water treatment on nematode m o r ta lity .. . . . . . . 196 (b ) E ffe c t of hot water treatment on yam storage . . . 197 (c ) E ffe c t of d iffe ren t hot water treatment on germination, growth and y ie ld o f water yam (D. a la ta ) . . . . . . . 198 (d ) P a la ta b ility and accep tab ility tests 198 17 PAGE 3.15 Cultural and chemical methods of control of the yam nematode Scutellonema hradys . . . 206 3.16 Prelim inary studies on the e f fe c t of gamma radiation (from a Cobalt 60 source) on storage l i f e o f nematode-infected white yam (D. rotund at a va r . e fo n ). • • • • • • 212 ( i ) E ffe c t of gamma radiation on yam storage 212 ( i i ) E ffe c t of gamma rad iation on nematode m ortality • m • • • • • • • • 213 ( i i i ) E ffe c t of gamma radiation on nematode- in fected ;7am peels o f D. :rotundata .. 213 CHAPTER k k . DISCUSSION • mm • • • • • • m m m 220 CHAPTER 5 5. SIMM ARX . • • • • • • • * • • mm 262 CHAPTER 6 6 . REFERENCES • mm * m • • • • m • • 273 APPENDIX A : STATISTICAL ANALYSES OF DATA.. 295 APPENDIX B: PUBLICATIONS.. . . . . . . 299 18 LIST 01? PLATES PLATE 1 . (a ) Yam tuber with no symptoms of nematode in fec tion . 0 0 Yam tuber with symptoms o f nematode in fec tion , (c ) Yam tuber w ith heavy symptoms (cracks on the skin) o f nematode in fec tion . 2 . A root-knot in fected water yam (D. a la ta ) from the Mid-West S tate, N igeria . 3. A ga lled tuber cut open. ij.. A root-knot female embedded in the yam tissue. Note formation o f giant c e lls and p ro life ra t io n o f c e l ls . 5. Root-knot females embedded in the yam tissue. 6 . A tra d it ion a l yam barn used for yam storage in Mid-Western State, N igeria . 7. Yam showing in fes ta tion by b e e t le . 8 . Yams inoculated in the middle. 9. A healthy yam tuber cut open. 10. Yam with symptoms of 'd ry -ro t ’ d isease. 1 1 . Yam with symptoms of 'w e t-ro t ' d isease. 19 1 2 . Transverse section of a healthy yam. 13. Transverse section of a dry-rotted yam. 1 i(.. Transverse section o f a wet-rotted yam. 15. In tra ce llu la r movement o f J3. bradys resu lts in c e l l rupture and necrosis. 16. Nematodes in cav ity formed inside the yam tissue. 17. Section showing in fe c t iv ity o f larvae and adults. 18. Robust appearance of adult nematode inside yam tissu e . 19. Head end (male) o f j3. bradys. 20. Head end (fem ale) o f S, bradys. 21 , T a il end (male) o f 3,. bradys. 22. T a il end (fem ale) o f S. bradys. 23. Invasion of yam by fhngal mycelium at the wet rot stage. Note gradual d is to rtion o f starch grains inside yam c e lls not yet reached by fungal mycelia, but already a ffected by th e ir secretions. 2k. Invasion of yam c e lls by fungal mycelium at the wet rot stage. Note complete disappearance o f starch grains and presence o f chlamydospores o f Fusariuro sp. 20 25. Starch grains inside the c e lls o f a healthy yam tuber. 26 . Starch grains (p a r t ia l ly d igested ) in the c e lls o f a dry-rotted yam. 27. Starch grains (com pletely d igested ) in a wet- rotted yam. 28. Longitudinal section of a yam root showing in fe c t iv it y "by nematodes. Note that the cortex is the favourite feeding s ite . 29. Autoanalyser equipment for amylose determination. 30. A technician operating the Varian T -60 N.M.R. Spectrometer. 31 . Apparatus for viscometric measurement o f pectinase enzyme. 32. A nematology greenhouse showing the experiment for the study of in terrela tionsh ips "between A. fliger and S . "bradvs. 33. Yam inoculated with Fusarium oxys-porum only. 3h. Yam inoculated with Penlei I l iu m s c lerotigenum only. 35. Yam inoculated with Aspergillus niger only. 36. Yam inoculated with S. "bradys and F. oxysporum. 21 37. 7am inoculated with S. brad.vs and P. sclerotigenum. 38. Yam inoculated with S. bradys and A. n ig e r . 39. Yam inoculated with S. bradys. F. oxysporum. P. sclerotigenum and A. n ig e r . 2+0. Yam inoculated with dry ro t ex tract. U1 . Yam inoculated with S* hradys only. 2+2. Culture o f A. n ig e r . 2+3. Culture of P. sclerotigenum. 2+2+. Culture o f F. oxysporum. 2+5. A nematode contro l t r ia l on v/ater yam (D. a la ta ) in the Crop C o llection Garden, Department of Agricu ltu ra l B iology, U n iversity o f Ibadan. 22 LIST OF FIGURES F ig . 1 . The yam zone of West A fr ica . 2 . Major yam growing areas in N igeria . 3. Map o f Mid-Western S tate, N igeria , showing the major areas of yam cu ltiva tion . Fluctuations in nematode population densities in stored tubers as a ffected by temperature and humidity. 5. Depth of penetration o f Scutellonema bradys in f iv e va r ie t ie s of D. rotundata P o ir . 6 . D istribution o f J3. bradys w ithin 12 mm o f the peridermal layer o f the yam tuber (D . rotund ata var efon ) . 7. Depth o f penetration in the top, middle, and bottom portions of nematode-infected tubers o f D. rotundata P o ir . 8. Standard curves o f some sugars (Monosaccharides) 9. Weight loss in healthy and nematode-infected yam tubers. 10. N.M.R. peaks o f deuterodimethyl sulphoxide (D6 ) 11 . N.M.R. peaks o f clean, dry rot and wet rot yam extracts. 23 1 2 . Some pharmacologically active constituents o f the yam tuber ( Dioscorea spp.) 13. Hydrolysis o f a 1p%ectin solution by homogenates of Scut ellonema bradys. 1k. Relationship between nematode homogenate (S . bradys) in ml and percentage drop in v is co s ity . 15 . E ffe c t o f Aspergillus n iger on the population o f Scutellonema bradys. 1 6 . A comparison of the possib le nematieidal properties o f A. n ig e r . P. sclerotigenum and 17. E ffec t of d irec t irrad ia tion on the m orta lity o f S. bradys. 18. E ffec t of d irec t irrad ia tion on the larvae and adults o f S. bradys. 19. E ffe c t o f gamma rad iation on S. brandys in yam peels, 24 LIST OP TABLES Table 1 . Plant parasitic nematodes found associated with yam tubers and yam rhizosphere in the Mid-Western State o f N igeria . 2 . Comparing the e ffic ien cy o f four methods o f surface s te r i l iz a t io n of nematodes (j3. bradys) . 3. Population build-up o f S. bradys in stored yams. k. Carbohydrate determination. 5. E ffec t o f Scutellonema bradys in fection on the free amino acids in three species o f D ioscorea. 6 . E ffe c t o f Scutellonema bradys in fection on the 0 protein amino acids in three species of Dioscorea. 7. E ffe c t o f Scutellonema bradys in fection on ille percentage protein nitrogen in three species o f Dioscorea. 8. Percentage weight loss in nematode-infected and uninfected tubers of Dioscorea. 9. Estimation o f the ed ible portions o f nematode- free and nematode-infected tubers of Dioscorea. Percentage peeling losses due to dry ro t disease associated with Scutellonema. bradys. 25 1 0 . Amino acids discharged in to 1 aqueous glucose solution "by su rfa ce -s te r ilized nematode S,. bradys incubated at 30°C for l\S hours. 1 1 . Amylase a c t iv ity o f homogenates of £>. bradys. 1 2 . Invertase a c t iv it ie s o f homogenates of S. bradys and in fected yam. 13. Fungi iso lated from dry ro t portions o f yams by d irect p lating method. 1U. Fungi iso la ted from in fected yam pieces kept inside humidity chambers for 1 U days and plated on to agar. 15. Mean fresh weights o f tubers in grammes. 16. Fungi iso la ted from yam tuber. 17. Fungi iso lated from yam roots. 1 8 . Host range studies of j3. bradys. 19. E ffe c t o f d iffe ren t hot water treatments on nematode m orta lity . 20. E ffe c t of the timing o f hot water treatment (50 - 55°C for kO mins) on the deterio ra tion o f tubers during subsequent storage. 21. E ffe c t of d iffe ren t hot water treatments on germination„growth and y ie ld of water yam (D. a la ta ) . 26 22. Comparison o f treatment means using Duncan's M ultip le Range te s t . 23. P a la ta b ility and accep tab ility te s ts . 2i+. Y ie ld of water yam (D. a la ta ) and nematode population as influenced by chemical and cu ltural methods o f nematode con tro l. 25. Comparison o f treatment means using Duncan's M ultip le Range te s t . 26. Prelim inary investigations into the e f fe c t o f gamma radiation on the storage l i f e o f nematode- in fected tubers o f white yam (D. rotundata var efon) . 27 CHAPTER I INTRODUCTION The fundamental problem which faces most o f the trop ica l regions of the world today is the feeding o f th eir increasing population. Despite the vagaries o f climate mentioned by Stamp (i960) in his fascinating book ’ Our Developing World’ , i t is be lieved that the trop ica l regions o f the world have tremendous poten tia ls for possible all-year-round production o f a va r ie ty o f food crops. Nevertheless, the practice o f agricu lture in the trop ics is fraught with many problems. Lack of technological s k i l l , pests and plant diseases are at the moment preventing the maximum exp lo ita tion and u t i l iz a t io n o f the ex istin g land resources. These and other sim ilar problems must therefore be resolved before the trop ica l regions o f the v/orld can be at par in production le v e ls with the technolog ica lly developed countries o f the world. The y/arm climate o f the trop ics which is by and large regarded as a b lessing is not without i t s disadvantages. 28 Many pests are able to reproduce for a longer period and more rapidly in the warmer clim ate. For this reason, nematode populations are believed to reach higher le v e ls in the warmer than in the cold clim ates. Unfortunately, most o f the nematodes that paras itise trop ica l crops have not received any s ign ifican t atten tion in the past. For example, except in a few cases lik e the research work which revealed the devastating e ffe c ts o f Radopholus s im ilis on banana, a study o f the paras itic nematodes associated with the food crops o f the tropics has only just begun. I t is recognised, however, that a study o f some aspects of parasitism on yams by nematodes has been progressing and in teresting questions are being raised. Yam cu ltiva tion is economically important in N igeria . Yam is eaten both by the r ich and the poor, and the demand fo r yam fa r exceeds supply. Unfortunately, not much is known about the b io logy o f the yam nematode. The knowledge o f i t s behaviour, con tro l, and in terrela tionsh ips with other- plant disease organisms is also inadequate. Nematodes are now. recognised as pests o f g lobal concern hence the need to research into nematode problems in the hope that the problems can be id en tified and e f fe c t iv e ly 29 solved in the near future. 1 .1 * The ob jectives o f the research. The aim o f th is study is to establish the ro le o f the yam nematode Scutellonema bradys in the formation o f ’ dry r o t ’ disease o f yams, to study the conditions favouring the occurrence o f the pathogen in yams and the possible ways o f con tro l. D etails o f the ob jectives are as follows s- ( i ) To evaluate ex isting methods of surface s te r i l iz a t io n or decontamination o f nematodes nematodes, with a view to recommending the best method for _in v itro studies. ( i i ) To study the behaviour o f Scutellonema bradys as regards su rviva l, rate of reproduction and rot development in re la tion to two prin cipa l environmental fac tors , i . e , temperature and humidity, ( i i i ) To study the depth o f penetration o f Scutellonema bradys in yam tubers. ( i v ) To study the histopathology o f the nematode-infected yam tissues. (v ) To estimate the economic losses due to Scutellonema bradys by 30 (a ) comparing the weight loss in nematode-free and nematode-infected tubers. (b ) comparing the food contents o f nematode-free and nematode-infected tubers. ( c ) determining the edible portion o f the nematode-infected tubers. ( v i ) To inoculate su r fa ce -s te r ilized nematodes into yams with a view to estab lish ing the ro le o f Scutellonema bradys in 'dry ro t ' formation. ( v i i ) To is o la te the fungi associated with 'dry r o t ’ disease o f yams. ( v i i i ) To study the rela tionsh ip between Scutellonema bradys and other microorganisms attacking yams in the disease complex. ( i x ) To id en tify substances discharged by the nematode which may be responsible for 'dry r o t ’ formation, (x ) To conduct greenhouse experiments on host-range studies o f Scutellonema bradys. ( x i ) To study the d is tr ib u tion , le v e ls o f in festa tion and seasonal incidence of yam nematodes in a major yam growing part o f the country. 31 ( x i i ) To attempt control o f Scutellonema bradys by the use o f cu ltural and other methods such as the use o f hot water treatment, nematicides, and gamma irrad ia tion . 32 CHAPTER 2 REVIEW OF LITERATURE PART I 2.1 Evidence for the occurrence o f nematodes in yam tuber. The occurrence of some paras itic nematodes in yam tuber has been long established by various workers a l l over the world. As early as 1880, de Man observed that yams from various trop ica l regions o f the world were attacked by the meadow nematode which he ca lled Anguillu lina n ratensis. Steiner (1931) published a b r ie f note on the occurrence o f disease in a yam tuber from Jamaica, with which there were associated large numbers o f a species o f nematode which he placed in the genus Honlolaimus. Steiner and LeHew (1933) published a description o f the nematode obtained from th is yam and gave i t the name brad.vs. In 1 93U, Steiner and Buhrer recorded the same parasite in a yam from Puerto R ico. T . G-oodey (1935)S using specimens from J. West mentioned the species Dioscorea a lata L inn ., D. Lam., and D. rotundata P o ir , as susceptible hosts in N igeria 33 In Guatemala, Schieber (1961) found that in several experimental p lantations, paras itic nematodes were found attacking three d iffe ren t species of Dioscorea - D. floribunda Mart, et G a l., D. composite Hemsl., ' D. s p icu lif lo ra Hemsl. The nematodes involved were Meloidogvne arenaria (N ea l) Chitwood; M. incognita (Kofoid and White) Chitwood and Heterodera sp. Caveness ( 1967 ) recorded seven species o f nematodes in fec tin g yams. Four species Scutellonema brad.vs (S teiner and LeHew), 3. clathricaudatum (Whitehead), Pratvlenchus brachvurus (Godfrey) F i l ip je v , Meloidogvne incognita (K ofo id and W hite), are endoparasitic in yam roots. Four species, S. bradvs. P. brachvurus. M. incognita and Rotvlenchulus reniform is L in ford and O live ira are endoparasitic in fresh ly harvested tubers. Of these species, only S. bradys pers ists in the tuber a fte r long periods o f storage. Several species o f these nematodes occur abundantly in trop ica l s o ils where they cause v is ib le damage not only to yams but other f ie ld crops. The yams are a ffected in various ways. F ir s t ly , the market value o f yams is decreased, the carbohydrate starchy tissue is destroyed 34 and in some cases, the whole plant is k i l le d . Nematode attack also resu lts in storage losses and there are instances where tubers reserved for ’ seed* may be so weakened resu lting in loss o f v ia b i l i t y or g iving r is e to unhealthy p lants. 2»2 B r ie f taxonomy o f the plant -parasitic nematodes o f economic importance. The plant paras itic nematodes comprise several hundred microscopic species o f the NEMATPDA - a la rge , s ize -variab le PHYLUM o f morphologically complex and d is tin c t invertebrate animals. This phylum is divided into two c lasses ; the PHASMIDIA and the APHASMIDIA. The class PHASMIDIA is further sub-divided into two ORDERS, f iv e SUB-ORDERS, th irteen SUPERFAMILIES and forty-seven EMILIES o f which two SUPER?AMILIES and four FAMILIES s feature plant paras itic nematodes o f economic importance. Some o f the s o il inhabiting species, a l l but a few o f the plant paras i t e s are grouped in the class PHASMIDIA. The class APHASMIDIA includes only a few plant p aras itic nematodes. The vast m ajority o f the plant parasitic nematodes are to be found in the order TYLENCHIDA, and also in 35 the super fam ilies DORYL AIM 0 IDE A and TYLENCHO IDEA. Two fam ilies in the superfamily TYLENCHOIDEA: - the HOFLOLAIMIDAE and the HETERODERIDAS include typ ica lly vagrant parasites o f plants* 2.3 Description o f Hoplolaimidae attacking yams. (a ) Scutellonema hradys (S te in er and LeHew). S. bradvs belongs to the subfamily Hoplolaiminae. This subfamily name was derived from the genus Hoplolaimus described e a r lie r by Steiner and LeHew (1933). Goodey (1935) named i t Angu illu lina and Rot.vlenchus (P i l ip je v , 1936). Andrassy (1958) gave the name Scutellonema to the genus which includes several other trop ica l species, namely 8. clathricaudatum ( ’Whitehead, 1959) and S. cavenessi (Sher, 1963). The la t te r two species are common in N igerian so ils (Bridge, P riva te Communication, 1972). S. bradys is stout. The body tapers s lig h t ly in the oesophageal region towards the head which is d is t in c t ly o ffs e t by a con stric tion . There is l i t t l e tapering o f the body p os ter io r ly and the female t a i l is broadly conical in shape. The male t a i l tapers steeply behind the cloaca! opening to a cloacal terminus which is completely surrounded by the rather voluminous bursa. Prominent glands 36 are present at the vu lva l opening. Body is normally stra igh t or s lig h t ly curved when relaxed. S. clathricaudatum and S. cavenessi are characterised by the absence o f vu lva l glands. The disease caused by Scutellonema bradys has been termed DRY ROT o f yams by West (193U). Apparently, the parasite confines its attack to the tubers, since up to the present, no recognisable symptoms o f attack has been reported in the a e r ia l portions o f the host p lants. This is somewhat sim ilar to that o f potatoes attacked by Ditvlenchus dipsaci (Kuhn, 1037) F i l ip je v , 1936 in which the main seat o f attack is also in the tubers. The diseased condition is revealed on peeling or l i f t in g the skin o f yam when small d iscrete areas are found which are at f i r s t yellow ish but la te r become brown or black in colour. In these areas, the parasites are found and are particu la rly numerous in older darker lesions just beneath the skin. The primary lesions caused by the parasite do not penetrate very deep (West 193U) but are confined to the sub-dermal layers . They a fford entry for various fungi, m ites, and other saprophytic organisms which gradually bring about the destruction o f the en tire substance o f the tuber. 37 Ob) Pratylenchus (F i l ip je v ) spp. Members o f th is genus "belong to the fam ily HOPLOLABIIDAE and subfamily PRATYLENCHINAE. About twenty-four species are described so fa r even though only a small proportion o f these attack yams. I t was o r ig in a lly ca lled the meadow nematode, but is now referred to as the les ion nematode because of the lesions i t forms on the host. While the species o f Pratylenchus are ty p ica lly root parasites, they may also attack other underground stems and tubers. They l iv e endoparasitica lly and produce large numbers in roots, rhizomes and tubers where they cause dark necrotic lesions on the in fested parts. Bridge (1972) found P . brachvurus in 9 o f the tubers he examined in the Western State o f N igeria . Other members o f the Hoplolaimidae found in yam so ils are Rotvlenchulus reniform is Lin ford and O live ira , Helicotylenchus pseudorobustus (S te in er ) Golden, Helicotylenchus dihvstera (Cobb), and Hoplolaimus proporicus (Goodey). Symptoms o f P. brachvurus attack include s p lit t in g o f the skin o f the tuber which gives i t a corky appearance and a dark brown ro t extending into the starchy tissue. Genus: Meloidogyne (G oeld i) spp. This nematode belongs to the subfamily HETlittODERINAE. In the older l ite ra tu re , the type o f the genus was e ith er Heterodera marionii (Cornu and Buhrer, 1938) or Heterodera rad ic ico la (M u eller ). About twenty species have been described. This is a universal pathogen with a tremendously wide host range and found attacking almost every crop. Root-knot nematodes were f i r s t reported on yams by Queva (1895) who noted that the nematodes were concerned with tissue m odification in some species o f D ioscorea. Young (1923) working in F lorida made a report o f th e ir attack on D. a la ta . Species o f Meloidogyne have also been observed in N igeria (Udeaja, 1961) while Caveness (1961) found &. arenaria in D. rotundata. M. incognita var. a c r ita was found in fes tin g yams in the Ivory Coast (Luc and de Guiran, 1961) Smit (1966), Schieber (1961), Jenkins and Bird (1962) also found root knot nematode associated with some w ild Dioscorea species in Guatemala. C h arac teris tica lly , species o f Meloidogyne form ga lls on tubers, but in some cases tubers may carry large 39 numbers o f females without showing knots or g a lls . Feeding o f the females occurs in plant tissue surrounding the head o f the nematode; such tissue produces giant c e l ls . P ro life ra t io n and hypertrophy o f the c o r t ic a l c e lls resu lt in the formation o f swellings or g a lls . The overa ll e ffe c ts o f root-knot nematodes on yams are sim ilar to those o f 3. bradys. Yellow to brown patches are found in the c o r t ic a l c e l ls o f the tuber. 2.5 Control o f .S, bradys in yams. The purpose of nematode control is to improve crop y ie ld s and qu a lity . Nematode contro l aims at maximising the e f f ic ie n t and e f fe c t iv e use o f arable lands to meet the increasing need fo r food and fib re throughout the world. As part o f this e f fo r t , the development and use o f an economical and e f fe c t iv e method o f con tro llin g the yam nematode would go a long way in so lv ing some o f the major problems associated with the yam industry in the trop ics . Unfortunately, such economical and e ffe c t iv e methods are not easy to come by. 2 .5a Chemical con tro l. There is l i t t l e information about chemical control o f S. bradys. Bridge (1972) did not aohieve e ffe c t iv e 40 control o f the nematode population by spot application o f D-D into yam h i l l s . He, th ere fore, recommended that a non-phytotoxic compound should be found that can be applied as a post-plant treatment at the stage nematodes are migrating from old in fected yams in to the s o il before invading the young developing tubers. Ayala and Acosta (1970 found D-D to be a very e f fe c t iv e s o i l fumigant fo r nematode control in a f ie ld t r ia l with Dioscorea a la ta . Ayala and Acosta ( 1971) found Dasanit (Bayer 25, 1̂ 1 ) at 1 ,250 ppm for 1 5 - 3 0 minutes, Nemafos (Ne 18,133) 625 ppm for 60 minutes and Nemagon 625 ppm for 30 minutes, e f fe c t iv e in bath treatments o f diseased propagation m aterial without a ffe c t in g its germination, Roth and Richardson ( 1965) reported that fumigation with methyl bromide was generally e f fe c t iv e in the control o f pest in fes ta tion on yams which showed a good tolerance to fumigation, but no data were s p e c if ic a lly provided on nematode con tro l. Thompson et a l . (1972) in Jamaica tested the e ffe c ts o f methyl bromide, hydrogen phosphide and ethylene dibromide as fumigants in con tro llin g the yam nematode in stored yams. Their resu lts showed that 41 fumigation with "both methyl bromide and hydrogen phosphide in i t ia l ly reduced the number o f nematodes, but a fte r 35 - 39 days in storage there was a steady increase in the nematode population in the tubers treated with hydrogen phosphide so that there were no s ign ifican t d ifferences from the con tro l. Thompson et al*. (1972) also concluded from their in vestiga tion that fumigation appeared to have l i t t l e p ra ctica l application because o f the damage that was caused to the tubers. The use of nematicides in the trop ics is beset with many fundamental problems. Nematicides are e:xpensive and, therefore, cannot be afforded by the m ajority o f farmers whose trad ition a l farming operations are characterised by 1ow productivity and very l i t t l e p ro f it margin. Besides, very l i t t l e is .known about the e ffec tiven ess , persistence and residue e ffe c ts of some o f the nematicides in the trop ics . (b ) Hot water treatment I t would appear that the f i r s t reported work on hot water treatment o f tubers as a measure o f nematode control was by Burk and Tennyson (19U1). They successfu lly d is in fested root-knot in fected sweet potatoes that were 42 to be used fo r propagation by trea ting them fo r 65 minutes at 116°]?. The treated roots produced good plants that were free from root-knot. This method has since been used in other parts o f the world to control yam nematodes. Control o f nematodes in seed yam tubers ( i . e . planting m ateria l) by immersion in water at b-60C for 60 minutes, 52°C for 7 - 1 7 minutes or 50°C fo r 15 minutes was described by Ayala and Acosta (1971 ) and at 51 °C fo r 30 minutes by Hawley (1956). Bridge (1972) also successfu lly achieved about 9Q% k i l l o f the yam nematodes using hot water treatment. His exposure time was UO minutes at water bath temperatures ranging between 50 and 55°C. ( c ) Cron ro ta t ion . Although nothing is yet reported in lite ra tu re about the use o f cu ltural methods fo r con tro llin g yam nematodes, the use o f crop rotation to reduce nematode population has been suggested as one o f the most e f fe c t iv e and most ?/idely used land management p ractices . To be an e f fe c t iv e control p ractice , crops that are unsuitable as hosts fo r the nematode must be included in the rotation and growth o f res istan t crops for two to four years must be encouraged. Planting two resistan t crops between susceptible crops may 43 give some measure o f con tro l, "but three to four years and with some nematodes seven to eight years, are sometimes necessary fo r e f fe c t iv e con tro l. The lim ita tion s are that resistan t crops grown in the ro ta tion may "be o f low value and consequently contribute l i t t l e to the farm income. Cultural control o f £>. bradys. however, by ro ta tion on non-susceptible crops is , not always possib le because o f it s d ispersal by in fected tubers (Bridge, 1972). (d ) B io log ica l con tro l. Although some investigators are optim istic about the poten tia l o f b io lo g ic a l control o f nematodes, very l i t t l e is reported in the lite ra tu re sib out the use o f such methods involving predaceous fungi, tox ic substances resu lting from crop residues or other b io lo g ic a l agents. Other methods such as irrad ia tion , e le c t r ic a l treatment or plasmolysis are known to be harmful or le th a l to nematodes, but th eir fu l l po ten tia l fo r e f fe c t iv e nematode control is yet to be investigated . 44 PART I I j THE HOST: YAM 2.2.1 Evolution o f Dioscoreaceae,: it s botanical fea tu res . Yams "belong to the genus Dioscorea named by- Linnaeus in 1937. They are angiosperms, and the genus contains about 600 species o f which only a small proportion have been studied in great d e ta il . The genus Dioscorea may be divided in to a number o f sections which are taxonomically recognised. The main food yams belong to the section Enantiophyllum comprising D. rotundata P o ir ., D. cayenensis Lam., D. a lata L ., D. onnosita Thurib. and D. .ianonica Thunb. Yams f i r s t occurred in the Jurassic when Proto- Dioscoreaceae evolved in Asia . They were o r ig in a lly hermaphroditic and rhizomatous* But i t was in the Cretaceous that the tuber began to evolve. The spread and d iv e rs ific a t io n o f Dioscorea with increasing tuber development, and separation o f A frican from A sia tic species o f Dioscorea probably occurred in the Miocene (B u rk ill, I960 ). The species in th is genus are typ ica lly monocotyledonous though some may have secondary cotyledons. They are 45 seasonally vege ta tive , producing annual shoots which are twining except in a few dwarf species. They p ers is t e ith er underground or at the surface of the ground by means o f storage organs developed in a va rie ty of forms. These storage organs are the tubers one or a few descending in to the s o i l , usually deeply, aris ing as a swelling or as swellings upon the base of the current year’ s stem. In most species, the base o f the stem becomes indurated and las ts in the s o i l beyond the end o f it s year. The so ft annually replaced tubers are protected by the depth of the s o i l over them. Annual stems are renewed from the hard woody knot. Although more than one tuber may be present, not uncommonly one stem alone is produced which drains the stored food from a l l the tubers. Storage o f food occurs in the parenchyma. Leaves are never compound and are always en tire . A l l the Dioscorea are dioecious, although occasionally both male and female flowers are borne on the same p lan t. Some cu ltivars flower only ra re ly . 2.2.2 Global yam -production. Asia and A fr ica are the continents in which yams are 46 Areas where yams are Areas where yam s a major food crop. are an important but secondary food crop. ____International Boundary -----------Regional Boundary THE YAM ZO N E’ OF W E S T AFRICA. FIG. 1. (Reproduced from ‘ Yarns* by D.G. Coursey, 1967) Longmans Green & Co. Ltd. 47 produced to the greatest extent. In Europe and North America, yam production is in s ign ifica n t. Edible yams are grown to a very lim ited extent probably as cu r io s it ie s The greatest yam growing areas in the world are found in the eastern part o f West A fr ica between the Central Ivory Coast and the Cameroun mountain chain, these areas produce more than two th irds o f a l l yams grown in the world. N igeria alone accounts fo r almost h a lf o f the g lobal figu re . The next in rank is South-East Asia. The most widely grown species here is water yam ( Dioscorea a la ta ) . This species is grown mainly in Burma, Laos, Thailand, Cambodia, Vietnam and Malaysia. In India, China and Indonesia, P. a lata is a lso most w idely grown (Coursey, 1967 ) . Yams are also grown in s ign ifican t quantities in North, Central and South America, where the to ta l production o f yams is estimated at about 7lJ+?000 metric tons per annum (P .A .O ., 1966). 2.2.3 Yam production in N igeria (a ) Acreage -planted to yarn and y ie ld per hectare. Yams are grown a l l over the country, but production is markedly concentrated in the Southern States. Yam is 48 predominantly produced in the East Central S tate. I t is la rge ly grown as a mixed crop in the Northern and Mid-West States, hut in the Western S tate, yams are mainly grown as sole crops. The to ta l area reported planted to yam in 1965/66 was approximately 1.7 m illion hectares. Excluding the East Central State, 0.7 m illion hectares were planted in 1966/67 and 0.8 m illion hectares in 1967/68. Assuming the area planted in the East Central State in 1965/66 remained about the same in the fo llow ing two years, the to ta l area planted would stand at 1.3 m illion hectares and 1 ,k m illion hectares in 1966/67 and 1967/68 resp ective ly . The average y ie ld s reported fo r the three years have been normal ranging between 3,633.8 and i+,1 05 kg per hectare. The y ie ld s recorded for Western State are, however, much higher than for the other states and ranged between U,235.8 to U,695.3 kg per hectare. In 1968/69, the to ta l area planted to yam was 1 .1 m illion hectares. The y ie ld in 1968/69 was 3,125.3 kg per hectare. In 1969/70, the number o f acreage planted to yam was 1 m illion and the y ie ld was 3,797 kg per hectare. 49 F I G . • M a jo r yam g row in g a r e a s in N i g e r i a . 50 In 1970/71 , the to ta l area planted to yam was approximately 1 m illion hectares with an average y ie ld of 4,027 leg per hectare. The to ta l area in 1971/72 was estimated at 1 .6 m illion hectares and the average y ie ld o f 3,223.9 leg per hectare. Y ie ld figu res from state to state are not given here, hut generally there is enormous varia tion in y ie ld between the sta tes . This is not surprising in view o f the d ifferences in clim atic conditions, cu ltiva rs , the nature o f cu ltu ra l p ractices , f e r t i l i z e r use, time o f p lanting and harvesting and other management methods. A l l these factors and practices are known to a f fe c t the y ie ld s obtained to a very great degree. 2.2.4 Consumption o f yam per head in N ig e r ia . A survey o f the weekly consumption o f yams per head conducted by the Federal O ffice o f S ta t is t ic s in the country in 1963/64 and 1965/66 shows that yams constitu te the main d ie t o f the people in most o f the s ta tes . The Benue-Plateau State tops the l i s t with a weekly consumption unit o f 13.39 kg. The figu res fo r South Eastern and East Central States are 6.05 and 3.98 kg per caput resp ec tive ly . Kwara and Mid-West States figu res stand 51 at h »b l kg.and 2.90 kg respective ly . Western State figu re stands at 2.38 kg per caput. Although the la s t consumption survey was conducted in 1965-1966* i t is generally assumed that consumption pattern in the rural areas has hardly changed over the years in the absence o f a rad ica l change in d ietary habits. 2.2.5 Economics of yam production. Probably because yam is a non-export crop, the knowledge o f the costs, e ffic ien cy and p ro fits o f yam production is fragmentary. The methods in use fo r yam cu ltiva tion by most farmers are in the main trad ition a l and require only simple home-made too ls . These methods require great labour inputs. The construction o f the large mounds which is very laborious occurs before planting and deep digging and carefu l handling are required at harvesting time. R e la t iv e ly , few studies o f the economics o f yam production under peasant farming conditions have been carried out. At Ejura, Northern Ashanti, Ghana, Hunter et a l , (1931) and Bray ('1958). estimated the labour requirement to be about 109 man-days per hectare per year, and the y ie ld o f saleable yams produced between 52 3763 and 6,272 leg per hectare per year. Other survey reports show that the labour requirement can vary from a l i t t l e as 36 to as much as 1 1 7 man-days per hectare per year. Later studies have also shown that the labour expended accounted for approximately two thirds o f the to ta l costs o f production. A combination o f a l l these investigations carried out under d iffe ren t conditions and with widely varying y ie ld s per hectare indicates that the labour required to produce 2,500 kg o f actual dry food m aterial in the form o f yam is around 1 50 - 200 man-days per kg. These figu res given in cash terms are meagre and indicate very low return fo r labour. In terms o f actual p roductiv ity , the figu res indicate that the labour o f one man expended on yam farming can provide food only fo r his immediate fam ily . 2.2.6 Important species grown. The most important yam species grown are the A s ia tic yam, D. a lata and the West A frican D. rotundata and 2* cavenensis. the f i r s t named in particu lar being cu ltivated almost throughout the trop ics . Wild species 53 o f D. rotundata are unknown but i t is be lieved that D. cavenensis was selected from w ild species. A ll these belong to the section Enantiophyllum o f the genus Dioscorea. and normally produce a single large tuber annually. The other A s ia tic yam, D. esculenta (Lou r.) Burk. (Section Combilium) which produces large numbers o f small tubers, in a manner s im ilar to the potato, is also cu ltiva ted w idely. This is not a pa rticu la r ly popular species in West A fr ica even though i t occurs to some extent in other yam growing areas o f the world. C u ltivation o f D. bulb i fe r a is comparatively lim ited . I t forms small a e r ia l tubers or b u lb ils in the le a f e x ils . This is an advantage because the a e r ia l tubers can be harvested with l i t t l e e f fo r ts . In many parts o f Asia, D. hispida Dennst (Section Lasiophyton) is grown to some extent, as is the very sim ilar D. dumetorum ( Kunth. ) Pax in A fr ica . D. t r i f id a L . (Section Macrogynodium) is confined la rge ly to i t s native Central America. This species also forms a number o f small tubers. Dioscorea opposita Thunb. and Dioscorea .japonica Thurib.- (Section Enantiophyllum) are grown in sub-tropical areas o f China and Japan. 54 2.2.7 Yam c u lt iv a t io n . Yams require well-drained heavy loam s o i l and about 100 - 135 cm o f r a in fa l l , Early planting is done in November while s o il is s t i l l moist and la te planting in February to A p r il. In the riverin e areas o f the country planting is done as soon as possib le a fte r the floods have receded. For seed yam production, close planting in May is the usual p ra ctice . I t is extremely important to se lect only healthy tubers espec ia lly for the November p lanting. Poor se lection may mean losses o f up to 100 per cent before the rains s ta r t . Whole tubers are the best planting m aterial but large tubers may be cut in to ’ s e t ts ’ for economy. Tops o f yams with buds are b e tte r than bottoms or middles. Setts are usually cut one or two days before planting and cut surfaces are l e f t to dry. Yam setts may be planted on the f la t without making any mounds but are usually planted in mounds or ridges. Covering the hidden se tt with a cap o f grass or leaves (mulching) is essen tia l fo r yams planted at the beginning or during the dry season and is desirable for a l l yams. The mulch is usually weighted down with a hoeful o f s o i l and helps in 55 reducing s o i l temperature in the region o f the s e t t . Germination starts 20 - 60 days depending on the r a in fa ll regime, but may take longer for November yams or fo r bottoms and middles. Staking o f the yam is essen tia l for good y ie ld s . L ive bush sticks are often used as stakes, with three or four stakes each carrying a d iffe ren t yam plant being tied together near the top to form a pyramidal structure. This gives greater r ig id i t y and provides a greater support fo r vine growth thus withstanding v io len t storms. 2.2.8 Storage p ractices . Like most other food crops, not a l l the yams harvested can be consumed immediately. Some have to be stored fo r fa i r ly long periods. However, the type o f storage practices varies from place to p lace, and within a single area, more than one technique may be used. The type of technique is often determined by the a v a ila b il i ty o f cap ita l resources. The peasant farmers o f the trop ics , unfortunately, have only lim ited resources and th e ir storage techniques are, th ere fore, very simple. Under these conditions, yamsare stored in the yam barn, which are constructed in a va r ie ty o f ways but 56 generally avoiding excessively high temperatures and providing adequate ven tila t io n . T ra d it ion a lly , peasant farmers who cannot a fford the manpower and the cost o f putting up yam "barns usually leave the tubers where they were grown u n til they are required for sale or food. This type of storage technique is "beset with set backs. The tubers are exposed to attack by agricu ltu ra l pests and p ilfe ra g e . In the event o f heavy rains, yams l e f t in the ground may ro t. On the other hand, i f the s o i l is very dry as i t is in the dry season, harvesting o f these yams when required may be d i f f i c u l t . Stacking yams in heaps is another form o f yam storage. Usually, each stack contains a few dozen yams. Yams stored under this condition, however, su ffer from lack o f adequate ven tila t io n , espec ia lly for the tubers in the in te r io r . Such yams are also susceptible to termite or rodent attack. Thatched shelters , shed or store rooms with adequate ven tila t io n are sometimes used to store yams. These prevent as much as possib le the spread o f ro ttin g organisms. The yam barn is the structure commonly used for yam 57 storage in many areas. This consists o f a v e r t ic a l framework to which the yam tubers are fastened ind iv idually by means o f s tr in g , or more commonly by means o f r a f f ia . In other p laces, however, a yam barn may consist o f a horizontal wooden platform where tubers are arranged in such a way that these yams are out o f contact with each other. 2.2.9 Magnitude o f storage lo sses . Because yams are stored mainly during the dry season and for a long period, substantial weight losses occur during storage. E a rlies t observations made by Williams (1925) on D. a lata grown in Trinidad showed that the va rie ty used lo s t about 1U. 5%of i t s weight during about four months storage. Weight loss in stored yams was ascribed to sprouting (Campbell jgt a l . , 1962) and microorganisms (Coursey, 1 961 ) . Waitt ( 1 9 6 1 ) suggested that the s ligh tes t cut or bruise may resu lt in the ro ttin g of yam tubers with substantial weight losses . In Puerto Rico species o f D. rotundata. v is ib le ro ttin g o f yams was associated with grea tly enhanced losses in weight during storage (Anon, 1937). The storage losses are valued at about H20 m illion annually (Anon, 1959). 58 Gooding (i960) showed that there is , however, much varia tion between cu ltiva rs . For instance, he recorded a weight loss o f 7.3% in 'b o t t le neck', a va rie ty o f D. a la ta ., and 32.2% in D. alata va r ie ty ' monn-Shine' . Despite these weight losses, however, i t has been suggested that yams normally store w ell unless ro ttin g organisms invade the yams during storage. 2.2.10 N u tritiona l value o f yams. Yams constitute the main d ie t o f the people in the trop ics not necessarily because o f i t s simple source o f ca lo ries but because i t is a source o f carbohydrate. Yams are also h igh ly s ign ifican t sources o f iron , thiamine and espec ia lly o f vitamin C, and minor, but s t i l l useful sources o f protein , calcium, r ib o fla v in and n ico tin ic acid . I t is as a source o f vitamin C (ascorbic acid) that yams are perhaps more important n u tr it ion a lly . No other vitamins are known to be present in yams in any s ign ifican t quantities. Coursey at a l . (1966) made a deta iled study o f the ascorbic acid content o f fo r ty d iffe ren t lo ca l v a r ie t ie s o f D. rotundata. D. cayenensis and D. a la ta . From the resu lts obtained, l i t t l e varia tion was found between samples 59 o f the same va r ie ty . Within individual tubers, i t was found that the highest concentrations o f ascorbic acid occurred near the bulbous end and also just beneath the skin, but the varia tion was not marked. 2.2.11 Uses other than as food . Besides using yam as a source o f food, the recent developments in the pharmaceutical industry employ various Dioscorea as sources o f b io lo g ic a lly active compounds. Some species contain diosgenin which is a valuable starting material for s tero id drugs such as cortisone, sex hormones and contraceptives. A lka lo ida l or s tero ida l toxins are present in a number o f species lik e D. dumetorum. D. h isp ida and D. drageana. The Zulus of South A fr ica are said to use D. dumetorum and D. drageana mixed with ba it fo r catching monkeys. D. hispida is s im ila rly used as a t ig e r poison in the Himalaya and as a fish and foY/1 poison in Indonesia. Many members of the Dioscorea are used in trad ition a l medicine in A fr ica , among the Chinese and other A s ia tic peoples. 60 CHAPTER 3 EXPERIMENTAL WORK AND RESULTS 3.-1 D istribution of nematode .parasites _of yam tubers in In th is study, the ob jectives o f the survey o f the major yam-storing and growing areas o f the Mid-Western State were:- ( i ) To determine the areas in the Mid-West State where plant pa ras itic nematodes are associated with Dioscorea species. ( i i ) To id en tify the more common nematodes of the yam rhizosphere and the tubers. ( i i i ) To estimate storage losses. ( i v ) To assess the e f fe c t o f the present farming systems and other cu ltu ral practices on the increase and spread o f nematodes. (v ) To suggest r e a l is t ic nematode control measures compatible with ex is tin g agricu ltu ra l p ractices . During the survey, about 2 kg o f s o i l and 9 yam tubers 61 o f the three main species grown (D. rotundnta. D. cayenensis and D. a la ta ) were sampled per sampling s ite in not less than 50 major yam-growing areas, yam haras and yam markets in the Mid-Western State. The s o i l vms placed in polythene hags and nematodes were la te r extracted from yam and s o i l samples using the tray m odification o f the Baermann funnel method (Whitehead and Hemming, 1965). The areas covered during the survey are shown in P ig .3 . The resu lts o f the survey o f species o f nematodes associated with yams in the Mid-Western State are summarised in Table 1. A to ta l o f 11 species were recorded, 2 in the yam tubers only, 7 in the rhizosphere only and 2 (£• hrachvurus and S. hradvs) in both rhizosphere and tubers. The dry- and wet-rot diseases o f yams were observed in a l l the yam barns and m arketsvisited. I t was found that about 100 hectares at I l e l e and Agbadu farms had been abandoned due to S. bradys and root-knot in fes ta tion . Symptoms o f nematode damage to tubers observed are sim ilar to those reported by Goodey (1935), West (l93h) and Bridge (1972). Cracking and flak ing o f the epidermal layers were generally noticeable as shown in P late 1. Storage losses estimated bet?/een 80 - 1 62 were observed in most o f the yam barns. Galled tubers caused by root-knot nematode species were also found to be common espec ia lly in D. a lata (water yam) (P la te 2 ). Sections through such ga lled tubers showed that yellow to brown patches are found in the cortex as shown in P la te 3. Nematode a c t iv it ie s also resu lted in c e l l p ro life ra t io n (hyperp lasia ) and formation o f giant c e lls (hypertrophy). These are shown in P la te U. Many root knot females were found embedded in the yam tissue (P la te 5 ). The yam barn technique o f yam storage was found commonly a l l over the Mid-Western State is shown in P la te 6. The yam barn consists o f covered v e r t ic a l framework and to th is framework, the tubers are fastened ind iv idua lly by means o f lo ca l cordage m ateria l. Examination o f f i f t y yam barns, each yam barn usually containing i+00 - 500 yams, revealed that th is method o f storage has one important setback. Because the tubers are in close contact with each other, the spread o f decay-inducing microorganisms was fa c il ita te d ; such in fections occur ea s ily through 'wounds’ . This probably accounts for the high incidence o f ro t observed. A less important method o f yam storage 63 found in a few places is the trad ition a l method of burying the yams where they were grown u n til required fo r food or sa le . Yams stored in th is way showed a high incidence o f both nematode and term ite attack. Yam propagation is by tuber cuttings (s e t ts ) and the use o f small tubers known as 'seeds’ . The ’ seed yam’ is obtained by harvesting the immature tuber early from the growing plant and leav ing the bulbous head and to continue growing. In due course, th is forms a new small tuber x¥hich may be used aa ’ seed’ for the folloT/ing season. The use o f yam seed was found to be advantageous in that the yam seeds examined and extracted were found to be comparatively nematode fre e . Localised in festa tion by yam b eetle was also observed (P la te 7) "but yam losses resu lting from their attack could not be compared with losses due to nematode in festa tion which was found everywhere. © AREAS COVERED DURING THE SURVEY FIG-. 3: MAP OF M ID -W ESTERN S T A T E , N IG E R IA SHOWING THE MAJOR AREAS OF YAM C U L T IV A T IO N EAST CENTRAL S TA TE 65 TABLE 1 ECOLOGICAL SURVEY ELAM) PARASITIC NIMATODB3 FOUND ASSOCIATED M i l TUBERS AND YAM RHIZOSPHERE IN THE MID-WESTERN STATE OF NIGERIA. ( 1974) Nematode species S o il Tub ers Criconemoides spp. X ilelicot.vlenchus spp. X Meloidogyne spp. X Meloidocvne incognita X Pratylenchus braehyurus X X Pratylenchus spp. X ; X X Scutellonema aberrans X Scutellonema clathricaudatum X Scutellonema spp. X Xiphinema spp. X 66 PLATE 1 : (a ) Yam tuber with no symptoms o f nematode in fec tion . (b ) Yam tuber with symptoms of nematode in fec tion . (c ) Yam tuber with heavy symptoms (cracks on the skin) o f nematode in fec tion . 67 GALLED tu b er \ PLATE 2: A root-knot in fected water yam (D. a la ta ) from the Mid-West S tate. N igeria GALLED TUBER PLATE 3; A ga lled tuber cut open.. 68 ELATE JU; A root-knot female embedded in the yam tissue. Note formation o f giant c e lls and p ro life ra t io n o f c e l ls . 69 ELATE 5: Root-knot females embedded in the yam tissue. 70 PLATE 6: A tra d it ion a l yam barn used for yam storage in Mid-Western State, N igeria . 71 PLATE 7: Yam showing in fes ta tion by b ee t le 72 3.2 SURFACE STERILIZATION 0? UMATODES. I t is essen tia l that nematodes "be cleaned or he made fu lly axenic before they can he used fo r in v itro studies. In order to obtain a good method o f surface s te r i l iz a t io n o f nematodes, four methods o f surface s te r i l iz a t io n were tested as fo llo w s :- Two hundred l iv e nematodes were f i r s t washed in s te r i le water and l e f t for 30 minutes. The nematodes were then transferred in to 0.1% streptomycin sulphate solution for 60 minutes and then immediately washed twice in s te r i le water (15 minutes each). The two hundred nematodes were then plated in agar (20 nematodes per agar p la te ). The p lates were incubated at room temperature and observed fo r a period o f two weeks fo r the number o f nematode a liv e and the percentage o f the agar p lates contaminated. ( i i ) Use o f 0.1% streptomycin sulphate and 20 ppm o f malachite green solutio n s , As above, two hundred l iv e nematodes were washed in s t e r i le water and l e f t fo r 30 minutes. The nematodes were then transferred to a known volume o f 0.1% streptomycin sulphate for 30 minutes; washed in s te r i le water fo r 30 minutes and then transferred to the same volume o f 20 ppm malachite green solution for 30 minutes. This was quickly followed by two washings in s te r i le water. 73 The nematodes were plated in agar, incubated at room temperature and observations carried out for a period o f 2 weeks as above. ( i i i ) Use o f 20 pom malachite green solution on ly. The same procedure as in the f i r s t method was followed but malachite green was substituted for streptomycin sulphate. ( iv ) Use o f 0.1 % mercuric ch lo rid e . As in ( i ) but mercuric chloride was substituted fo r streptomycin sulphate. The nematodes used for th is and subsequent experiments were extracted from yams using the tray m odification o f the Baermann funnel method as in (3 .1 ). 50 g o f yam, peel were cut into small pieces and spread on ’ Kleenex’ tissues supported on gauze in a p la s tic coated w ire basket standing in a p la s tic photographic developing dish. Water was added to the c o lle c t in g dish and the pieces of yam were kept constantly moist. A fter U-8 hours, most o f the nematodes would have moved in to the water in the photographic dish, ready to be used. The nematodes were then withdrawn into specia l dropping funnels containing each o f the s te r ila n ts . The nematodes were kept in the 74 sterH ants for 30 minutes to fa c i l i t a t e su ffic ien t cleaning. The nematodes having se ttled at the "bottom o f the funnel were ca re fu lly withdrawn into agar p la tes. Potato dextrose agar (PDA) was found to be a su itab le medium for culturing nematodes. I t was prepared by mixing 39 g o f the PDA powder with a l i t r e o f d is t i l le d water. This mixture was autoclaved u n til a clear and homogeneous solution was obtained. This was then poured into s te r i le p e tr i dishes. A ll other handling too ls used fo r this experiment were s te r il is e d by exposure to U.V. lig h t before use. The resu lts obtained are shown in Table 2. An attempt has been made in th is investiga tion to determine q u a lita tiv e ly and qu an tita tive ly the e ffic ien cy o f four methods o f surface s te r i l iz a t io n o f S. bradys. The c r it e r ia for e ffic ien cy o f each o f the methods are (a ) a b il i t y o f the nematodes to remain a liv e a fte r various treatments in the s te r ila n t and (b ) the a b i l i t y o f each s te r ila n t to keep away contaminants during the period o f observation. The s ign ificance o f (a ) and (b ) is to fa c i l i t a t e correct in terpreta tion o f results obtained from s te r i le inoculation. Prom this in vestiga tion , i t 75 appears that the method involving the use of streptomycin sulphate gave very re lia b le resu lts and can best be recommended. Nematodes were observed to be generally more active a fte r treatment in th is s te r ila n t than in other methods. Malachite green did! not appear to be a good s te r ila n t . The recommended strength was found to be too concentrated and the nematodes had to be rinsed several times with d is t i l le d water. Even at lower concentrations, nematode survival was poor. I t was only in its combination with streptomycin sulphate that k i l l o f the nematodes was obtained as opposed to 20%> k i l l when only Malachite green was used at 20 ppm. With Malachite green alone s ix ty per cent of the plates were contaminated. The use o f 0.01^ mercuric ch loride gave sim ilar resu lts with method ( i i ) , but with only o f the p lates contaminated. 76 TABLE 2 COM PAT? TNG THE EFFICIENCY OF POUR METHODS OF SURFACE STERILIZATION OF .NEMATODES (S . BRADYS) . A B c D S terilan ts Method o f surface % nematodes % p lates s te r i l iz a t io n k il le d or Immobile* contaminated Rating i . 0.1 % streptomycin sulphate solution 3.5 30 Good i i . 1 : 1 ra tio o f a mixture o f 0.1% streptomycin sulphate and 20 ppm malachite green 13.5 50 Fair i i i . 20 ppm malachite green 20 60 Poor iv . 0.01% mercuric 13 ko chloride N.B. ^Results show means o f ten rep lica tes . A: Recommended strengths o f the s te r ila n ts . B and C: Period of observation was two weeks. - * HS3* 77 3.3 STUDIES ON POPUL.T ION BUILD-UP OF S. BRADTS IN STQRAGE. ( i ) Population b u ild-up o f S. brad.vs. The population build-up o f S. bradyjg in storage was investigated using 3 species of Dioscorea (D. rotundata. Di cavenensis and D. a la ta ) . F ive nematode-free tubers o f each species were surface cleaned by using s te r i le d is t i l le d water and alcohol as described by Bridge (1973). About 1+00 - 500 surface s te r il is e d nematodes were Inoculated. Inoculation was done using s te r i le p ipettes through one cy lin d rica l core made by a cork borer in the middle o f each yam tuber. The s ite o f inoculation was then sealed with wax and wrapped in adhesive tap e .(p la te 8) The tubers were stored in a yam barn for s ix months (October -M arch) and the ambient temperatur e and humidity were recorded using a weekly Cassela-type thermohygrograph. Control was provided by inoculating nematode-free tubers with s te r i le water. A fter s ix months, the yam tubers were cut open through the s ite o f inoculation and 50 g from each o f the surrounding areas were extracted. Estimation o f the nematode population was done using P e te r 's counting dish 78 under a stereomicroscope. ( i i ) The e f fe c t o f temperature and humidity on nematode population de n s it ies in stored tubers . This experiment was undertaken to determine the e f fe c t o f temperature and humidity on nematode population densities in stored tubers. About 200 tubers of white (£ . rotundata) and water yam (D. a la ta ) showing symptoms o f nematode in fection were c la s s ifie d into hatches as fo llo w s : 50 tubers o f white yam (D. rotundata) 50 tubers of water yam (B. a la ta ) BATCH A 50 tubers o f whate yam (D. rotundata) ............ ' ' BATCH B 50 tubers o f water yam (B. a la ta ) The tubers in BATCH A were stored under ambient conditions (23 - 32°C; 1+0 - 85$ R.H.) fo r 5 months and those in BATCH B were kept in the cold store with temperatures ranging between 16 - 18°C and 80 - 85$ R*H. Ten tubers from each treatment, i . e . f iv e from white yam and f iv e from water yam were selected at random and extracted fo r nematodes every month for f iv e consecutive months. About 50 g o f yam tissue from each tuber were extracted and 79 nematodes were counted as described b e fo re . The resu lts o f studies on the build-up o f S. b radys population in stored yams are presented in Table 3. There were substantial increases in nematode populations in a l l the three yam species. There were 9 -fo ld , 8 -fo ld and 5 -fo ld increases in the tubers o f D. rotundata. D. cayenensis and D. a lata resp ec tive ly . The dry rot symptoms previously described by Ytest (193U) were observed in 12 out o f the 15 tubers inoculated and the severity o f the dry ro t disease was rela ted to the number o f nematodes extracted. No dry rot symptom was observed in the 5 tubers inoculated with s te r i le water. These findings confirm that the dry ro t was caused only by nematodes. Figure k shows the results obtained from nematode- in fected tubers stoned over a 5 month period under ambient conditions and at low temperatures ranging between 16 — 18°C. The results obtained are sim ilar to those from experiment ( i ) . With storage at ambient temperatures (23 - 32°C) nematode populations in tubers rose to exceedingly high le v e ls , but the low temperatures (16 - 18°C) had a s ligh t depressing e f fe c t on nematode populations even though the in i t ia l nematode count o f the 80 PLATE 8: Yams inoculated in the middle 81 tubers stored at low temperatures was lower than those stored under ambient conditions. The lower temperatures also completely inh ib ited sprouting. TABLE 3 POPULATION BUILD-UP OF S. BRADYS IN STCRED YAMS. Final count Yam species Inoculum le v e l a fte r 6 months/ Populati on 50 g tuber** increase S .if. D. rotundata U00-500 nematodes U,600+67.8 9x app (Male & Female) i|.00-500 nematodes 3,980+63 8x app (Male & Female) D. alata 1+00-500 nematodes 2,765+32.6 5x app (Male & Female) ** Means o f 3 rep lica tes . S.E. Standard error STORAGE TEMP 3 R. H. 0---- ---------- - TUBERS OF D - rot undot a 1 1BATCH A (23 - 32° C JVA .“ _— i D. alata 1 ( 10 - 85%R A A A UQi 10,000 - D. rot undot a 1| BATCH B T16 - 18° c l K. A — — A -------A B i D. data 1 ( 80-85 Vo RH.O £ N; 8,000 Ijj Uj § 6,000 sUj 1,000 uo. fUtj CQ 2000 A--------- § t- j1 _ __________2i™ ________ ___3__L.r ________1i _5________ i . MONTHS INS TORAGE FIG. 4 - FLUCTUATIONS IN NEMATODE POPULATION DENSITIES STORED TUBERS AS A F F E C T E D BY TEM PERATURE AND HUM IDITY 83 3 .U DEPTH 0? PEHCTRAT ION . The depth to which nematodes penetrate yam tissues gives a good ind ication of the extent o f damage caused by nematodes. Experiments were therefore designed to find out: ( i ) Whether the depth o f penetration varies with d iffe ren t cu ltivars o f D. rotundata. ( i i ) The actual concentration o f nematodes w ith in the periderm,and ( i i i ) Whether there is any varia tion in depth o f penetration o f nematodes among the three portions o f yam, i . e . apical (to p s ), middle (m iddles) and d is ta l (bottom s). These investigations, i t was hoped would provide information \ ,f for the se lection o f nematode-free planting materials and aid in yam nematode con tro l. The yam tubers used fo r th is and subsequent analyses were harvested from a p lo t in the Crop C ollection Garden o f the Department o f Agricu ltu ra l B iology, University o f Ibadan, Ibadan, which was previously a r t i f i c ia l l y inoculated with S. bradys. The tubers were washed, dried and stored in a yam barn for 3 months a fte r which only those tubers showing d is t in c t ly recognisable symptoms o f 84 in fec tion were used for analyses. Clean tubers showing no apparent symptoms o f nematode in fection and stored fo r the same period were also analysed and used as control* ( i ) V arie ta l s u s c ep tib ility t The in vestiga tion was conducted using 5 d iffe ren t cu ltivars of D. rotundata. namely; akosu. omifunfun. esinm irin. honiribonin and efon . These cu ltivars were harvested from an a r t i f i c ia l l y inoculated p lot in the University Crop C ollection Garden. Using 2.5 cm diameter cork borer, 10 samples were taken from each cu lt iva r . Each p iece, usually about 3.5 cm in length was cut into 0.5 cm pieces from the peridermal la ye r. Each h a lf centimetre piece was cut into smaller p ieces, macerated, and extracted into small p e tr i dishes using sieves o f nylon gauze embedded in r ig id polyethylene supporting rings. Most o f the nematodes were recovered a fte r 48 hours. ( i i ) D istribution o f nematodes w ithin the periderm. Using one o f the susceptible cu ltiva rs - e fon . a deta iled study o f the actual d is tribu tion o f nematodes w ithin the periderm was made. This was done by taking 10 samples from the in fected portion o f the tuber using a cork borer. Each sample was cut in to 2 m illim etre 85 p ieces. Bach piece was macerated and extracted as above and nematode counts o f these were made, bottom portions o f -the yam tuber. Nematode in fected white yams (D. rotundata) were divided into 3 portions: top, middle and bottom ends and from each of "these portions, 10 g samples o f yam tissues were taken at t i e fo llow ing depths: 0-2; 2-U; U-6; 6-8; 8-10; 10-12; and 12-1U mm. Each sample was taken starting from the periderm to the central cylinder o f the yam tuber. Each piece was cut into smaller p ieces, macerated and extracted in to small p e tr i dishes using sieves of nylon gauze embedded in r ig id polyethylene supporting rings. Nematodes ?/ere counted a fte r U8 hours under a stereomicroscope. Each determination was rep licated ten times. The resu lts o f studies on the v a r ie ta l su scep tib ility are shown in P ig . 5. Prom the resu lts obtained, i t appears there is some degree o f va ria tion in the depth of penetration o f nematodes between cu ltiva rs o f the same species. The va r ie ty efon was found to be particu la r ly susceptible to nematode penetration. Larger number o f 86 nematodes were extracted between 0 - 0.5 cm depth and 0.5 - 1*0 cm depth than any o f the other cu ltivars and in 8 o f the ten samples, nematodes penetrated to a depth o f 1.5 cm. The va r ie ty omifunfun was found to be leas t penetrated by nematodes. The v a r ie t ie s esinm irin and akosu were found to be only moderately penetrated by S . bradvs. Perhaps the d ifferences in the thickness o f peridermal layer might be a factor responsible for the d ifferences in the degree of penetration o f these cu ltivars by the nematode. Pigure 6 shows the resu lts o f the deta iled analysis o f the numbers of S. bradvs to a depth o f 1 0 mm of the periderm. The majority o f nematodes appear to be concentrated in the f i r s t 6 mm and that the bulk o f th is are to be found within 2 - h mm. The resu lts o f investigations o f the d iffe ren t portions o f the yam tuber are shown in P ig . 7« The results showed that nematodes were concentrated in the top (a p ica l) portion than either the middle or the bottom (d is ta l ) portions. Higher nematode populations were found in the 2 - h mm zone. Only few nematodes in zone 6 - 8 mm and only in top and middle; none in bottom. Ho nematodes were found beyond a depth o f 1 2 mm. DEPTH FROM THE PERIDERMAL LAYER IN (C M ) FIG. 5. DEPTH OF PENETRATION OF bSradys IN FIVE VARIETIES OF D. rotundataPcir. NUMBER OF NEMATODES EXTRACTED FIG. 6. DISTRIBUTION OF S- brgdys within 12 m m O F THE LAYER OF TH E YAM TUBER ( rD. otundata var e fon) FIG. 7 D E P T H O F P E N E TR A TIO N O F S. TH E TOP, MIDDLE, A N D BOTTOM PORTIONS OF N E M A TO D E - IN F E C TE D TUB ERS O F rot un data Poir. 90 3.5 HIST0PATH0L0GY STUDIES OF THE WHITS Y AM ( DI03C0REA ROTUNDATA POIR. ) INFECTED WITH SCUTELLONEMA BRADYS. In th is in vestiga tion , a h is to lo g ic a l study of nematode relationsh ips within the tissue and. c e lls o f the yam tuber was carried out. Observations were nade on the ove ra ll e ffe c ts o f th is nematode on the tissues and its probable ro le in aiding subsequent invasion by secondary microorganisms lik e fungi and bacteria was a lso studied. Carefu lly selected portions o f healthy and nematode- in fected yam tubers with early symptoms o f dry ro t and those with early and la te symptoms o f wet rot were cut into pieces and sectioned at on a s lid in g microtome. The transverse and longitudinal sections obtained were dehydrated in Butyl alcohol series and stained e ith er in Eosin and Haematoxylin or in Safranin and Past green. The sections were mounted in Canada balsam, observed under high and low powers of the microscope and the information studied and interpreted in re la tion to the condition o f the yams sectioned. Photomicrographs o f these sections were taken. The resu lts o f the observations are presented in 91 photographs on P lates 9 - 2 8 . ( i ) Sections across dry roi Microscopic examination of "both transverse and longitudinal sections o f yam tuber parasitised by Scutellonema bradvs reveals extensive d is in tegra tion o f the epidermis (P la te 13). These nematodes seem to confine th e ir a c t iv it ie s to the tissues ly in g writhin the periderm layer and those immediately beneath the periderm (P la te 13)* Darkened and thickened c e lls were noted only in areas where nenatodes had fed (P la te 15 ). C avities which may serve as in fection s ites fo r other invading microorganisms were present both in the epidermis and the cortex (P la te 13). Nematode a c t iv it ie s disrupted the yam tissue by emptying the c e l l contents and breaking the c e l l w a lls . Rupture o f c e l l walls seems to fo llow every forv/ard movement of the nematode. Wherever a nematode was found in the section, broken c e l l walls were seen around i t , but farther away, a l l c e lls we in tact (P la te 15). In te rce llu la r spaces are usually very small or non­ existen t in yam tissu e, Where they e x is t , they are smaller than the width of adult £>. bradvs. This is 92 probably one o f the reasons why nematode migration w ithin the tissue resu lts in c e l l w a ll "breakdown. C avities are the resu lt o f rupture o f several c e lls in the same lo c a l i t y (P la te 16 ). Several adults and larvae were found in such cav itie s (P la te 17). Even in stained sections, i t was easy to d iffe ren tia te the larvae and the adults, espec ia lly when they ex is t side "by side in the same cav ity (P la te 17). No eggs were recognised in the stained sections. They may have dropped out during processing "because eggs and a l l the la rva l stages were found with male and female adults whenever a yellow ish portion o f the yarn tuber was teased out under the stereomicroscope (P la tes 19, 20, 21 and 22). Observations in nearly a l l the sections made revealed that nematode movement is in tra ce llu la r , not in te rce llu la r (P la tes 15, 17 18). No fungi were found in early dry rot sections (P la tes (15> 16, 17 and 18) ind icating that fungi are c le a r ly not associated with th is stage o f disease condition . ( i i ) Section across wet rot area. When early wet-rotted pieces of yam were teased under the stereomicroscope, "both nematodes arfl fungi were 93 observed, but th is stage o f yam decay seems to be very short and tran s ition a l and ra re ly encountered. Advanced stage of wet rot seems to fo llow th is ’ early wet r o t ’ condition very fa s t , probably w ithin a matter o f hours, not days. In ’ early wet r o t ’ the yam tissue turns yellowish-brown to brown and both nematodes and fungi were present. In ’ la te wet r o t ’ , dark-brown to black are the p reva ilin g symptomatic colours and no nematodes were found. When portions o f tuber with ’ la te wet ro t ' symptoms were teased out and examined under the compound microscope, fungi, bacteria and mites were found. The cause o f the complete disappearance o f S. bradvs in ’ la te wet r o t ’ is s t i l l under in vestiga tion but i t is thought that some o f the fungi associated with ’ wet r o t ’ e .g . Aspergillus niger probably produce substances antagonistic or toxic to the nematodes. Another conspicuous d ifferen ce observed between sections o f 'e a r ly wet r o t ' and ’ la te wet r o t ' was that ■ the former had many c e lls in tact with mycelia around their c e l l walls as w e ll as in the lumen o f the c e l ls . Haustoria were v is ib le on these mycelia (P la te 21+). Apparently, not every invasion o f the fungus is associated with c e l l 94 rupture. On the other hand, individual c e l ls are hard to recognise in ’ la te wet r o t ’ stage. Instead, what is v is ib le in the stained sections of ’ la te wet r o t ’ is a completely d isin tegrated mass of watery tissue (P la te 1h). The c e l ls were dead and have sh rive lled together and have brown to black deposits on th eir w a lls . Numerous strands o f mycelia were v is ib le in the ca v it ie s formed in the yam tissues. Chlamydospores o f Fusarium. probably F. oxysporium were found in ’ la te wet r o t ’ areas (P la tes 23, 2h)• No starch grains were found. Secretions from the fungi probably d iffu se scores of c e lls ahead o f the growing mycelia because th e ir d isco loration and d is in tegra tion e ffe c ts on c e lls and starch grains respective ly were evident fa r away from mycelia, more so than the e ffe c ts o f nematode secretions (P la te 27). ( i i i ) Longitudinal sections through yam roots (P la te 28). Longitudinal sections through yam roots show that the root cortex is the favourite feeding s it e . Nematodes are embedded within a cavity in c o r t ic a l c e l ls . This cav ity is a resu lt o f the destruction o f parenchymatous c e lls by the nematode. From close examination o f these sections, i t appears that nematode entered the roots 95 PLATE 9 : A h ea lth y yam tuber cut open. : : 96 PLATE 10: Yam with symptoms o f ’ d r y - r o t ’ d ise a se 97 PLATE 11 iYam with symptoms o f ’ w e t - r o t ' d ise a se 98 FLJffiE 12: Transverse se c t io n o f a h ea lth y yam. 99 PLATE 13: T ran sverse se c t io n o f a d ry -ro t te d yam. 100 PLATE lU: Transverse section o f a v/et-rotted yam 101 PLATE 15: In trace llu la r movement o f S. bradys resu lts in c e l l rupture and necrosis. 102 PLATE 16: Nematodes in c av ity formed in s id e tbs yam t is s u e 103 PLATE 17: S ec tion showing in f e c t iv i t y o f la rv a e and a d u lts 104 PLATE 18: Robust appearance o f adult nematode inside yam tissue. 105 PLi-JTE 19: Head end (m a le ) o f ,3. Hrac{3£JL* 106 PLATE 20s Head end (fe m a le ) o f 3 . Lrad&s, 107 PLATE 21 T a i l end (m a le ) o f S... ~bradys.. 108 ELATE 22: T a i l end (fe m a le ) o f 3, b radvs 109 PLATE 23: Invasion o f yam "by fungal mycelium at the wet rot stage. Note gradual d is to rtion o f starch grains inside yam c e lls not yet reached by fungal mycelia, but already a ffected by th e ir secretion s. 110 PLATE 2h: Invasion of yam c e lls by fungal mycelium at the wet ro t stage. Note complete disappearance o f starch grains and presence o f chiamydospores o f Fusarium sp. Ill PLATE 25: Starch grains insicle the c e lls o f a healthy yam tuber 112 PLATE 26: Starch grains (p a r t ia l ly d igested ) in the c e lls o f a dry-rotted yam. 113 PLATE 27: Starch grains (oompleoely d igested ) wet-rotted yam. 114 k ELATE 28: Longitudinal section o f a yam root showing in fe c t iv it y "by nematodes. Note that the cortex is the favou rite feeding s it e . 115 through, le n t ic e ls . Since photosynthates are stored in the parenchyma c e l ls , such tissues would provide excellen t environment for the development, growth and reproduction o f Scute11onema bradys. 3.6 CHANGES IN CARBOHYDRATE CONSTITUENTS INDUCED IN DIOSCOREA ROTUNDA! A VAR. EPQN BY SCUTELLONEMA BRADIS. Although a considerable number of analyses for proximate composition o f yam tuber have been carried out in d ifferen t parts of the world, comparatively l i t t l e information is ava ilab le on the changes in carbohydrate constituents o f yam tubers resu lting from nematode attack. I t is necessary to study the action o f nematodes on the starch and sugar content, since the food value o f yams is determined prim arily by th e ir carbohydrate content. In th is in vestiga tion , the carbohydrate constituents o f the yam tuber at the dry and wet rot stages are c ompared with those of apparently healthy yam tuber from the same source, and using the w idely used white yam ( Dioscorea rotundata var. efon ) . ( i ) Preparation o f samples. About 50 g samples were removed from 5 mm depth below the suberised epidermis of uninfected tubers and those 116 showing symptoms o f dry and wet ro t . These tubers were separately cut in to smaller pieces and oven-dried for 2h hours in an e le c tr ic oven pre-set at 80°C to reduce moisture content s u ff ic ie n t ly to in h ib it further changes and fa c i l i t a t e grinding. Bach o f the samples was la te r ground to powder using a 'Moulinex1' blender. ( i i ) Quantitative estimation o f starch. An aliquot sample, weighing 0.2 g from each of the ground yam samples was put in to separate 50 ml centrifuge tubes. Twenty-five ml o f hot ethanol was added, s tirred thoroughly and centrifuged a fte r f iv e minutes. The a lcoholic solution in each tube was decanted and discarded. Th irty ml o f fresh ly heated 80%o ethanol was again added to each of the tubes, s tirred and centrifuged as be fo re . The a lcoholic solution (containing soluble sugars) was again discarded. This washing treatment was repeated many times u n til a tes t with anthrone was negative. The residue was used for the estimation o f starch using the method o f McCready et a l . (1950). By th is method, starch is estimated and extracted by the Glucose - Anthrone Sulphuric Acid reaction . 117 ( i i i ) Be The percentage amylose in each yam sample was estimated by a m odification o f the autoanalyser procedure or s im p lified amylose procedure o f Williams at a l . (1958). Ten milligrams o f pov/dered samples were weighed into a fia sk . Each sample was then wetted with about 10 drops o f 95% ethanol and 2 ml o f 1 N sodium hydroxide. The mixture was heated for f iv e minutes in a b o ilin g water bath to g e la tin ize the starch, and th is was transferred quan tita tive ly with water washings into a 100 ml volumetric fla sk . The flask , with its contents was cooled and the sample was brought up to volume with water. The starch solutions were transferred in to sample cups o f the autoanalyser for amylose determination. Calibration was done using yam samples o f pre-determined amylose content. About 20mg o f ground yam sample was measured into a beaker to which UO ml o f 80% ethanol was added and l e f t in a water bath for one hour with occasional s t ir r in g . The sample was allowed to se ttle and the supernatant liqu id was decanted in to a 1 00 ml volumetric fla sk . Proteins were prec ip ita ted from the sample by the addition 118 of 1 ml o f saturated lead acetate . The sample was shaken properly and l e f t to stand for 15 minutes. About 2 g sodium bicarbonate was added to 1he sample and the sample was shaken and made upto 100 ml with ethanol. A fte r cen trifu g in g, the sample was decanted. The samples in the sugar solution were analysed using the "Dubois Phenol-Sulphuric Acid Colorim etric Method" (Dubois _et a l . 1956). The resu lts o f the e f fe c t o f nematode in fec tion , the dry and wet rot phases o f the disease on starch and sugar leve ls in in fected yams are shown in Table A. Starch decreased from 60.8% to 39.5% in dry rotted tubers and to 22.2% in wet rotted tubers. Amylose also decreased from 27% to 20% in the dry rotted tuber and to 2% in the wet rotted tuber. Percentages amylopectin also decreased from 33.8 in the healthy yam to 19.5 in the dry rotted tuber and to 20.2 in the wet rotted tuber. These resu lts showed that at these two stages o f in fec tion there is a degradation o f starch to low polymer sugars aided in the f i r s t instance by the presence o f nematodes and la te r by fungi and bacteria . 119 Results o f a l l the analyses showed that a l l the three yam samples, i . e , healthy yam, and those with symptoms of dry and wet rot disease contain sucrose, glucose, fructose, and galactose. Small, though noticeable increases in the sugars were generally observed in the in fected tubers. Sucrose and glucose increased from in the healthy yam to 0.3%° in the dry rotted tubers and to O.hfo in the wet rotted tubers. Galactose increased from 0.3% to O.U% in the dry ro tted tubers but f e l l back to 0.3% in the wet rotted tubers. Fructose was unchanged by in fec tion . TABLE 1+ CARB OHYDRATE DETERM f f l j ION I I I I I I IV V VI V II V I I I £ •H -OP C CD CDOO§• - iH OP t2it ft H 6 0 .8 27 33.8 0.3 0.3 1 0.3 0.1 D 39.5 20 19.5 0.5 0.5 o.k 0.1 W 2 2 .2 2 2 0 .2 o .U o.i* 0.3 0f1 N.B. Results show means o f three rep lica tes . I - H: Healthy yam D: Dry rotted yam W: Wet rotted yam IV - The values o f % amylopectin were obtained by the d ifferen ce between % starch and % amylose. Type o f yam % Starch % Amylose . ____________ 1 % Sucrose % Glucose ! L _______________ j I » 1 > | % Galactose \1 j! FIG . 122 PLATE 29: e q u ip m e n t f o r a m y lo s e d e t e r m i n a t i o n .A u t o a n a l y s e r 123 3.7 QUALITATIVE AND QUANT ITiiT IYECHANUBS IN THE FREE AND PROTEIN AMINO ACIDS IN THE HEALTHY’ AM) M i f f ODE - —IN—F—E .C..T..E..D.. ..T..U..B ER.S IN ..T.H..R..E..E.. ..S..P..E..C. .I.E..S . .O.. F ■ jD-aIOaS-BCiOuRsEr-Ai, ( i ) Extraction o f free amino acids. Samples o f healthy and nematode-infected yam species (D, rotundata. D. cavenensis and D. a la ta ) were analysed for their amino acid constituents hy the method described by Oke at a l. (1973). About 100 g samples were removed from 5 mm depth below the suberised epidermis o f healthy and in fected tubers. These were cut into small p ieces, mixed with 100 ml o f d is t i l le d water and homogenised in a blender. To prevent foaming, a few drops o f amyl alcohol was added. The supernatant was co llected and made up roughly 70% alcohol by the addition o f ethanol. The extract was desiccated in a vacuum and la te r vacuum dried. Id en tific a t io n o f the various amino acids was e ffec ted by means o f paper chromatography (2-dimensional ascending chromatography) using Whatman No. 1 papers. The spots were separated in n-butanol; acetic acid and water a t the ra tio o f 12 : 3 s 5 by volume as the f i r s t dimension, and phenol : ?/ater at the ra tio o f 80 : 20 w/v for the 124 second dimension. The chromatogram was sprayed with ninhydrin to locate the acids. ( i i ) Extraction o f prote in - amino acids hy column ^omatograt The Perkin Elmer automatic amino acid analyser was used to qu an tita tive ly determine the amino acid constituents of yam samples using the method o f Moore and Stein (1951). About 100 mg o f the freeze-d ried yam were hydrolysed with 1 0 ml o f 6 N NCI in a pyrex tes t tube. The a ir in the te s t tube was quickly replaced with nitrogen to prevent oxidative decomposition o f some amino acids. Hydrolysis o f the yam samples was accomplished by placing the sealed te s t tubes containing the samples in an oven equipped with a mechanical fan and operating at 110°C for 22+ hours. At the end of the heat treatment, the tes t tubes ?/ere cooled and the hydrolysate f i l t e r e d . About 5 ml o f the hydrolysate was evaporated jLn vacuo to get r id o f the HC1. The film o f amino acid in the vacuum flask was then taken up in b u ffe r . An aliquot o f th is solution was placed on the column fo r separation o f the amino acid mixture. The peaks obtained were then compared with those o f standard amino acids and the 125 necessary calcu lations carried out. Tryptophan is extensively destroyed "by acid hydrolysis and so could not he estimated hy th is method. The resu lts o f the e f fe c t o f S. hradvs in fec tion on the free amino acids, protein amino acids and protein nitrogen in three species of Dioscorea are shown in Tables 5? 6 and 7 resp ec tive ly . (a ) Free amino acids. In th is in vestiga tion , 13 ninhydrin-positive amino acids were extracted from the uninfected white yam (D. rotundata) . These were g lyc in e , argin ine, isoleucine, leucine, ly s in e , p ro lin e , serine, threonine, alanine, h is tid in e , aspartic acid, glutamic acid and amino acid mixture. In the nematode-infected white yam, there was a sim ilar d istribu tion o f amino acids but there were limn three fewerAin the healthy yam. Lysine, leucine and isoleucine were not detected by paper chromatography. In D. cayenensis. 11 anino acids were detected in the uninfected yellow yam and only 10 amino acids in the nematode-infected tuber. However, unlike in D. rotundata. leucine and isoleucine were present. In the healthy tuber of water yam (D. a la ta ) , 10 ninhydrin-positive amino acids were 126 detected. These include two 'essen tia l' amino acids - lysine and tyrosine, Other essen tia l amino acids lik e leucine and isoleucine were absent. In the in fected tuber the number o f amino acids was only two fewer than those detected in the healthy tuber. The nematode- in fected water yam was d e fic ien t in almost a l l 'e s s en tia l ' amino acids except h is tid in e . (b ) Protein amino ac id s . Eighteen ninhydrin-positive amino acids were detected in the protein hydrolysate. There was no corre la tion between the to ta l number o f amino acids found in the free amino acid pool and the to ta l number found in the protein hydrolysate. Methionine peaks were In su ffic ien tly resolved to be measurable in the healthy yam samples o f both D, rotundata and D, cavenensis. Pro line peak was also in su ffic ien t ly resolved in the healthy yam sample o f yellow yam (E>. cavenensis) . Tryptophan and cystine are extensively destroyed by acid hydrolysis and so could not be estimated by th is method. Generally, the amounts o f each protein amino acid from both healthy and in fected tubers varied considerably with each yam species. In the white yam (D. rotundata) . 127 a l l amino acids detected except g lyc ine, methionine, and ammonia, decreased in the in fected tuber. Serine was unchanged "by in fec tion . Ammonia, g lycine, and methionine, however, increased in the infected tuber. In the yellow yam (D. cayenensis) . the reverse was the case, a l l the amino acids detected increased in the in fected tuber with the exception o f arginine which was unchanged hy in fec tion . In the water yam the trend was sim ilar to that o f yellow yam. A l l the amino acids increased as a resu lt o f in fection except glutamio acid, va lin e , isoleucine, and phenyl alanine which were not changed by in fec tion . Methionine, leucine, and arginine decreased in the in fected tuber. In both the white yam and yellow yam, methionine was not detected in the healthy tubers, but was detected in the in fected tubers. Pro line was not detected in the healthy sample of yellow yam, but was substantia lly detected in the in fected yam. Ammonia, alanine and g lyc ine increased as a resu lt o f in fec tion in a l l the three species o f D ioscorea. Except in the white yam, the percentage protein increased as a resu lt o f in fec tion . Sim ilar trends were also observed for the protein nitrogen determined. TABLE 5 EFFECT 0? SCUTLLLONEMA BRACK’S I NFECTION ON TIB FREE AMINO ACIDS IN THREE SPECIES OP .D..I.O...S.C. OREA White yam Yellow yam Water yam D, rotundata D. cayenensis D. alata Healthy Nematode Healthy Nematode Healthy Nematode in fected infected infected Glycine + + + 4 4 4 Arginine (Monohydrochloride) 4* - + - + 4 DL - i s ol euc i ne 4* - + 4 — - DL-leucine + - + 4 — — DL-lysine (Monohydrochloride) + - + - 4 - Tryptophane - •Mt - - - - L-tyrosine - - - - 4 — DL-valine - 4* - - - - Hydroxy ino lacetic acid - - - - - - Methionine — - — - - - Phenylalanine - - - - - - DL-proline + - - 4 - - DL-serine Jr - 4 4 4 Threonine + + 4 4 - - Alanine 4* 4* 4 4 + 4 DL-histidine 4* + 4 4 4 4 Aspartic acid + + 4 4 4* 4 L-cystine - H- - - - - L-glutamic acid + 4- 4 - 4 4- Amino acid mixture + + + 4 + 1 + N„B. + (Amino acid detected) - (Amino acid not detected) 128 TABLE 6 EFFECT OF SCUTELLONEMA BRADY'S INFECTION ON TIE PROTEIN AMINO ACIDS IN THREE SPECIES OF DIOSCORSA Percentage Dioscorea rotundata Dioscorea cayenensis Dioscorea alata Amino acids; moisture Healthy Nematode Healthy Nematode Healthy Nematode and protein in fected infected infected Aspartic acid 0.58 0.50 0.49 0.64 0.72 0.74. Threonine 0.19 0.18 0 .16 0.23 0.25 0,29. Serine 0.25 0.25 0.20 0.35 l 0.39 0.41 Glutamic acid 0.67 0.55 0.48 0 .6? 0.90 0.90 Proline 0.18 0 .16 * 0.22 0.27 0.28 Glycine 0.18 0.21 0.13 0.23 0.23 0.26 AALanine 0.20 0.21 0.14 0.26 0.27 0.30 Cystine N.D. N.D. N.D. N.D.' N.D. N.D.. • Valine 0.21 0.19 0.1 2 0.26 0.31 0.31 Methionine & 0.05 $ 0.06 0.08 0.07 Iso-leucine 0 . 17 0.16 0*11 0.21 0.26 0.26 Leucine 0.33 0.29 0; 21 0.39 0.49 0.48 Tyrosine 0.16 0.14 0.08 0.19 0.22 0.20 Phenylalanine 0.32 0.21 0.33 0.36 0.37 0.37 H istid ine 0.11 0.09 0.09 0.12 0.13 0.14 Lys ine 0.29 0.23 0.21 0.28 0.33 0.35 .Ammonia 0.18 0.22 0.1 0 0.30 0.24 0.28 Arginine 0.65 0.31 0.32 0.32 0.74 0.61 Tryptophane N.D. N.D. N.D. N.D. N.D. N.D. Protein 6.5 4.9 5.9 6.4 7.4 7.9 Moisture 11.6 11 .9 13.5 14.2 10.5 11 .3 * Peaks in su ffic ien tly resolved or formed to be measurable. N.D. Not determined. Results are expressed as % amino acid ( i . e . g/100 g of sample). 129 TABLE 7 EXPECT 0? SQUTELLONEMA BRADYS INPECTION ON TIE PERCENT AGE PROTEIN NITROGEN IN THREE SPECIES "OP DIOSCOREA “ Dioscorea rotundata Dioscorea ŷcxy c iiO-iio x o Dioscorea alata Amino acids as % Healthy Nematod e Healthy Nematode Healthy Nematode nitrogen infected infected in fected Aspartic acid 0.06 0.05 0.05 0.07 0.08 0.08 Threonine 0.02 0.02 0.02 0.03 0.03 0.03 Serine 0.03 0.03 0.03 0.05 0.05 0.06 Glutamic acid 0.06 0.05 0.05 0.06 0.09 0.09 Proline 0.02 0.02 * 0.03 0.03 0.03, Glycine 0.03 0 .01+ 0.02 0.0k 0.01+ 0.05 Alanine 0.03 0.03 0.02 0.0k 0.01+ 0.05 Valine 0.03 0.02 0.01 0.03 0.01+ 0.01+ Methionine * 0.005 0.01 0.01 0.01 Iso-leucine 0.02 0.02 0.01 0.02 0.03 0.03 Leucine 0.03 0..03 0.02 0.0k • 0.05 0.05 Tyrosine 0.01 0.01 0.01 0.01 0.02 0.02 Phenylalanine 0.03 0.02 0.03 0.03 0.03 0.03 H istid ine 0.02 0.02 0.02 0.03 0.03 0.03 Lysine 0.06 0*01+ 0.0k 0.05 0.06 0.07 Ammonia 0.15 0.18 0.08 0.25 0.28 0.23 Arginine 0.21 0.10 0.10 0.10 0.21+ 0.19 Total nitrogen 0.81 0.68 0.51 0.90 1.07 1 .09 % nitrogen 1 .Ok 0.785 0.93 1 .02 1.18 1 .26 % recovery 87 55 88 91 87 i_____ 71 N.B. fa ) The results are expressed as %n itrogen ( i . e . g/lOO g of sample), (h ) The poor recovery o f nitrogen from Dioscorea rotundata (healthy) and Dioscorea cayenensis (healthy) is partly due to the low le v e ls o f pro line and methionine and th eir consequent poor resolu tion . (c ) Non-protein nitrogen was not determined. 130 131 3.8 THE EFFECT Off NEMATODE INFECTION ON PERCENTAGE WEIGHT LOSS AND EDIBLE PORTIONS IN THREE SPECIES 01? DI03C0REA. ( i ) Weight Loss. This investiga tion was carried out using 3 species o f Dioscorea (D. rotundata. D. cayenensis. and D. a la ta ) . Yams with symptoms of nematode in fec tion and non-infected yam tubers were stored for 12 weeks in a yam barn. The in fected and non-infected yam tubers were divided and la id out as fo llow s : D. rotundata (un in fected) - 20 tubers) D. cayenensis " - 20 " j BATCH A D. a lata " - 20 " ) D. rotundata (nematode-infected) - 20 tubers) D. ccayenensis " " - 20 " ) BATCH B D. a lata H ,r - 2 0 ” ) Weight losses were determined by weighing each o f the tubers weekly and recording the weekly loss in weight. Records o f temperature and humidity were taken in the yam barn with a weekly Cassela-type thermohygrograph. 132 ( i i ) Estimation of*.edible portions In nematocle- ln fected tubers. 60 in fected yam tubers made up o f 20 o f D. rotundata. 20 o f D. cayenensis and 20 o f D. alata were weighed in d iv idu a lly . The tubers were then peeled u n til the nematode- damaged portions had "been removed, and only the ed ib le portions remained. The tubers were again 'weigaed. The d ifferences in weight were recorded. 20 clean yam tubers of each va r ie ty were also weighed and peeled in the same way; the data obtained from these were used as control data. The resu lts o f these investigations are shown in Tables 8 and 9 and P ig . 9. I t is noteworthy that there is considerable varia tion in weight losses between and w ith in the three d iffe ren t yam species stored under the same environmental conditions. Table 8 sho’ws the means o f wei^it loss for both the uninfected and nematode-infected tubers as 19.1, 22.7 and 12.2 fo r the in fected tubers of D. rotundata. D. cayenensis . and D. a la ta respective ly even though weight loss as high as 5U,2% was recorded for one o f the in fected tubers o f 133 D. rotundata. In the healthy tub ers , the means o f to ta l weight loss were '12.7$* 16.6$ and 8.9$ for D. rotundata. D. cavenensis and D. a la ta resp ec tive ly . The d ifferences in the mean percentage weight loss "between the nematode- in fected ani uninfected tubex»s o f D. rotundata and D. oavenensis were s ta t is t ic a l ly s ign ifican t hut not in D. a la ta . I t can also he seen that the lea s t percentage weight loss was recorded in D. a la ta and highest was recorded in D. cayenensis. The resu lts o f the cumulative percentage wei^it loss fo r the three d iffe ren t yam species (uninfected and nematode-infected) are shown in P ig , 9. The results show that there is considerable varia tion in the cumulative percentage ?/eight loss among the three d iffe ren t species and between the uninfected and the nematode-infected yam tubers. Cumulative percentage -weight losses o f 1 h%9 13.9$ and 8.8$ were recorded for the uninfected yam tubers and 2k%9 22.3$ and 11.9$ for the nematode-infected yam tubers of D. rotundata. D. cavenensis and D. a lata resp ective ly . Table 9 shows the resu lts o f the estimation o f percentage edible portions in nematode-infected and 134 uninfected tubers'1 of Dios cor ea. The resu lts showed peeling losses as 28.1, 26.2 and 26.3 for the in fected tubers o f D. rotundata. D» cayenensis and D. a la ta respective ly even though peeling loss as high as 57% was recorded for one of the tubers o f D. cayenensis. In the healthy tubers * the means o f peeling losses were 9%, 9% and 6 . 9 % fo r D. rotundata. D. cayenensis and D. a lata respective ly . TABLE 8 PERCENTAGE-WEIGHT LOSS JIT IOtATODE-IN7LCTED AiSDftUNIN?ECTEI)''''TUBER3 0? DIOSCOREA Dioscorea rotundata Dioscorea cayenensis Dios cores, a la ta Mean o f Mean o f Mean o f Range to ta l to ta l to ta l weight Range weight Range weight lo ss ** loss*# loos#* Nematode in fected yam tubers (2.99-5U.20) 19.07+1.71 (i0 .U 0 -38 .90 ) 22.65+1.66 ( 3 .20-26.50) 1 2 . 20+1-.70 Uninfected yam tubers (0 -33.30 ) 1 2 .70+1 .71 ( 5.70-28.60) 16.55+1 .90 (3.U0-1U.10) 8.93+0.50 i * * Mean o f twenty tubers + standard erro r Level o f s ign ifican ce between the healthy and nematode-infected tubers o f ( i ) D. rotundata = P O.OS^ - s ig n ific a n t ( i i ) D. cayenensis= P 0 .0 5 ^ - s ig n if ic a n t ( i i i ) D# a la ta = P 0 .0 5 ^ - not s ig n if ic a n t . 135 TABLE 9 ESTIMATION 0? THE EDIBLE PORTIONS OR NEMATODE-FREE AND NEMATODE-INEECTED TUBERS OP DIOS GORE A. PERCENTAGE PEELING LOS333 DUS TO DRY ROT DI3EASE ASSOCIATED WITH SCUTELLONEMA BR*L)YS. ------------------ ----- D. rotundata D. cayenensis D, a la ta Range Mean** Range Mean*-* Range Mean** ( " ' - Nem atode-in fectea ( a ) ( c ) yaqi tubers (16.60-51 .70) 28.05+2.1 2 (13.30-57.10) 26.19+2. k l <15.60-50.0) 26.30+2.25 Uninfected yam (b ) (d ) I t ) tubefs (3.1+0-20.00) 9.01+1 .05 (3 .60 -19 .00 ) 9.02+0.85 (3 .2 -1 2 .3 ) 6.88+0.57 ** Mean o f twenty tubers + Standal'd E rro r. Level o f s ign ificance between (a & b ) = P 0 .0 5 < - S ign ific an t. (c & d) = P 0 .05< - S ign ific an t (e & f ) = P 0 . 0 5 Si gni f i cant . 136 HEALTHY INFECTED W EEK S IN STO R A G E FIG. 9. W EIGHT LOSS IN HEALTHY AN D YAM TUBERS 138 3.9 SUBSTANCES DISCHARGED BY THIS YAM NEMATODE S, BRADYS In th is study, the substances discharged by the yam nematode were investigated by methods described by Krusberg and Myers (l96h) and by spectrometric analysis, a previously unexplored method, ( i ) Sources o f the nematodes and extraction procedure Nematodes were extracted from in fected yam tubers using the tray m odification o f the Baermann funnel method. Since contaminating microorganisms might metabolise substances discharged by nematodes and secrete other m etabolites, the nematodes were su r fa ce -s te r ilized using 0,1% streptomycin sulphate. The nematodes were further ir r iga ted with a fine mist o f s te r i le d is t i l le d water. The f in a l d is t i l le d water rinse was repeated f iv e times a fte r which the nematodes were used in experiments. ( i i ) Preparation o f the surface s te r i l iz e d nematodes- fo r microchemical te s ts . The surface s te r i l iz e d nematodes were placed in flasks containing d is t i l le d water and 1% glucose adjusted with 1 N HC1 to pH 5.0 and incubated in a water bath shaker at 30°C for i+8 hours. The incubation solution was checked for m icrobial contamination by p ip ettin g about 139 1 ml o f the solution in to melted potato dextrose agar (PDA) in a te s t tube. The P.D .A. and the incubation solution ?/ere mixed and poured in to p e tr i dishes. The p e tr i dishes were incubated at 30°C fo r l+8 hours a fte r which microbial colonies were counted. A fte r Aj.8 hours, the counts o f m icrobial colonies were found to be n eg lig ib le (le s s than 200) and the incubation solution was used immediately. The solution was divided into two portions. In the f i r s t portion , the nematodes were f i lt e r e d o f f and the second portion contained a l l the nematodes together with a b i t o f the incubation solu tion . A few drops o f the f i r s t solution were chromatographed and the second solution containing the nematodes was used for spectrometric analys is . ( i i i ) Chromatographic analysis o f incubation so lu tion . Nematodes incubation solutions were tested microchemically to determine the classes of compounds present. A few drops o f the incubation solu tion (about 0.1 ml) were used in each tes t fo r amino acids, urea and other amides. A two dimensional ascending technique with n-butanol: acetic acid and water (12 : 3 : 5) as 140 the f i r s t dimension and phenols water (80 : 20 w/v) as the second dimension. The papers were l e f t for 2I+. hours and then sprayed with ninhydrin and dried at 80 - 1 00°C fo r some minutes when most of the amino acids showed up b r igh t ly . ( iv ) Preparation o f nematode homogenate for spectrometric analysis . The concentrated suspensions o f nematodes were p e lle ted by cen trifugation , and the supernatant removed with a p ip ette . To elim inate most bacteria and soluble substances present in the suspensions, the nematodes were re-suspended and centrifuged for a few seconds at 680 G several times with d is t i l le d water. A fter each washing, the supernatant was discarded and the process repeated with fresh solution. These massed nematodes were ground to prepare homogenates. The nematode homogenate was evaporated on a rotary evaporator (attached to a vacuum pump) u n til i t was reduced to a powdered form. The powdered extract was again mixed with benzene, which removed excess water leaving the extract completely dry. The resu lting extract v/as then mixed with N iyol and run on the In fra red and U.V. equipment. The remainder o f 141 the o r ig in a l extract ( i . e . without N iyo l) was dissolved in D6 (Deuterodimethylsulphoxide) and run on the Nuclear Magnetic Resonance (N.M.R.) equipment. As a form of contro l, the solu tion containing the extracted nematode, i . e . dry rot extract was s im ila r ly treated as the nematode homogenate ( i v ) . The dry rot solution was evaporated on a rotary evaporator, mixed with N iyol and run on the In fra-red and la te r the Nuclear Magnetic Resonance equipment as above. (v ) Enzymes of S. bradys. ir ea— im. m \ i»r mm am mmmtm mm tmasmtmmmmmmtgppt nmvsmism About 5 ml o f the massed nematodes (obtained from i ) in 1% sodium chloride solution were placed and homogenized fo r 30 minutes in a 1 5 ml Ten-Broeck ground glass tissue grinder held in an ice bath. Microscopic examination revealed that a l l the nematodes were macerated. The homogenates were then f i l t e r e d , and the clear nematode extract was assayed for pectinases, ce llu lases , amylases and invertases. ( v i ) Assay systems for hydrolytic enzymes. C e llu lo ly t ic and p ec to ly tic engymes v/ere separately assayed by viscom etric methods (Levinson et a l .. 1950). Solutions of 1 % carboxymethylcellulose 142 (C ellu lose gum Type CM32) and 1% pectin in 0.03 M potassium phosphate b u ffe r , were prepared as substrates and a small amount o f toluene was added to inh ib it b ac te r ia l a c t iv ity . The pH o f the so lu tion for the ce llu lose gum was 5.0 and that o f pectin was adjusted to 6.0. About 1 0 ml of the substrate were added into a Volac No. 0507 v is co s ity p ipette which was supported in a constant temperature water bath at 27 + 0.5- oC. The in i t ia l time flow o f the substrate alone was measured. Then 3 ml o f the enzyme were added and thoroughly mixed with the substrate in the v is co s ity p ip e tte . Time flow measurements were therea fter made at f iv e minute in terva ls . Extracts from in fected yam (from which nematodes had been extracted ) clean yam, and 0.1 M Nacl were tested in the same way. The clean yam extract was used as con tro l. ( v i i ) Relationship betv/een nematode homogenate in ml and percentage drop in v is c o s ity . The a b i l i t y o f the extracts of nematode homogenate to degrade pectin using various quantities o f the nematode homogenate was tested v iscom etr ica lly . F ive d iffe ren t quantities of the enzyme were used 2.5? 5, 7.5? 10, and 12 .5 m l. About 2 .5 ml o f the enzyme w ere 143 pipetted each time in to the viscometer p ip ette containing 10 ml o f the substrate mixed thoroughly with the substrate and therea fter v iscos ity measurements were made at f iv e minutes in te rva l. The substrates for the amylase and invertase assays consisted o f 1 g of starch in 100 ml 0.02 M potassium phosphate bu ffer (pH 6 .9 )» and 5 g o f glucose in 100 ml 0.02 M potassium phosphate bu ffer (pH 5.0) resp ec tive ly . To 2 ml o f each o f the substrates, 2 ml of the enzyme from the nematode extracts and in fected yam were separately added and each mixture was incubated at between - 28°C fo r two hours. Controls o f 0.1 M Nacl solution and homogenate b o iled fo r f iv e minutes (B ' HOM) were incubated along with others. A colorim etric technique using 3 , 5 d in itro s a lic y lic acid reagent (D .N .S .A .) which reacts with reduc ing sugars to g ive a coloured solution was employed to measure amylase (Bern feld , 1955) and invertase a c t iv it ie s (Sumner and Somers, 1953). Approximately, 2 ml o f the reaction mixtures were combined with 2 ml of D.N.S.A. reagent, bo iled for 5 minutes, made up to 2b ml with d is t i l le d water and read on a colorim eter 144 operating at 525 nm. The controls were treated in the same manner. The absorbance of D.N.S.A. was subtracted from a l l readings, and the amounts o f enzymes were determined by reference to standard curves previously constructed for maltose (amylase) and glucose (in v e r ta s e ). N.B. The D.N.S .A, reagent used was prepared by d isso lv ing 1 g o f 3? 5, d in itro s a lic y lic acid in 20 ml o f 2 N NacH and 50 ml o f water. About 30 g o f Rochelle sa lt (sodium- potassium ta r tra te ) were added and the f in a l volume was made to 100 ml with d is t i l le d water. Table 10 l is t s f iv e amino acids found to be discharged by the yam nematode £>. brad.ys... By a series o f chromatographic analyses, the amino acids detected in incubation solution were iso -leu c in e , leucine, aspartic acid, hydroxyinol acetic acid, and phenylalanine. Phenylalanine was found to be present in appreciable amounts. From th is in vestiga tion , i t appears that _S. bradvs discharges both essen tia l and non-essential amino acids. The essen tia l amino acids discharged were leucine and iso-leucine whereas the non-essential amino acids discharged were aspartic, hydroxyinol acetic acid and phenylalanine. 145 TABLE '10 SUBSTANCES DISCHARGED BY S , BRAD! 5 AMINO ACIDS DISCHARGED INTO AQUEOUS GLUCOSE SOLUTION BY SURFACE-STERILIZED NEMATCDES 3 , BRADY’S INCUB/JED AT 30°C FOR U8 HOURS Amino acid Scutellonema bradys H istid in e - Isoleucine + Leuc ine Arginine - Alanine - V aline - Glycine * Aspartic acid + Tyrosine — Threonine — Methionine — Hydroxy p ro line - Lysine - Serine Glutamic acid - P ro lin e - Tryptophane - Hydroxyl in o l acetic acid + Phenyl alanine ++ Urea - N#B. * + Amino acid present +4- Amino acid present in appreciable amount - Amino acid absent,* 146 Because In su ffic ien t quantities of dry rot and nematode extracts contributed to the fa ilu re of the U.V. and I.R . spectra to g ive meaningful resu lts , extracts from clean, nematode-infeeted, and wet rotted yams had to he examined for other classes o f compounds using the N.M,R. equipment. The resu lts o f the various peaks obtained are shown in Figs 10 and 11, The solvent peaks are shown in F ig , 10 and the peaks obtained from the freeze-dried yam extracts are shown in F ig . 11 , In the clean yam extract, there is a peak at 1.0 (.05) li varfasn 60 MHs K&8 JW>*»y(tc*« in»tn#m *nt d.vtiton .6l.\ SnCTOUM WO.. PIG. 10: H.M.R. PEAKE 0E DEUTERODIMETHYLSULPHOXIDE (Dg) FIG. 12. SOME PHARMACOLOGICALLY A C TIVE CONSTITUENTS OF THE YAM TUBER (DJOSCOREA SPP.) FIG. 13. HYDROLYSIS OF A 7% PECTIN SOLUTION BY HOM OGENATES O F Scutellonema o eo r 70 - § 60 ‘o 8 50 “ & § 40 - Uj Uoj cUtj 20 ■0. 10 - _l__________ I_______ _ __ Jl__________ I____________ I 0 2 5 5 75 10 125 NEMATODE HOMOGENATE IN ML FIG. 74. RELATIONSHIP BETWEEN NEMATODE HOMOGENATE (S. brodys) IN ML AND PERCENTAGE DROP IN VISCOSITY 154 TABLE 11 Amylase a c t iv ity o f homogenates of j3̂ . bradys D is t il le d Nacl D.N.S.A, Homogenate Boiled Enzyme source water (blank) (blank) (starch - homogenate (blank) substrate) (starch-Sub) 1 . Scutellonema bradys ( i ) 0.00 0.01 0.076 0.^0 0.1+1 Cii) 0.00 0.01 0.076 0.1+6 0.1+6 2. In fected yam ( i ) 0.00 0.01 0.076 1 .15 1 .10 ( i i ) 0.00 0.01 0.076 1 .25 1 .20 155 TABLE 12 Invertase a c t iv it ie s of homogenates o f S, bradys and Infected yam D is t i l le d Nacl D.N.S.A. Homogem te Boiled Enzyme source water (blank) (blank) ( suerose- homogena te sub) (sue rose-sub) 1. Scutellonema bradys ( i ) 0.00 0.01 0.076 0.00125 0.00125 ( i i ) 0.00 0.01 0.076 0.00125 0.00125 2. In fected yam ( i ) 0.00 0.01 0.076 0.005 0.005 ( i i ) 0.00 0-.01 0.076 0.005 0.005 156 PLATE 30: A technician operating the Varian T-60 N.M.R. Spectrometer. 157 Apparatus for viscom etric measurement o f PLATE 31 : pectinase enzyme. 158 3.1 0 FUNG-1 ASSOCIATED WITH THE DRY HOT DISEASE OF YAM TUBERS. To iso la te the fungi ’which may be associated with ’ dry r o t ’ disease o f yams, tubers o f D, rotundata. D. alata and D. cavenensis showing recognisable symptoms o f dry rot disease were investigated . As a control healthy tubers were also used. The iso la tion o f fungi was done in two ways. ( i ) Small pieces o f diseased and healthy tissue were removed a sep tica lly (using a flamed s ca lp e l), p lated on potato dextrose agar (P.D .A .) in oven-dried p e tr i dishes and incubated at 25°C for 14 days. The fungi that grew were sub cultured on P.D. A . to obtain pure culture o f the is o la te s . These fungi were la te r id en tifie d as fa r as possib le, ( i i ) The second method involved keeping the yam pieces in oven-dried p e tr i dishes inside humidity chambers. During 14 days of incubation, the fungi that grew were subcultured on to agar p lates and id en tifie d . The resu lts are showi in Tables 13 and 14. 159 I t is very in teres tin g to note from this experiment that the two methods used in iso la tin g the fungi gave d iffe ren t resu lts . By the d irect p la ting o f diseased portions o f the dry rot tissue on agar, only two fungi were iso la ted even though profuse m ycelial growth was observed. Fusarium oxvsporum Schlecht and Rhiz o p u s nigricans Ehrerib. were iso la ted by th is method. From the other method involving keeping the yams in humidity chambers for 1 kdays before p la ting the diseased tissues on agar, the fo llow ing fungi were is o la ted ;- Aspergillus niger van T iegh j Botr.vodiplodia theobromae P a t., Fusarium moniliforme var subolutinans Woron and Reinking., Pen icillium sclerotigenum Yamamoto,, Trichoderma v ir id e Link ex F r ., and Rhiz o p u s nigricans Ehrerib. A sperg illu s . PenciIlium . Rhiz o p u s and Fusarium sp. were the most commonly iso la ted . Pen icillium sp. were more commonly iso la ted from yellow yam (D. cayenensis ) and the two Aspergillus sp. were not host s p e c if ic . The fungus was iso la ted from a l l the three yam species. 160 TABLE 13 FUNGI ASSOCIATED WITH DRY ROT DISEASE OF YAMS FUNGI ISOLATED FROM DRY ROT PORTIONS Off YAMS BY DIRECT PLATING METHOD Fungi D. rotundata D. cavenensis D. alata Aspergillus niger : - - - - Aspergillus sp. - - - Botrvodiplodia theobromae - - - Fusarium moniliforme - - - Fusarium oxysporum + + Pen lc illium sclerotigenum - - Pen icillium sp. - - - Trichoderma v ir id e - — - Rhizoous nigricans + + + Fungi iso la ted - Not iso la ted Profuse m ycelial growth without spores observed on most p la tes . 161 TABLE 1 k INFECTED YAM PIECES KEPT INSIDE HUMIDITY CHAMBERS FOR 1U DAYS AND PLATED OH TO ACtAR Fungi D. rotundata D. cayenensis D. a lata m ii i , i, , i, , t m 'm i -in * * — . i i . « , " , Aspergillus n iger + 4 4 Aspergillus sp. 4 4 4 Botryodiplodia theobromae 4 - - Fusarium moniliforme 4 - - Fusarium oxysporum 4 4 4 Pen icillium solerotigenum - 4 - Penicillium sp. 4 4 4 Trichoderma v ir id e 4 - 4 Rhizopus nigricans + 4 4 + Fungi iso la ted . Fungi not iso la ted 162 3.11 THE IHT ERRBLATI ON SH IPS OP SCUTSLLOmiA BRADYS AHD gum I AS 3 PC I/JED WITH WET ROT Off YAMS . Plant p a ras itic nematodes are often regarded so le ly as pathogens in their own r igh t, capable of producing a recognizable disease condition. Such a concept may be v a lid in some cases but not in a l l cases. There is now an increasing awareness espec ia lly amongst those concerned with plant protection , that symptom expression o f a particu lar disease in plants may not be a ttribu tab le to one pathogen only but that often a group o f concurrent pathogenic in fections may produce disease conditions. By ca re fu lly contro lled experiments, possib le in teraction between J3. bradvs and some fungi previously iso la ted from yam were studied with the hope that the resu lts would elucidate the ro le o f various organisms and such complex interactions and indicate the basis fo r the development o f an e f fe c t iv e disease control programme. ( i ) Greenhouse Experiment. One greenhouse experiment was conducted to study the e f fe c t o f the in teraction of S. bradvs and Aspergillus niger on the growth o f water yam (D. a la ta ) 163 and the fungus were tested on water yam as fo llow s: Nematode-free yam setts of water yam ( Dioscorea ala t a ) o f approximately equal weights (50 g ) previously surface s te r il is e d fo r 20 minutes in 1 :1 0 commercial bleach (C lo rox ), and rinsed in s t e r i le d is t i l le d water several times, were planted in au toc lave-ster ilised sandy loam s o i l . The autoclaved pots were wrapped up in s ilv e ry paper and maintained in a d is in fected greehouse. S te r ile water was used for watering the pots using sp ec ia lly adapted s te r i le tes t tubes embedded inside the pots. The tops of the te s t tubes irere covered with absorbent cotton wool to le t in a ir , and to prevent or minimize the entry of contaminants. The fo llow in g treatments were applied to the pots at three treatment times, i . e . at p lanting; two months a fte r p lanting and three months a fte r p lanting. Bach treatment was rep licated s ix times. Treatment A: Pots were inoculated with approximately 500 males and 500 females o f Scutellonema bradvs alone. Treatment B: Pots were inoculated with the fungus Aspergillus n ig e r . 164 Treatment C: Pots were inoculated with "both was inoculated three weeks a fte r the nematodes). Treatment K - Control: No nematode, no fungus. For the nematode inoculation, a nematode suspension containing roughly 1000 surface s te r i l iz e d male and female adults o f j3. bradys was p ipetted into the s o i l through the embedded tes t tubes. The emerging vines o f the tuber were supported with twines in place of the usual yam stakes. A fte r s ix months, the yams were harvested and assessed for the fo llow ing: (a ) Incidence o f dry ro t. (b ) Tuberization and dry ro t in teractions. (c ) The e f fe c t o f the nematode/fungal in teraction on nematode development. (d ) The e f fe c t o f the nematode/fungal in teraction on the establishment of the fungus. ( i i ) Stora This experiment was conducted with UO healthy tubers o f D* rotundata. The yam tubers were previously surface s te r i l iz e d by three washings in 1 :1 0 commercial 165 Clorox. Three fungal species - namely, n ic e r . Pen icillium sclerotigenum and Fusarium oxysporum. previously iso la ted from ro ttin g yam, and the yam nematode £>. hradvs were tested for pathogenicity by inoculation into healthy yam tubers according to the method described by Okafor (1966). One cy lin d rica l core was removed at the middle o f each yam tuber with a s te r i le 2 cm cork borer. F ive mm discs of J-d.ny old fungal cultures were placed into the holes in the tubers, and the cores o f yam from the tubers were replaced. Inoculation o f the surface s te r i l is e d nematodes was done using small micro p ip ettes . The replaced cores were then waxed and further sealed with adhesive tapes. The fo llow ing treatments were applied to the yam tubers: 1 . Inoculation with the nematode Scutellonema bradys alone. 2. Inoculation with the nematode Scutellonema bradys and Pen icillium sclerotigenum. 3. Inoculation with the nematode Scutellonema bradys and Fusarium oxysporum. h. Inoculation with the nematode and A sperg illu s n ig e r . 5. Inoculation with the nematode _S. bradys + Pen icillium + Fusarium and A sp erg illus. 166 6. Inoculation with the nematode Fusarium oxysporum alone. 7. Inoculation with the nematode Pen icillium 8. Inoculation with the nematode A sperg illu s n iger alone. 9. Inoculation with dry ro t extract. 10. Inoculation with s te r i le d is t i l le d water as con tro l. Bach of these treatments was rep lica ted four times. (One spot was inoculated per tuber and four tubers were used fo r each treatm ent). The yams were then stored in a yam barn for three months and afterwards an assessment o f the fo llow in g was made: (a ) The ro le o f Q. bradys in dry rot formation. (b ) The ro le o f fungi in dry rot formation. (c ) The combined e ffe c ts o f the nematode and each o f the fungus, and a l l the three fungi A fter 3 months o f storage, the tubers were cut open from the s ite o f inoculation and photographs o f the in fected areas Yrere taken. P lates 33 3k> and 35 the yams that have been inoculated with Fusarium P e n ic il l iu m sc lerotigenum. and Asp. and Plates 36, 37 9 and 38 show the yams that have been 167 inoculated with the nematode S. bradys and Fusarium oxysporum. nematode + Pen icillium sc1erot ig e num and nematode + Aspergillus n ig e r . P late 39 shows yams that were inoculated with S. bradys and a l l the three fungi, and P la te 1+0 shows the yam that was inoculated with the dry ro t extract only, P late U1 shows the yam that was inoculated with 3. bradys alone. In the f i r s t case- where each fungus was inoculated alone, penetration o f fungal mycelium was res tr ic ted despite the fa c t that a form o f ’ wound' had been caused a r t i f i c ia l l y using a cork borer, a general m ottling o f the yam was, however, observed. But in the second case, where both the nematode and fungus were inoculated in pairs simultaneously the in fection spread was much greater espec ia lly where nematodes were inoculated with P en ic iliu m and Ausarium species. Where the nematodes and a l l the three fungi were inoculated, the depth o f penetration was also much enhanced. But with A. n ig e r , the presence o f the nematodes did not influence the depth o f penetration o f the fungus in any p os itive way. In th is case where the nematodes were inoculated alone, in fec tion was sim ilar to that previously described as ’ dry r o t ’ recognised by the yellow ish necrotic lesions res tr ic ted to the c e lls beneath the 168 ELATE 32. A nematolo^r greenhouse showing the experiment for the study o f in terrela tionsh ips between A. n iger and 3. bradys. 169 170 PLATE 3b. Yam in o c u la te d w ith P a n ic i l l iu m sc le ro tig en u m o n ly . 171 PLATE 35. Yam in ocu la ted w ith A s p e r g i l lu s n i o n ly . 172 PLATE 36. Yam in o cu la ted w ith 3 . b rad^s and F . oxysnomm. 173 PLATE 37. Yam in ocu la ted w ith 3 . Lradys and P . sc le ro t igen u m . 174 PLATE 38. Yam in ocu la ted w ith S . hradys ard A . n ig e r . 175 PLATE 39. Yam in o c u la te d witti £». L r.a d ^ s, oxysmaffl) P . s c 1 e r o t ig e num and A. n ig e r . 176 PLATE 1+0. Yam in ocu la ted w ith dry r o t e x t r a c t . 177 PLATS U1 . Yam in o cu la ted w ith j3. h radys o n ly . 178 periderm (P la te U1 ) . The mean fresh weights o f tubers obtained in the greenhouse experiment are shov/n in Table 15. The weights o f tubers from each treatment were subjected to s ta t is t ic a l analysis (completely randomized block design) and the resu lts show that there were no s ta t is t ic a l ly s ign ifican t d ifferences in weights o f tubers from each treatment. 179 TABLE 1 5 MEAN FRESH WEIGHT OV TUBERS IN GRAMMES GREENHOUSE EXPERIMENT -..- i r....... Treatments A B C D ■ E F urt- H I K Mean fresh weight 137 121 115 1 26 1 22 133 139 131 117 11*1 in G* *Mean of s ix rep lica tes . No s ign ifican t d ifferen ce among mean fresh weights o f tubers at a l l le v e ls in the analysis of variance. Treatments. A Yam inoculated vdth S. bradys at p lanting. B tf tf tt A. n iger at p lanting. C I f tf tt S. bradvs and A. n^ger at p lanting. D tf tt ft S. bradvs 2 months a fte r p lanting. E tf tf ft A. n iger 2 months a fte r p lanting. P tf tf ft S. bradys and. A. n iger 2 months a fte r p lan ting. G tf ft ft S. bradys 3 months a fte r planting. H tt tf It A. n iger 3 months a fte r p lanting. I tt tf ft S. bradys and A. n iger 3 months a fte r p lanting. /m _ i _ _ -i • t t f____ ______r _ 180 Nematodes were extracted from the tubers, roots and s o i l a fte r harvest. Nematodes were extracted from 50 g o f tuber from each treatment using the method described ea r lie r* . Nematode extraction from the roots was done by cutting 10 g fresh roots from each treatment, into m illim etre pieces and these were macerated in 100 ml o f water fo r 3 minutes in a Waring blender. These were la te r extracted into small p e tr i dishes using sieves o f nylon gauze embedded in r ig id polyethylene supporting rings. Most nematodes were recovered a fte r 2+8 hours, and only one species o f nematode (,3. bradys) was recovered from yam roots extracted. The s o il extraction procedure was sim ilar to that used for the tubers, but in th is case, 200 cc o f s o il from each treatment were extracted fo r 2+8 hours. The resu lts o f nematode population in the tuber, root and s o il fo r each o f the treatments are presented in F ig . 15. In treatments A, D, and G, i . e . where nematodes were inoculated at p lanting, 2 months a fte r planting and 3 months a fte r p lanting resp ec tive ly , fewer nematodes were extracted from the s o i l , and greater numbers o f nematodes were extracted from the tubers and the roots. I t was c lea r that 181 m ajority o f the nematodes entered the roots and tubers and reproduced in them. The average population increase was o f the order o f 3.8x, 3x and 3x fo r treatments A, D, and G, resp ec tive ly . But in C, F, and I , i . e . where the nematodes and A . niger were inoculated at p lanting, 2 months a fte r planting and 3 months a fte r p lanting, much greater numbers o f nematodes were extracted from the soi}.. This implies that fe?/er numbers of nematodes entered and reproduced in the roots and tubers. No nematodes were extracted in the tubers, roots and s o il from control p o ts . Fungi were iso la ted from yam tubers and roots by p la ting small pieces o f surface s te r i l iz e d yam roots and tubers from a l l the treatments and control on agar (P .D .A .) and incubated at 25°C for a period o f two weeks. Some o f the p lates contained a mixed population of fungi and were subcultured and incubated for a further period o f two weeks before id en tific a t io n . The l i s t s o f fungi iso la ted are shown in Tables 16 and 17. 182 Fungi were iso lated from s o i l by two methods: (a ) S o il d ilu tion and (b ) D irect p lating methods In the s o i l d ilu tion method, 1 0 cc o f s o i l from each treatment were thoroughly mixed in 100 ml o f water in a volumetric fla sk and afterwards the fo llow ing s o il _1 d ilu tion series were prepared from th is , namely, 10 , 10“ 2, 10-3, . . . . . . 10-7. Using graduated p ip ettes , 1 ml o f each o f these d ilu tions was p ipetted into agar and incubated at 30°C fo r two weeks. In the d irec t p la ting method, about 0,5 g of s o il (from each treatment) was made in to a paste, sprinkled in each agar p late and incubated for two weeks. By both the s o il d ilu tion and d irec t p la tin g methods, two species o f fungi were found predominantly in the p lates and these were Aspergillus niger and Fusarium oxysporum. TREA TM EM IS A Y a m inoculated with nematodes at planting. 5.000- C'rYam inoculated with nematodes and fungus (A - ruger)a t 4.500- D:-Yam inoculated with nematodes 2 months 4.000 after planting. to UQj 3500- F:-Yam inoculated with nematodes and 0 t - . fungus (A - )re2ngi months after planting. 3.000- Lu G'-Yam inoculated 'with nematodes 3 months 2.500- after planting. 1 Yam inoculated with nematodes and Oc 2.000 fungus (A - nicer) 3months after plantingUCOl 1500- 1 :y§1.000- Soil Tuber 50 0 - : I Root A T R E A T M E N T S FIG. 15. E F F E C T O F ASPER GILLUS M G E R ON THE POPULATION O F SCUTELLONEMA 184 TABLE 16 m i ISOLATED FROM YAM TUBER Treatments ‘’ungi iso la ted A Rhizopus spp. and Fusarium sp. B Aspergillus n iger and Rhizopus sp. C Aspergillus n ig e r„ Pusarium sp. and Rhizopus sp, Rhizopus spp, and Fusarium sp. E Aspergillus n iger and Rhizopus spp, F Aspergillus n iger and Fusarium G Rhizopus spp. H Rhizopus spp, and Fusarium sp. I Aspergillus n iger J Rhizopus spp. and Fusarium spp. 185 TABLS 17 PUHII ISOLATED FROM YAM ROOTS 186 3.12 COMPARISON OF POSSIBLE NEMATICIDAL EFFECTS OP A. NIGER„ P. SCLEROT IGENUM AND F. OXYSPORUH. In the previous experiment involving Aspergillus niger and S. bradvs„ the fungus did not support the development o f a large population o f the nematode hence i t ?/as thought that th is was due to a possib le an tib io tic action o f A. n ig e r . To investigate th is further, a study was carried out to compare the anti-nematode e ffe c ts o f three fungi previously iso la ted from yams - A. n iger (P la te I+2), P. sclerotigenum (P la te 3k) , and F . oxysporun (P la te The fungi ?/ere groT/n in Potato Dextrose Agar (P .D .A .) for 21 days and f i l t r a t e s o f the fungi were obtained by gently washing the spores into s te r i le d is t i l le d water. The spores were diluted with d is t i l le d water to obtain the fo llow ing f iv e d ilu tions (20,000, 15*000, 10,000, 5*000, and 1 ,0 0 0 spores per m l). Suspensions o f SI. bradys were added to the d iffe ren t spore concentrations in v ia ls and incubated at 2S°G. Three rep lica tes o f each spore concentration were prepared for each fungus and a control series o f d is t i l le d water. The percentages o f immobilised nematodes were determined a fte r 12 hours. The resu lts o f 187 th is investiga tion are shown in F ig . 1 6 . A fter 12 hours incubation, the nematodes were examined under a stereomicroscope and the percentage number of nematodes k il le d or immobilized was determined. Of the three fungi, Aspergillus was most active at spore concentrations between 15»000 and 20,000 when and 55%> o f the nematodes were k ille d resp ec tive ly . The maximum percentage numbers of nematodes immobilized by Pen icillium and Fusarium species were and 25% respective ly . Immobilized nematodes did not recover when removed from Aspergillus niger spores and la te r d ied. The le th a l and immobilizing e ffe c ts o f spores o f Aspergillus on JsU bradvs may be due to some tox ic substances being produced by the fungus. w 60 s -oc a fe c 50O i fc 40 Cb A: Vo. JO *X i 10 q----- • Control ~im ~ l[6ob 10,000 k 'oqo No of spores per ml. FIG. 16. AC OMPARISON O F THE POSSIBLE PROPERTIES O F A- niger, P. sclerotigenum AND JF- oxyspcrum 5 20 ' i •0S c1: Si f £ 189 PLATE 1+2. C u ltu re o f A . j+ lg e r . 190 PLATE 2+3. C u ltu re o f P . s_slg r g t lEgBBffl. 191 PLATE U4. C u ltu re o f F . 192 3.13 HOST RANGE STUDIES OF THE YAM NEMATODE - SCUTELLONSMA BRADYS. Autoclaved sandy loam, consisting o f 3 parts o f loam and one part o f aand were thoroughly mixed, potted and planted to 30 test plants. The seedlings o f these tes t plants were inoculated each around the roots with a suspension o f about 500 - 1000 eggs and larvae o f S. bradys. A l l plants were maintained in p la s tic pots in the greenhouse. A fter 30 days, the roots of the plants were harvested, washed and 1 0 g from each tes t plant was communited in a Waring blender fo r 30 seconds. The communited roots were then extracted into small p e tr i dishes using sieves o f nylon gauze embedded in r ig id polyethylene supporting rings. Estimation o f nematodes was made a fte r 1+8 hours. Table 18 l i s t s the tes t plants in descending order o f nematode population. Host plant e ffic ien cy was rated in terms o f the highest nematode population increase. Twenty out o f th ir ty te s t plants were found to be su itable hosts for J3* bradys even though only 3 plants supported a s ign ifican t population increase. Substantial population increase were obtained with Benniseed, Cowpea (var New Era) and Cowpea (var I fe Brown). The high rate of reproduction on cowpea 193 is o f considerable importance. Four other legumes were found to be moderate hosts to th is nematode. These were Yam bean, Green gram, Pigeon pea and Pueraria. Of equal in terest is that tobacco, cotton, groundnut and maize did not support the nematode. TABLE 18 HOST RANGE STUDIES OF S. BRADYS No. o f Host Common names nematodes plant o f plants S c ie n t ific names per 10 g e ffic ien cyroot tissue rating 1. Benniseed Sesamum indicum Linn. 2,5U0 Good 2. Cowpea ( var ’ Nev. Era’ ) Vigna unguiculata (L „ ) Wain. 1 ,661+ -do- 3. Cowpea (var ’ I fe Brown’ ) Vigna unguiculata (L . ) Walp. 1,196 -do- k Sriosema Eriosema nsoraleoides (Lam.) G. Don 31+2 Moderate 5. Siam weed Eunatorium odoratum Linn. 252 -do- 6. Yam bean Sphenostylis stenocarpa ■(Hochst ex A. Rich) Harms 239 -do- 7. Synedrella (s common weed) Synedrella nodiflora Gaertn. 208 -do~ 8. Green gram Vigna aureus Xl TT ” 172 -do- 9. Mallow |Urena lobata Linn. 157 -do- 10. Pigeon pea Ca.ianus ca.lan (L . ) Druce 152 -do- 11 . Okra Abelmuschus esculentus Linn. 1 1+3 -do~ 12. Kenaf Hibiscus cannabinus Linn. 11+0 -do- 13. Loofa gourd Luffa aegyntiaca M ill 135 -do- 1 k Soko Celosia argentia Linn. 131 -do- 15. Tomato Solanum lyconersicum Linn. 1 29 -do- 16. Melon Cucurbita neno L. 1 28 -do- 17. Pueraria Pueraria nhaseoloides (Roxh.) Benth. 118 -do- 18. Roselle Hibiscus sabdariffa Linn. 1 16 -do- 19. Jute Corchorus o lite r iu s L. 97 Poor 20. Guinea corn Sorghum spp. 96 -do- 21 . Mai 7e Zea mays - Non-host 22. Tobacco Nicotiana tabacum Linn. - -do- 23. Indian spinach. Basella alba Linn. - -do- 2k. Cotton Gossypium hirsutum Linn. - -do- 25. Groundnut Arachis hypogea L. -do- TABLS 18 (c o n t ’ d ) No. o f Host Common names S c ien t ific names nematodes planto f plants per 10 g e ffic ien cy root tissue rating 26. Water le a f Talinum triangulare (Jacq.) Wild -do-* 27. Tridax Tridax procumbens Linn. i - -do*- 28. Hot pepper Capsicum frutescens Linn. — -do- 29. Snaxe gourd Tricosanthes anguina - -do- 30. Green tete Amaranthus v ir id is Linn. -do- N.B. Host plant e ffic ien cy rating Good host a number increase o f 2 - 3 times Moderate entry accomplished Poor entry just accomplished Non-hcst entry o f nematode into the root not accomplished. 195 196 3.1 h CONTROL PI* SCUTELLOHEMh BRADYS BY HOT ViL̂ ER TREATMENT c Because of the s im p lic ity and inexpensive app lication o f hot water treatment, th is method has been proposed by a number o f workers as a means o f elim inating nematodes from yam tubers (Bridge, 1972). Treatment is carried out by the immersion of the tuber in hot water at a known temperature fo r a spec ified length o f time using a therm ostatically contro lled hot p la te . Besides the e ffe c ts o f hot water treatment on nematode m orta lity , i t is important to determine the e ffe c ts of such treatment on germination, growth, y ie ld , p a la ta b ility and storage o f yam tubers, ( a ) E ffe c t o f hot water treatment on nematode m ortalit: The in i t ia l countsof nematode population were made using 50 g o f nemo tod e-in fee ted tubers o f water yam (£ . a la ta ) and yellow yam (D. cayenensis) , Each extraction was rep lica ted f iv e times. Therea fter, the in fected tubers were subjected to the fo llow ing treatment temperatures, ( i ) Heating from cold to 50°C ( i i ) Hot water at 55 - 55°C for UO minutes ( i i i ) Hot water at 55 - 60°C fo r kO minutes. 197 A fter treatment, the tubers were allowed to cool fo r 2k hours, then and the resu lting live-nematode count was taken. These treatment temperatures were selected from the in i t ia l laboratory screening experiments conducted on the e f fe c t o f hot water dip on germination and growth of yam tubers. (b ) E ffe c t o f hot water treatment on storage. Hot water treatment at 50 - 55°G for i+O minutes gave consistently good results of nematode k i l l without apparent danger to tuber growth, hence i t was selected fo r th is in vestiga tion . Eighty yam tubers of D. rotundata were subjected to hot water treatment at 50 - 55°C between October 1972 and March 1973, Twenty yams each were treated on four d iffe ren t occasions as fo llow s: ( i ) Immediately a fte r harvest in October. ( i i ) Immediately before the November planting (Early p lan tin g ). ( i i i ) In January, i . e . a fte r k months o f storage and before February p lanting (Late p lan tin g ). ( i v ) A fte r dormancy has broken - March i . e . a fte r 6 months o f storage. Twenty untreated yams were stored under the same conditions as con tro l. The treated and untreated yams were 193 stored in the yam tarn and observations on ro ttin g and other e ffe c ts were carried out weekly. Arabient temperature and humidity in the yam barn were'"recorded using the Cassela-type thermohygrograph. (c ) E ffe c t of d iffe ren t hot water treatments on germination, growth and y i e l d o f water yam . 1 ( D. a la ta ) t The three treatment temperatures in (a ) were employed in th is experiment. A fte r trea ting the tubers of water yam at these temperatures, the tubers were cut in to setts weighing approximately 1 50 g. These yam setts v/ere then 2 planted on a 99 m p lo t in the Teaching and Research Earm. The layout was a completely randomised block design. (d ) P a la ta b ilitv and accep tab ility te s ts . A fte r harvest, the tubers from (c ) were assessed using a panel o f ten tasters and assessors. The fo llow ing qu a lities were assessed - ( i ) external appearance o f Y/hole tubers ( i i ) in terna l appearance when cut, ( i i i ) taste and appearance of yams when bo iled with s a lt , ( i v ) pounding qua lity , and (v ) peeling losses . The resu lts o f the e f fe c t o f d iffe ren t hot water treatments on nematode m orta lity are shown in Table 19. Hot water 199 treatment o f nematode-infected tubers o f D. alata and D. cayenensis at temperatures between 50 and 60 fo r i+0 minutes completely elim inated the nematode population. Heating yam tubers from cold to 50°C a lso s ign ific a n tly reduced nematode population, but did not completely d is in fes t the tubers. EFFECT 0? DIFFERED HOT WATER TRT^JgHTS ON ITTL'-TODT: MORTALITY Mean nos o f nematodes per 50 g tuber** ( + Standard E rro r) Percentage k i l l E ffic ien cy rating Treatment D. a la ta D. cavenensis D. a la ta D# cavenensis (/») Heating to 50°C from c o l l 200+1 U.1 5Q+7 99.98 99.99 Good (B ) 50 - 55°C fo r kO mins N il N il 100 100 Y . Good (C ) 55 - 60°C N il N il 1 00 - 1 00 V . Good (D ) Control 1 0,000+1 00 U ,500+67 - - =f*Means o f f iv e rep lica tes 201 Table 20 shows the e f fe c t o f the timing o f hot water treatment (50 - 55°C fo r kO mins) on the deterioration o f tubers. A l l the yam tubers which were treated with hot water immediately a fte r harvest, i . e . in October rotted completely before the end o f the storage period. Those tubers treated a fte r 2 and 6 months o f storage showed very l i t t l e signs o f deterio ra tion , and hot water did not appear to a ffe c t th e ir sprouting p o ten tia l. TABLE 20 EFFECT OF THE TIMING 0? HOT WATER TREATMENT (50 - 55°C FOR hO MINS) ON THE DETERIORATION OF TUBER3 DURING- SUBSEQUENT STORAGE T Percentage no, o f D iffe ren t treatment times rotted yams at Percentagethe end o f sprouting storage period ( ! ) October (immediately a fte r harvest) 1 00 1 00 ( I I ) i . e . Late November before early planting i . e . a fte r 2 months o f storage 20 1 00 ( i l l ) January i . e . a fte r h months o f storage 20 1 00 ( IV) March (a f t e r dormancy „ >, has broken i . e . a fte r 6 months o f storage 5 1 00 (V ) Control i* e . untreated 15 1 00 202 The resu lts o f the e f fe c t of d iffe ren t hot water treatments on germination, growth and y ie ld of water yam are shown in Table 21. A comparison o f the treatment means using Duncan’ s M ultip le Range Test is shown in Table 22, and the table o f the analysis of variance is shown in Appendix 1 . A l l the treatment temperatures except those between 55 and 60°C did not have any adverse e f fe c t on percentage emergence, growth and y ie ld o f tubers o f water yam (D. a la ta ) . Treatment with hot water at 55 - 60°C completely inh ib ited emergence and no y ie ld s were obtained from th is treatment. TABLE 21 EEPECT 0? PI1?™EREIN'T HOT WATER TREATMENTS ON GERMINATION. GRO.VTH,, AND YIELD 0 ̂ WATER YAM(JD. RLATA) Treatments Percentage Mean y ie ld in Mean nos o f nematodes emergence (k g )* * per 50 g tuber Rating+ S.E. (A ) Heating to 50°C from cold 75 16.5 280+16.7 Good (B) 50-55nC 90 25.1 N il V. Good (C) 55-60°C 0 0 - Poor (D) Control i . e . Untreated 95 35.9 3,i4+0+58.7 Poor *Means o f f iv e rep lica tes . **Means of twenty tubers. 203 204 TABLE 22 COMPARISON 0? TREATMENT MEANS USING DUNCAN’ S MULTIPLE RANGE TEST D = 9.0a B = 6.3b A = A.1b Means followed by the same le t t e r are not s ign ific a n tly d iffe ren t at 5% le v e l . 205 Table 23 shows that yams treated at temperatures between 50 and 55°C fo r 1+0 minutes were accepted by m ajority of ta s te rs . Those tubers heated from cold to 50°C were rated next. The untreated contro l were the lea s t acceptable and were re jected by most people because o f the unattractive appearance, taste and high percentage of ined ib le portions. TABLE 23 PAL AT ABILITY AND ACCEPTABILITY TESTS Grading Heat from cold 50 - 55°C to 50°C fo r 1+0 minutes Control A 86 92 - B 8 - C - - 17 D - - 83 Results are expressed as percentage of tasters A - Very Good B - Good C - Pair D Poor 206 3.15 CULTUEkl, .JMD CHAMICAL METHODS 0? CONTROL 07 THE YAM NEMATODE SCUTELLONSMA BRAIDS. This t r ia l was set up in the Crop C o llection Garden of the Department o f Agricu ltura l B iology, University of 2 Ibadan. The p lo t used was approximately 851 m containing 120 yam heaps. The heaps were set approximately 1 m apart. Each o f these heaps had e a r lie r been a r t i f i c ia l l y inoculated with peels o f nematode-infected yam tubers. The heaps were watered da ily to fa c i l i t a t e the establishment o f the nematodes. A fte r a week, various treatments for nematode control were applied. There were s ix treatments each of which was rep licated twenty times in a completely randomized design. The fo llow ing treatments were applied to the heaps. 1 . D-D Mixture (1 , 2-dichloropropane and 1 , 3-Uichloropropene in the liqu id form was applied at a depth o f 1 5 cm at the rate of 25.3 l i t r e s a . i . per hectare using the conventional ch isel-type app lica to r ). 2. Organic manure: 1.5 kg o f organic manure was (Cow dung) thoroughly mixed with each heap (1886.3 kg/ha. ) . 207 3. Wood Ash: This was used to coat yam setts before p lanting. 2+. Nemagon (D .B .C .P .): Mixture o f 1 , 2-dibromo-3-chioro propane in the granular form, was applied at -the rate of 112+ g per heap (35.2 kg a . i ./ h a .). This was also thoroughly mixed with the s o i l . 5. NPK f e r t i l i z e r : Nitrogen (in form of NH^(S0^)2) was applied at the ra te of 100.8 kg/ha. Phosphate (in form o f PgO^) was applied at the ra te o f 67.2 kg/ha and Potassium (in form of KNO )̂ was applied at the rate of 33.6 kg/ha. 6. Control. The s ix treatments except (3 ) were applied in advance o f planting to g ive time for the tox ic materials to d iffu se out o f the s o il and for the organic matter to decompose. Yam setts were planted two v/eeks a fte r applying these treatments. The y ie ld o f water yam (D. a la ta ) and nematode population as influenced by chemical and cu ltural methods o f control are presented in Table 22+ and a comparison o f 208 the treatment means using Duncan’ s M ultiple Range Test is shown in Table 25. Treatment with organic manure gave the highest y ie ld , followed by wood ash. The control was ranked th ird and D-D, N.P.K. and Nemagon came fourth, f i f t h and sixth , resp ec tive ly . In th is in vestiga tion , the y ie ld o f water yam was increased, and the nematode population depressed by the use of organic manure. D-D was in e ffe c t iv e both in terms o f y ie ld and reduction o f nematode population. Wood ash was e f fe c t iv e in terms o f y ie ld but only s lig h t ly e f fe c t iv e in reducing nematode populations. Application o f N.P.K. considerably reduced nematode population but surprisingly did not increase y ie ld . Although nemagon was very e f fe c t iv e in reducing the nematode populations i t a lso caused s ign ifican t depression o f y ie ld s . This suggests that nemagon may be phyto-toxic to yams at the le ve ls used. TABLE 2k YIELD 07 M iR Y.AM (DI03C0REA ALATA) AM) NBI1'-TPPL POPULATION AS INFLUENCED BY CHEMICAL AM) CULTURAL METHODS 0? NEMATODE CONTROL. Treatments Rate o f Y ie ld in Mean nos o f nematodes application kg/hectare' + S.B./(50 g tuber)*** (1 ) D-D. Mixture o f 1 , 2-dichloropane and 25.3 litres/h a . 2326.k U?870+6U.79 1 , 3-dichloropropene (liq u id form) (2 ) Organic manure 1.5 kg/heap 3357.5 1 ,U1 0+37.55 (1886.3 kg/ha) (3 ) A/ood ash. To coat yam ’ s e tts ’ before 2628.2 3,350+57.8 planting (k ) Nemagon Mixture o f 1 j 2-dibromo-3- 35.22 kg/ha chloropane (granular form) 1169.5 260+16.1 2 (5 ) N.P.K. (Mixed as N=1 00.8 kg/ha f e r t i l i z e r ) P= 67.2 kg/ha 21 00 81 0+28. U6 K= 33.6 kg/ha (6 ) Control Untreated 2A6U.7 1h,U80+120.3 **:. bradys has always been referred to in the older lite ra tu re as the yam nematode., i t is gettin g c lear in recent surveys carried out in various parts o f the world that the nematode genus or species associated with dry ro t o f yam tubers varies from one geographical location to the other. West (1 93 )̂> Steiner and Buhrer (1934) associated 3. bradys with yam rot in N igeria and West Indies resp ec tive ly . Schieber and Lassmann* (1961 ) found that the root-knot nematode was the p rin c ipa l cause o f damage to yam tubers in Guatemala. Unny and Jerath (1965) found S. bradys. M. incogn ita , and M. .javanica as the most important endoparasitic nematodes o f yams in Eastern N igeria . Bridge (1973) found _S. bradys as the only nematode of economic importance in Western N igeria . On the other hand, Thompson et a l . (1973) reported P, coffeae as the prin cipa l nematode associated v/ith yam 221 tuber decay in the West Ind ies. The survey resu lts from th is s tudy reveal that both S. bradys and M. incognita are important in yam tuber decay in the Mid-Western State o f N igeria and that th is degree o f importance varies w ith lo c a lity and yam species or cu lt iv a r . Reference to S. bradvs as the ’ yam nematode’ is therefore l ik e ly to assume only lo ca l s ign ificance or become obsolete as further surveys may reveal other nematode genera which are equally important in yam decay. The Mid-Western State probably has the poten tia l to produce more yams but pest and disease problems are lim itin g yam production. Despite the fac t that the proceeds from the yam farms form tbs bulk o f the farmers’ annual income (Consolidated Results of Crop Estimation Surveys, 1968-1970 th is problem is not being given serious consideration. Storage losses are serious and could in h ib it the development o f the yam industry i f not prevented or con tro lled . The costs of nematicides are p roh ib itive and as has been shown in th is study nematicides may not necessarily be e f fe c t iv e and cannot be recommended to the peasant farmer. The use o f nematode-free yam ’ seeds’ should be encouraged. Mixed cropping with other 222 susceptible crops lik e cowpea and beniseed should be avoided as they are good a ltern a tive hosts to the yam nematode; successive cropping o f yam should be avoided. Fallow or planting of non-host plants l ik e maize and tobacco for one growing season w i l l starve the remaining yam nematodes. I t should be brought home to the farmers that yam barns with b e tte r ven tila tion and storage f a c i l i t i e s are l ik e ly to enhance not only the storage o f, but also the economic returns from th e ir yam production. Methods of surface s t e r i l i z a t io n. Many workers have attempted using one method or the other to obtain axenic cultures o f nematodes, but i t appears that very few have attempted to estimate the e ffic ien cy o f th e ir treatments q u a lita tiv e ly and qu an tita tive ly . Mountain (1955) placed single females o f Prat.vlenchus penetrans in successive drops o f s te r i le water and then la te r transferred the nematodes in to streptomycin sulphate for about f i f t e e n minutes, followed by further washing in s t e r i le water. He claimed that he obtained axenic cu ltures. Zuckerman and Brzeski (1966) obtained axenic nematodes by using 0.5% Hibitane d iacetate followed by rinsing with s t e r i le water. D o lliv e r et a l. (1962) and 223 Myers (1967) independently axenised Aphelenchoides ritzemabosi (Schwartz) S teiner, and Aphelenchoides sacchari Hooper, respective ly using 0.01 aqueous mercuric ch loride. Peacock (1959) also claimed to have obtained axenic larvae from Meloidogyne egg masses by washing the larvae fo r f iv e minutes in 0.1 % le tav lon (ce ty ltr im eth y l ammonium bromide), followed by carefu l rinsing in s t e r i le water, immersion in 0.5% Hibitane diacetate fo r f i f t e e n minutes and f in a lly washed in d is t i l le d water. S te r ile inoculation o f nematodes is one o f the s c ie n t if ic methods for determining the ro le of plant nematodes as causative agents o f d iseases. There is convincing evidence that by the app lication o f Koch’ s postu lates, the complex host-parasite relationsh ip invo lving nematodes can be b e tte r understood. Obtaining axenic cultures o f nematodes is also useful in morphological and morphometric measurements which are diagnostic too ls employed in taxonomic studies. Build u p o f population. From these investigations and sim ilar investigations by Bridge (1973)* there is overwhelming evidence that 224 Scutellonema bradys has the a b il ity to reproduce fast in yams stored under ambient conditions. In Bridge’ s in vestiga tion s, increases in nematode population were recorded in 6 out o f 8 tubers o f D, rotundata inoculated with J3. brad v s . In two o f the tubers, a 1i-|.-fold increase was recorded a fte r 5 months of storage. In the second experiment, resu lts obtained for the in fected tubers were s im ilar to those obtained in the inoculated ones. Increases in nematode numbers were recorded throughout the five-month period despite the fact that the in fected tubers were selected for nematode count at random. Cool conditions i . e , 16 - 18°C did not have any depressing e f fe c t on nematode population fo r the f i r s t 3 months when compared with the tubers stored under ambient conditions. But a fte r the f i r s t 3 months, whereas the nematode number of tubers stored under ambient conditions was on the increase, the number in tubers stored under cool conditions began to show noticeable decreases. The e f fe c t o f the cool conditions began to show a fte r the f i r s t 3 months. Of equal in teres t is the e f fe c t o f the cool conditions in suppressing sprouting whereas those tubers stored at ambient conditions sprouted luxurian tly . The suppressing e f fe c t o f the cool conditions on nematode 225 number a f t e r the t h i r d month, and on s p r o u t in g ap p ea rs to be an advantage in th a t tu b e rs under such c o o l c o n d i t io n s w i l l keep f o r c o n s id e r a b ly lo n g e r p e r i o d s . W a l la c e (1 9 6 3 ) d iv id e d in to f i v e a r b i t a r y phases the in f lu e n c e o f tem peratu re on p l a n t nematodes namely (1 ) n o n - l e t h a l ..low tem pera tu res a t which a c t i v i t y i s i n h i b i t e d . (2 ) optimum temperatures, (3 ) non-lethal high temperatures at which a c t iv ity is inh ib ited . (U) le th a l low temperatures,and (5 ) le th a l high temperatures. Th eore tica lly , i t is believed that every species should have a temperature range corresponding to each of these categories, but complete data on any one species are lack ing. The temperature lim its at which a plant paras itic species reproduces or is k il le d are variab le and depend to a large extent on the host plant and time o f exposure. Results of studies on temperature rela tions of nematode reproduction in plants are sometimes d i f f ic u l t to in terprete because the host plant i t s e l f may often be a ffected by temperature. The a b i l i t y o f 3. bradys to survive and reproduce at 226 trop ica l ambient conditions in yam tubers confers some degree of success on th is nematode. The e f fe c t o f the feeding and reproduction o f S . bradys on stored tubers is the major problem in yam storage. With such high numbers o f nematodes produced at the end o f storage period, the quality of such in f ected-tuber s drops resu lting in tubers which are v ir tu a lly inedible and also unsuitable as planting m aterials. Denth o f penetration . In a l l the 5 cu ltivars o f D. rotundata examined, the bulk o f the nematode population was found mainly under the skin i . e . to a depth o f between 0 to 0.5 cm. This is in general agreement with observations made by West (1934)* G-oodey (1935) and Bridge (1972). Penetration may extend beyond the 1 cm depth as was found in three v a r ie t ie s namely * akosu *, * esinm irin* , and efon . In these v a r ie t ie s , nematodes y/ere extracted beyond the 1 cm depth in most o f the samples. In thus confining th eir destruction to the surface layer o f the yam tissues the nature o f attack o f £3. bradys resembles that o f A, dinsaci when paras itiz in g potato tubers. This parasite also does not penetrate very deeply below the skin o f potato. 227 Within the tubers, the population o f j3. hradys builds up considerably with time. This is shown by the fact that from each f iv e milimetre piece o f in fected portions o f the yam tissu e, several hundreds o f _S. bradys were extracted. These nematodes were found to occur mainly between 0-k mm o f the peridermal layer o f yam where they cause extensive c e l l damage. As a resu lt o f this in fec tion , cracks are developed on the skin and a fte r some time, i t peels o f f ea s ily . The apparent preference of j3. bradys fo r the oldest part o f the tuber i . e . top end seems to be simply because i t was the f i r s t to be formed during the process o f tuberization and remains longer under the s o il surface and probably in constant contact with the nematodes. I t is only in the la te r growth o f the yam tuber that other portions are in fected . At harvest, the top end must have been severely attacked by nematodes whereas at the middle or bottom portions, the nematode populations remain low. Information obtained from these experiments are very usefu l in many respects. Since i t is now established that S. "bradys is capable o f penetrating only to a depth o f about 1.5 cm, eradication of the nematodes in the yam tuber may be achieved by chemical or hot water d ip . Knowing 228 that the nematodes p re fe r the oldest portion o f the yam also aids in the selection o f planting m ateria l. The top end may not he used for propagation i f in fected even though i t germinates fa s te s t. The middle and bottom ends can be used for propagation, and although they tend to germinate slow ly. These two portions i . e . middle and bottom are more l ik e ly to produce b e tte r quality yam tubers than the top end. Histonathology stud ies. Most of the findings in this work are in perfect agreement with the observations and conclusions made by Goodey (1935). In Western N igeria , as many as 100,000 nematodes are found in an average dry rotted tuber. Early nematode in fections o f yam, especia lly by 3 . bradys appear symptomless to the naked eye, even though the nematodes are there in hundreds at that stage. By the time the symptoms become obvious (which is usually a fte r some months in storage ), the nematodes are there in thousands or hundreds o f thousands per tuber. This is in agreement with the recent observations by Bridge (1972) who recorded a maximum nematode population of 62,000 per 1 0 g o f tuber. Observations of diseased pieces of yam tuber under 2 2 9 the microscope and the h is to lo g ica l studies indicated that yam decay is a disease process which can he separated conveniently in to 2 main types ’ dry r o t ’ and ’ wet r o t ’ each having an early and an advanced stage, fo rtu n ate ly , the in fec tion o f IS. hradvs on white yam (D. rotundata) can he distinguish©d hy the ch aracteris tic colour i t imparts on d iffe ren t stages of yam deterio ra tion . ’ Early dry r o t ’ is associated with cream and lig h t yellow colour while 'la t e dry r o t ’ ranges from dark yellow to lig h t hrown. 'Early wet r o t ’ usually has hrown to dark "brown colour, hut ’ la te wet r o t ’ ranges from dark hrown to hlack. Throughout the course o f these in vestiga tions, only nematodes were found in stained sections o f dry rotted tuhers and fungi in wet rot sections. In the pathogenicity tests carried out hy Bridge (1973) s, the dry rot symptoms have heen reproduced experimentally hy inoculating surface s te r i l iz e d Scutellonema hradvs into clean yam tuhers. This symptom was not produced when fungi were s im ila r ly inoculated in to clean yam. The fact that Aden iji (i9 7 0 ); Ogundana (1970) found that the fungal iso la tes used could not estab lish on yams in the absence of wounds also lends support to the argument that 230 yam decay is in it ia te d by nematodes. The nematodes are good m odifiers of the yam substrate which is starch. This is reduced to simple sugars and in the presence o f these sugars the fungi grow a c t iv e ly . The 'dry r o t ’ stage is a slow process compared with the 'wet r o t ’ stage. Once the ’ wet rot* stage s ta rts , yam decay becomes very fa s t and nematodes seem to disappear. The periods ?/hen nematodes and fungi can be found side by side, in yam tissue - ’ la te dry r o t ’ or ’ early wet ro t ' are transitiona l and very short. Whereas the nematodes are confined to the outer 1.5 cm of tuber,-fungi are found in the deepest region o f the in fected tuber. Besides, fungal secretions are found several cms ahead o f th e ir advancing mycelia. Oxygen requirement may account for the more su perfic ia l penetration o f the nematodes. From the observations and explanations above, i t is obvious that yam decay is a disease syndrome involving microorganisms o f both plant and animal o r ig in . I t is also obvious that i t involves a process that can be separated into 1+ main stages 'e a r ly dry r o t ' , ’la te dry r o t ’ , ’ early wet ro t ' and ’ la te wet ro t* . Bach o f these stages can be recognised by the naked eye and is 231 a s s o c ia t e d w ith d e f i n i t e c o lo u r a t io n and c e r t a i n m ic roo rgan ism s . D is e a s e i n t e r a c t i o n s in which nematodes a re f o l lo w e d b y fu n g i to p roduce an a g g ra v a te d d i s e a s e c o n d i t io n a r e a l r e a d y w e l l documented in o th e r p l a n t t i s s u e s ( P o w e l l , 1 9 6 3 ). S tu d ie s on d e te rm in a t io n o f c a rb o h y d ra te c o n s t i t u e n t s o f the yam tu b e r have b een re p o r t e d e a r l i e r . A number o f a n a ly s e s r e p o r t e d by Oyenuga (1 955-1939), and recent o b s e rv a t io n s made in N i g e r i a b y Ketilcu and Oyenuga (1970) have in d ic a t e d th a t in the c a s e o f D. r o t u n d a t a . th e re a re two p r i n c i p a l su g a rs p r e s e n t , su c ro se and g lu c o s e and th a t su c ro se in p re sen t in g r e a t e r amounts than g lu c o s e . The r e s u l t s o f t h i s i n v e s t i g a t i o n a g ree w ith th e se o b s e r v a t io n s . A few a n a l y t i c a l r e s u l t s so f a r a v a i l a b l e a l s o con firm th a t d u r in g s t o r a g e o f yams, th e re i s m e tab o lic breakdown o f the p r i n c i p a l component, s t a r c h , hence s w e e t is h t a s t e i s d e v e lo p e d , s u g g e s t iv e o f the accum u lat ion o f su g a rs in the t i s s u e s . But in none o f th ese s t u d ie s was the accum m ulation o f s u g a rs a s s o c ia t e d w ith the p re se n c e o f d e c a y o rgan ism s nam ely , nematodes, fu n g i and b a c t e r i a which n o rm a lly a t t a c k yams in s t o r a g e . The p re s e n t study goes f u r t h e r to show th a t the 232 intereonversions o f starch to soluble sugars may be influenced by the presence o f nematodes and secondary invaders l ik e fungi and bacteria which attack yams in storage. The subsequent decrease in sugar le v e ls in the v/et rotted yam when compared with the dry rot phase indicates the possible conversion and u t ilis a t io n o f some sugars by nematodes. The conversion o f starch to simple sugars induced by the presence o f nematodes has fa r reaching consequences on the yam tuber. The sugar increases recorded in the nematode-infected yam tuber explains one o f the ways by which nematode may pre-dispose yam to in fection by secondary invaders lik e fungi and bacteria . This pre­ d isposition is done by the provision of food in form o f simple sugars (monosaccharides) for the fungi which may be associated with the dry rot o f yam. These simple sugars have been shown to support the growth o f fungal iso la tes (Ogundana et aL. 1970). In the presence o f these sugars, the fungal spores grow a c tiv e ly and la te r invade the yam tissu e. Their a c t iv it ie s resu lt in progressive deteriora tion o f the en tire substance o f the yam tubers. The invasion by fungi and bacteria may subsequently speed up other ph ysio log ica l processes l ik e resp iration and 233 water lo ss . These processes can a ffe c t the storage l i f e of the tubers adversely. There are other ways in which the nematode-induced hydrolysis o f high polymer carbohydrates to low polymer sugars in yam tuber could lead to economic losses. The low polymer sugarslike sucrose, glucose and galactose are water soluble, and therefore can be leached out o f the yam tubers which are exposed to rain in the open barn. Secondly, these sugars would be lo s t eas ily during the usual processing steps lik e washing and b o ilin g in water before eating, and one is f in a lly l e f t with more fibrous and less nourishing pieces of yam. Q ualitative and quantitative amino acids. The free amino acids in plant may be regarded as a pool which is o f great importance in nitrogen metabolism. This component constitutes usually about 70 - 80% o f the non-protein nitrogen, and in some cases, these amino acids act as a reservo ir from which protein may be formed. The resu lts obtained show that, although the r e la t iv e number of amino acids was not m ateria lly reduced fo llow ing in fection by Scutellonema bradvs. qu a lita tive d ifferences may ex is t in the number of ’’e ssen tia l5’ amino acids. In 234 th is study, fewer number o f "essen tia l” amino acids was consisten tly detected in the in fected tuber than in the uninfected. Perhaps, in the nematode-infected tubers the "essen tia l" amino acids are u t il iz e d more fay the nematode than the "non essen tia l" ones. Prom the resu lts obtained in the protein amino acids, increases in amino acids were generally observed in the in fected tubers except in the case of white yam (D. rotundata) . I f one b e lieves previous calcu lations based upon known concentration of amino acids in larvae ° f M. incognita (Mcclure et a l . , 1973) which showed that the d irec t contribution of nematodes to the amino acid composition to the root was not s ign ifican t (le s s than 0.1^), then the greater quantity o f protein amino acids in the in fected than in the uninfected tissues indicates a plant response to nematode in fec tion . However, Howell et a l» , (1966) and Saxena (1972) have remarked that in terpreta tion o f biochemical changes in host-parasite complexes is d i f f ic u lt espec ia lly where ob ligate endoparasites are involved. Increased le v e ls o f amino acids upon in fec tion by root-knot nematodes have been noted by Owens ert a l. (1966). They found that free amino acids increase very sharply in the ga lls o f tomato. 235 Increased le v e ls o f amino acids have also "been found in the ga lls o f a l fa l fa caused by D. dipsaci (Howell et_ a l .«, 1966) and in g a lls in it ia ted by Longidorus africanus on grape vine roots (Epstein jst jaL, 1 9 7 1 ). An increase in proteins with a concomitant decrease in free amino acids has also been observed for black rot o f sweet potato ( Iuomea batatas L . ) caused by Ceratoc.vstis fim briata E l l is and Halst (U r itan i, 1 961 ) . The small increase recorded in p ro te in , and protein amino acids in the in fected tubers of yellow and water yam could conceivably a rise d ire c t ly from a stimulation in nitrogen metabolism of the host, or from oxidation o f certa in phenols by the host to form quinones that combine with and thereby inactiva te some proteins. Howell et a l.. (1966) noted that increase in leve ls o f free amino acids in ga lled when compared with healthy tissues o f a l fa l fa and pea could be due to (a ) increased translocation into the areas o f in fec tion (b ) increased rates o f synthesis, and (c ) decreased rates of translocation out o f the g a ll and/or decreased rates o f breakdown. The resu lts o f these investigations show that the amounts o f prote in , protein amino acids and fre e amino acids may a lte r as a resu lt o f nematode in fec tion , but ho?/ 236 th is is "brought about is s t i l l la rge ly a matter of speculation. In view o f the substantial contribution T/hich may be made to prote in nu trition and to pharmaceutical industries generally by amino acids in yams, i t is h ighly desirable that much more extensive investigation on the amino acid composition o f yam proteins should be undertaken. Weight lo sses . Many workers have reported that substantial weight losses in yam tubers do occur during storage. The ea r lie s t observations were made by Williams (1925) who found that the species o f D. a lata he used lo s t weight to the extent o f 1^.5% during about four months o f storage. Gooding (i960 ) in Trinidad showed that varia tions in water loss do occur among d iffe ren t species and cu ltiva rs o f the same species. Observations in the Caribbean area in Puerto Rico showed that v is ib le ro ttin g was associated with greatly enhanced losses in weight during storage (Anon, 1937). Weight losses of 7 .&%» 11 .0^, and 28% were recorded fo r healthy, s lig h t ly in fected , and badly in fected tubers resp ec tiv e ly . The resu lts o f th is study are in agreement with the above findings that there is a considerable va ria tion in 237 the weight loss amongst d iffe ren t species and between the healthy and in fected tubers. Because yams are l iv in g storage organs, basic metabolic processes lik e resp iration and carbohydrate metabolism are going on a l l the time in the tuber. The process o f resp ira tion converts the carbohydrates in the yam to carbon dioxide and water, the la t t e r being lo s t by evaporation. I t is , th erefore, reasonable to assume that there w i l l be a corre la tion between the resp iratory rate and weight loss during storage. I t is also reasonable to assume that the presence o f microorganisms lik e nematodes w i l l make a d e fin ite contribution to a higher resp iratory a c t iv ity in the nematode-infected yam tuber and hence, a higher rate of weight loss than in the healthy yam. This fact is w e ll documented by Coursey et a l t (1969) in decayed tubers in fected with Botr.vodipl od.ia theobromae Pat. and Pen icillium c.v c l opium Westling. ??rom the data obtained fo r both the means of to ta l weight loss and the ’ cumulative percentage weight lo s s ’ x\ » for the uninfected yam tubers, i t appears that by using yam barns with b e tte r ven tila tion and avoiding the rotten ing organisms l ik e nematodes and fungi, yams can be stored fo r longer periods without any major loss o f qu a lity . The higher 238 degree ofweight loss resu lting in the greater loss o f qua lity in the neon tod e in fected tubers than in the uninfected is an indication of the re la t iv e economic importance o f the yam nematode - Scutellonema bradys in yam storage in N igeria . Estimation o f the ed ible portion . The primary importance of nematodes as pests o f yams is in the reduction in market value and ed ib le portion which resu lt from th e ir in fes ta tion (Smit, 1967). This in vestiga tion showed that more than a quarter o f the fresh weight o f the nematode-infected yam tuber can be lo s t or rendered in ed ib le . Figures recorded for the mean o f 20 tubers were 28.1 %,26.2$, and 26.3$ for in fected D. r otund at a . D. cayenensis and D. a lata resp ec tive ly . At the la te r stage of dry rot d isease, more severe losses o f the ed ib le portion can occur. The highest peeling loss recorded for one of the species at an advanced stage was 57$. \\ U In the nematode free tubers, figures recorded for the mean o f 20 tubers were 9$, 9% and 6.9$ fo r D. rotundata. D. cavenensis and D. a la ta resp ec tive ly . The d ifferen ces in peeling losses between the nematode-infected and nematode free yams were found to be s ta t is t ic a l ly s ign ifica n t 239 at the 5% le v e l . Although control measures involving the use of hot water treatment, chemical and cu ltu ral methods have been used to suppress the population o f th is nematode in yam tubers, further investiga tion on how to reduce such peeling losses are essen tia l to ass ist the small scale farmers who derive th e ir main income from yams. The cumulative amount of losses due to S, bradys represents a staggering to ta l which the teeming masses of our under-nourished people cannot a ffo rd . Substances d ischur g ed b y J3. b r a d y s . By a series of chromatographic analyses, f iv e n inhydrin-positive amino acids were discharged by a mixed population (adults and larvae ) o f 3. b radys. These were iso-leu c in e , leucine, aspartic acid, hydroxy ino l acetic acid and phenyl alanine. Phenyl alanine appeared to be a c tiv e ly secreted or excreted by th is nematode because i t gave a rather deep purple colour with ninhydrin. Discharged substances probably represent secretory and excretory products, and those lo s t due to in e ffic ien cy o f body function. The synthesis and discharge o f amino acids by p lant- paras itic nematodes are pa rticu la rly in terestin g because 240 o f th e ir possib le function in the nematode and host tissues. Myers and Krusberg C1965) studied the rsmino acids discharged by three p lan t-paras itic nematodes, D. d insaci. P. penetrans and Meloidogyne incognita . Aspartic acid, glutamic acid , serine, g lyc ine, ornith ine, threonine, alanine, methionine sulphoxide, asparagine, ly s in e , arginine and iso-leucine/leucine were discharged by D. d i ia sac i and M. i n c o g n i t a . P . p en e t ran s d is c h a r g e d a l l except asparagine, ly s in e , argin ine, and iso-leucine/ leucine. Anders (1961 ) suggested that tryptophan, lysine and h istid in e in aphid sa liva were responsible for plant ga lls caused by these insects. Myuge (1957) suggested that ammonia discharged by D. destructor Thorne. into in fected plant tissues was -the major cause of tissue damage. Tryptophan - C 1 h was produced when roo t- knot nematodes were incubated in acetate C 1 h . The discharge of phenyl alanine in appreciable amounts by S. bradys is in terestin g in that th is amino acid may possibly contribute to phenol metabolism in host tissues. I t is , th ere fore, l ik e ly that the amino acids discharged "by bradys w i l l contribute to the physio log ica l and biochemical changes and deterio ra tion induced in in fected 241 yam tissues. Id en tific a t io n o f substances discharged by plant p a ras itic nematodes is therefore important in many ways. The substances released by nematodes into parasitized plant tissues w i l l very l ik e ly contribute to symptoms of d isease. Interactions between plant nematodes and other plant pathogens may also be b e tte r understood in re la tion to substances discharged by them. Enzymes o f Si. bradys. Nematode homogenates .and the nematode in fected yam tissues were tested for the a c t iv it ie s of four enzymes, two of which were detected. P ecto ly tic a c t iv ity was detected in the nematode homogenate and the in fected yam but was not detected in the healthy yam. No ce llu lase a c t iv ity was detected in a l l the extracts. The presence o f pec t ic enzymes in nematode homogenate and in the in fected yam tissue elucidates the ro le o f S. bradys in c e l l w all separation. In the past, the mechanisms by which nematodes in jure host plants have been la rg e ly a matter o f speculation. Myuge (1957)? claimed that a protopectinase excreted by D itv lenchus a l l i i (B e ije r in ck ) F i l . and Sch. Stek. d issolved in te rce llu la r protopectinaceous layers and caused tissue maceration. 242 Supportive h is to lo g ic a l and histochemical evidence was, however, not presented. Mountain (i960) hypothesised that since pectic compounds are important structural components o f the middle lam ella , p ec to ly tic enzymes might he agents o f th e ir d isso lu tion . But there is l i t t l e published evidence that pectinases are e f fe c t iv e in destruction o f the middle lam ella . One o f the mechanisms of action o f S. bradys as demonstrated by th is study is that the nematode secretes into the yam tissues pectio enzymes responsible fo r breaking down the cohesiveness between c e l ls . The pectinase detected in v it r o must have been produced by the feeding a c t iv it ie s of the nematode since healthy yam did. not cause any decrease in v is co s ity o f the pectin . The absence o f ce llu lase a c t iv ity further lends support to the argument that in tra ce llu la r movement o f 8. bradys leads to a mechanical c e l l wall separation and not to a d isso lu tion o f the ce llu lose c e l l w a ll. Amylase a c t iv ity was demonstrated in both the nematode homogenate and in the nematode in fected yam tissue. From this in vestiga tion , greater amylase a c t iv ity was detected in the in fected yam than in the nematode. Perhaps the nematode in terferes with plant metabolism by causing a 243 further release o f the enzyme "by the p lant. The detection o f amylase agrees with e a r lie r findings (Adesiyan _et a l . , 1975) that in the nematode-infected yeans some hydrolysis o f starch to glucose by th is enzyme takes p lace. Incomplete hydrolysis of starch gives maltose. /□.though the presence o f invertase enzyme in some plant paras itic nematodes has been demonstrated by various workers (Z ino jev, 1957)? it s presence was not detected in th is study. I t is possib le that th is enzyme was present in the nematode but that i t s a c t iv ity was in terfered with at the pH used, or probably, a c t iv ity was lo s t during the preparation o f the extracts . There are many unknown factors in extracts that can in te r fe re with the a c t iv ity o f some enzymes. Hov/ever, th is study illu s tra te s the importance o f pectinases and amylases in the pathogenicity o f S. bradys. Yam tissue extracts examined for other compounds. The N.M.R. absorption at 1.0 is thought to be due to the a liph a tic or s tero ida l group o f compounds. This peak is sh ifted to about 8.2 cfni both the dry rot and wet rot yam extracts. Absorption at this peak is due to the aromatic group o f compounds. The biochemical a lte ra tion 244 o f the s te ro id a l to aromatic compounds in the in fected yams is very in teresting because of the e ffe c ts th is might have on the use o f yams in the pharmaceutical industry. There has been considerable in teres t in recent years on yams (D ioscorea sp .) by pharmaceutical industry overseas. Yams have been found to contain the stero id diosgenin which is eas ily converted to the cortisone group o f drugs. Thus we propose that the absence o f s tero id group of compounds in the in fected tubers appears to be d isease-re la ted as evidenced by it s appearance in the healthy tubers. The appearance o f the aromatic group o f compounds in the in fected tubers may probably help to estab lish a basis fo r further investigations as these groups of compounds may be involved in modifying host su scep tib ility . Fungi associated with the dry rot o f yams . This prelim inary study on the iso la tion o f fungi associated with th e ’dry rot* o f yam gave basic information and resu lts on which further investigations were based. Only two fungi were iso la ted from the dry rot portion by the ’ d irect p la t in g ’ method and these were id en tified as Fusarium oxvsporum and Rhizopus n ig r ic ans. Fusarium sp. 245 and Rhizopus nigricans caused no decay o f yam under experimental conditions („,deni;ji, 1970). The fa ilu re of other fungi to germinate may he due to other factors l ik e pH o f the medium, e ffe c t of toxic secretions hy other organisms, or spore dormancy, ’Then these yams were, however, transferred in to humidity chambers, many fungi grew out. P e n ic i l l iu m sp., T r ichoderma v ir id e were most commonly iso la ted . I t appears from this in vestiga tion that keeping the yam pieces f i r s t in humidity chambers before p la tin g them in agar created favourable conditions for germination. Investigations carried out by Aden iji (1970) and Ogundana jet a l . (1970) showed that fungi were eas ily iso la ted from decayed portions of yams by p la tin g in agar without necessarily keeping them f i r s t in humidity chamber. Perhaps the stage o f the yam decay was important in determining the type o f fungi that would be iso la ted , .ationsnj In experiments involving in teractions o f two organisms o f proven pathogen icity, many factors need to be considered c r i t ic a l ly , e .g , (a ) The e f fe c t o f the nematode on the fungus. (b ) The e f fe c t of the fungus on the nematode. 246 (c ) The additive e ffe c ts of the two pathogenic components on th eir host. E ffec t o f nematodes on the fungus. In these experiments, the presence o f nematodes seemed to have "brought about grea ter depth of penetration o f mycelia o f Pen icillium sclerotigenum and Fusarium oxvsnorum in yam tissues than the absence o f nematodes. Aden iji (1970) was able to te s t the pathogenicity o f fungi lik e A.snergillus niger and Bo try od ip lodia theobromae on yams only a fte r 'h o les ’ had been made on the tubers. Ogundana (1970) also found that no fungal penetration o f tubers occurred in the absence o f wounds. In th is study, despite the 'wounds’ in i t ia l ly caused on the tubers before inoculating the fungi, penetration by mycelium was res tr ic ted when fungi were inoculated alone. The presence of nematodes seemed to have increased the degree of pathogenicity or in fec tion in these two cases. But the presence o f nematodes did not seem to have helped the penetration o f A. n ig e r . A l l plant pa ras itic nematodes wound in some degree e ith er by a simple micro-puncture or by rupturing or separating c e l ls . These micro-punctures aid the penetration 247 of pathogens incapable o f s e l f establishment. Besides creating micro-punctures, the nematodes can aid in the d igestion of host tissues which in this case is starch. The presence o f nematodes has been shown to a id the d igestion o f starch to sugars. These simple sugars form a food base for the ftingij the food base rein forces the invasive poten tia l of fungi and bacteria . Such is thought to be the way in which the yam nematode S cut e l 1 onem a bradys aids in the in fec tion o f the yam tuber by secondary pathogens l ik e Pen icillium and Pusarium species. E ffe c t of the Ihngus on the nematode. The e f fe c t o f the fungus on the nematode in the storage and greenhouse experiments can be viewed from two angles; (a ) E ffe c t on survival o f the nematode and (b ) Its e f fe c t on reproduction. Because m ajority o f plant paras itic nematodes are ob ligate parasites and because they are prim arily the in it ia to r or in c itan t o f such a complex, they are espec ia lly vulnerable to competition. The balance may s h ift with time, the nematode eventually becoming worse o f f when the competitive organisms become dominant. The nematodes 248 begin to disappear due to lack o f food and because the environment has been made unsuitable by toxic secretions by the invading secondary organisms. In the greenhouse experiment, the in teraction between S. bradys and A. niger was disadvantageous to the nematode. The presence o f the fungus seemed to have some e f fe c t on the number o f nematodes that invaded the roots and tubers and subsequently on nematode development. This may be due to a possib le an tib io tic action of A. n iger on j3. bradys. The a n tib io tic action may have had a le th a l immobilizing e f fe c t on • S. bradys. Such a n tib io tic action o f A. n iger was previously reported by Mankau (1969) and Murad (1966). This incom patib ility between plant p a ras itic nematodes and many decay promoting organisms has been reported by many workers. Davis (1962) and Davis and Jenkins (1963) noted that the presence of fungal hyphae prevented maturation o f female Meloidogyne spp. James (1968) found that fungi lowered the hatch o f Heterodera r ostochiensis . decreased the number o f cysts produced by the nematode and la rva l invasion o f tomato roots. In some cases, where the nematode is not an ob ligate plant paras ite , i t may b en e fit by feeding on the mycelium (Mankau and Mankau 1963; Baker et a l . . 195U) • 241? The additive e ffe c ts of t wo -pathogens on the host. I t was c lear from visual observation o f the tubers that association of n iger and the nematode did not produce an aggravated condition. The d ifferen ce betv/een the weights o f tubers from the inoculated pots and the asep tica lly grown and uninoculated ones was comparatively small, ind icating that the fungus and nematodes added to the s o i l were not capable of independently causing a s ign ifican t growth reduction. ?rom these in vestiga tion s, i t is evident that jS. bradys obviously has a profound influence on the development o f dry rot disease of yam. I t is the primary pathogen in this complex. I t is , therefore, unnecessary to use fungicidal pestic ides for economic control o f the d isease. Although fungi may be associated with ’ dry rot* o f yam they are not a party to it s formation. There is also some evidence from these investigations that m odification of host’ s tissues is an in tegra l basic component of in teraction and that this may even be more important than mechanical in ju ries . The ro le of fungus in nematode diseases has been l i t t l e studied. But in th is experiment, i t appears fungi are secondary invading organisms which aggravate the nematode- 250 induced ro t . They are, th ere fore, secondary pathogens in yam decay. Although the in teraction "between S. "bradvs and A. nijger on yams is disadvantageous to 3. bradys i t is not su ffic ien t fo r useful economic suppression o f nematode in yams. *omparative nematicidal a c t iv it ie s of A. n ig e r . P. sc l er o t ig enum and P . opryspopqq. Of a l l the three fungi, only Aspergillus n iger was found to be nematicidal at high spore concentration - 20,000 spores per ml. /it th is high concentration, only about 53% o f the nematode population was immobilized. Fusarium and Pen ic illium species v?ere not as nematicidal, even though about 25% of the nematode population was immobilized at the same spore concentration. Many fungi are known to support the development o f a large population o f nematodes, but some fungus iso la tes sustain very lim ited reproduction, and others allow l i t t l e or no increase in the numbers of the nematode. The p o s s ib ility that such fungi produced an tib io tics has been demonstrated by some workers. Mankau (1969) found that Aspergillus n iger culture f i l t r a t e was nematicidal to Anhelenchus avenae Bastian at as low a concentration as 251 10/3, whereas Fusarium sp. was not nematicidal at a l l concentrations. Sim ilar studies were carried out by Murad (1966) who showed that black Asperg illu s species had an an tib io tic action on a nematode Polodera chitwoodi (Bassen) Dougherty and that the ’ tox in ’ produced by th is fungus had sim ilar e ffe c ts on rhabditiform nematodes. Mankau (1969) in his experiment, detected that toxic amounts o f oxalic acid were produced in s o i l under conditions favourable for rapid or extensive growth o f Aspergillus n iger . and concluded that the le th a l immobilizing of A. avenae by culture f i l t r a t e s of A. n iger may be due to tox ic concentrations o f oxalic acid produced by the fungus. Host range stud ies. A few workers in the past three years have investigated the survival of Scutellonema bradys in crops other than yam. Bridge ( i 973) reported that yam is the only good host to _3. bradys and that the nematode survived endoparasitica lly in the roots o f melon, sorghum, okra, tomato, kenaf and pigeon pea. He lis te d cassava, maize, hot-pepper, pineapple, pawpaw, cocoyam, o i l palm, groundntit, yam bean, ro s e lle , and r ic e as non-hosts. Odihirin and Ososami (unpublished data, 1 97k) found cowpea, and 252 groundnut to be "very good" hosts. Tomato, yam bean, and Eupatorium were rated as " fa ir ly good" hosts. C elos ia . ro s e lle , maize and Tridax were among the plants l is te d as non-hosts. The resu lts of th is investigation showed that beniseed, cowpea ( ’ New Bra* and ’ I fe Brown* v a r ia t ie s ) are good hosts to Scutellonema brad.vs because about 2-3 fo ld population increase was recorded in these hosts in 30 days. Small populations o f j3. bradys just survived end op ar as i t i c a lly in the roots o f 17 plants including kenaf, ro s e lle , tomato, melon, Svnedrella and Eupatorium. Ten plants were rated non-hosts and these included maize, tobacco, T rid ax and water le a f (Talinum triangu lare ) . The results obtained in some respect agree with the resu lts obtained by Bridge (1973) and Odihirin and Ososami (1974). Roselle and yam bean were found to be fa ir ly susceptib le. However, groundnut was not found to be a host. For the f i r s t time, beniseed _Sesjunurn indie urn is being reported as a good host and tobacco as a non-host. Because o f the economic importance o f 3. bradys on yams, 253 the need to search for other crops that harbour th is nematode cannot be over-emphasised. Such a ltern a tive hosts can be excluded in ro ta tion sequence. Rotation o f non­ hosts l ik e maize, tobacco or cotton with susceptible hosts may give an e ffe c t iv e control o f nematode population in the s o i l . Such non-hosts w i l l be a su itable a ltern a tive to nematicides which cannot be afforded by m ajority o f the farmers in the trop ics , Even where farmers can a fford nematicides, an e f fe c t iv e crop rotation invo lving the use o f non-hosts is also necessary so that the nematodes which escape from the nematicide are deprived of any host and thereby starved to death. However, such non-hosts must be high value crops to compensate the farmers for income derived from the sale of yams. The high rate of reproduction of 3. bradys on legumes is pa rticu la rly d isturbing. Squally, disturbing is the fac t that some weeds lik e Eupatorium odoraturn and Synedrella and some vegetables l ik e jute CCorchorus o lito r iu s L . ) and soko ( Celosia a rgen tia ) commonly used in mixed cropping a l l support populations of jS. br adys. These weeds and vegetables growing in rows with highly susceptible crops l ik e yams, cowpea and beniseed with th eir roots intermingled,, would encourage the build-up o f large nematode populations 254 and perhaps lead to greater damage and loss . I t is , however, in teresting to note that crops l ik e tobacco, cotton, and maize did not support the reproduction o f the nematode. These crops may he o f value in rotations. The resu lts reported here show that S. hradvs is able to reproduce on a considerable range of p lants. These crops should not therefore be used in rotation without supplementary chemical control where th is nematode is a problem. Weed control and exclusion of legumes in yam p lots are very important in preventing population increase and the use o f non-hosts may also aid in population reduction. Control o f S. bradvs by hot f/ater treatment. I t is obvious from the resu lts obtained in th is investiga tion and other investigations conducted by Hawley (1956), Ayala _et a l; (1971) and Bridge (1973) that there is a great deal o f po ten tia l in the use of hot water treatment as a means o f elim inating nematodes from yam tubers. Dipping nematode-infected tubers in hot water maintained at temperatures between 50 and 60°C fo r 1+0 minutes completely suppressed nematode population. Although heating yam tubers from cold to a temperature o f 255 50°G also s ign ific a n tly suppressed the nematodes, the nematodes were not en tire ly elim inated. Temperatures between 50 - 55°C did not adversely a ffe c t germination and p a la ta b ility o f water yam (D. a la ta ) . but y ie ld was s ign ific a n tly reduced at 5%when compared with the h con tro l. However, nematode population in the treated was completely suppressed and th is has a long term advantage over the con tro l. Despite the lite ra tu re on the usefulness o f hot water treatment in suppressing nematode populations, very l i t t l e work has been done on the e f fe c t o f hot water treatment on yams at d iffe ren t stages o f storage. Hrom this in vestiga tion , however, i t was observed that o f a l l the treatment times, only yams treated immediately a fte r harvest rotted com pletely. Only a small proportion o f the yam tubers treated between November and March rotted and hot water did not a f fe c t th e ir sprouting p o ten tia l. The high percentage of rotted yams recorded for those treated in October is to be expected in that the new yams are immature, the skin being so ft and fr a g i le , hot water probably penetrated deep into the tissues causing physio log ica l damage. Because o f i t s a b i l i t y to suppress nematode populations 256 in in fected tubers, hot water treatment was considered useful in prolonging the storage l i f e o f nematode- in fected tubers. This method is p a rticu la rly successful on nematodes because they do not penetrate deep into the tubers. Hot water dip also attempts to ’ clean ' yam setts before planting, but the method cannot be expected to be e f fe c t iv e i f treated yam setts are planted in nematode-infected s o ils . I t can, hov/ever, be used in combination with other control measures. I t appears that from the resu lts obtained from these in vestiga tion s, hot water treatment as a means of nematode contro l in tubers has a great deal of po ten tia l for the fu tu re . Chemical and cu ltural methods for control o f j3. bradys. There had been reports on the use o f orge~nic amendments in the control o f plant diseases (Walker, 1969 Morgan and C o llin s , 196U; Krusberg, 1 961 Johnson et, a l . , 1967). These reports indicate that there is stimulation o f m icrobial a c t iv ity or an increase in the antagonism o f microorganisms in s o il by the addition o f organic amendments. Laan (1956) observed that organic manuring suppressed the rate o f in fes ta tion and reproduction o f Heterodera rostoch iensis . Singh ( l 96U) postulated that 257 reduction in nematode populations "by organic matter added to the s o i l may he due to to x ic ity to the nematodes, in terference with i t s resp iration and a lte ra tion o f the oxygen, nitrogen and pH status o f the s o i l . Organic amendment is also thought to influence the increase o f nematophagous fungi l ik e Arthrohotrys oligospora (F res ) and Dact.vlaria Candida (Nees) Sacc. and the mononchid types of nematodes genera lly . Factors a ffe c t in g the decline o f plant paras itic nematodes in s o i l during degradation of organic m aterial are not w e ll understood. But i t is thought that simple nitrogenous compounds which are intermediate breakdown products might he responsible for reducing nematode populations. On the other hand, with the addition of organic manure the plants are kept growing vigorously. Nutrients are released fas t in to the s o il by the organic manure and these probably contribute to rapid tuberization . From the resu lts obtained in this study, i t appears that organic manure plays a double ro le o f increasing y ie ld o f the tuber and suppressing the nematode. Th eore tica lly , the addition o f f e r t i l i z e r s should promote healthy, well-nourished plants which are able to 258 withstand some kind o f diseases* But in th is case fe r t i l i z e r s as applied in th is 7/ork did not seem to have any remarkable improvement on y ie ld o f water yam, but the nematode population was suppressed considerably. Heald and Burton (1968) reported that n itrogen f e r t i l i z e r components are detrimental to nematodes. Results obtained from th is experiment suggest that one b en e fit o f n itrogen, phosphate and potassium fe r t i l i z a t io n might be reduction o f populations of £3. bradys in s o i l . The use o f wood ash to coat yam 's e t t s ’ before planting is a trad ition a l practice amongst yam growers in the trop ics , but there are at present no published data on it s b en e fic ia l e ffe c ts on yams. Prom th is study, i t was observed that tuberization was enhanced with wood ash. The reason for this might be due to supply o f nutrients to the s o il as v/as the case w ith organic manure. Wood ash also caused a s lig h t suppression o f nematode population. Although application of nematicides has made i t possible to reduce nematode population in s o i l to low le v e ls , success in usage depends to a large extent on several factors l ik e , dose, method o f application and the type o f crop. Nemagon was espec ia lly e f fe c t iv e against S. bradys 259 in th is experiment, hut yam appears sen sitive and probably susceptible to phytoxicity and did not do w ell with nemagon. E a r lie r work in the Netherlands has, however, shown that tobacco and potato are also sen s itive to nemagon. Although some degree o f nematode control was achieved by the use o f these treatments when compared with the con tro l, no single treatment seemed to have achieved complete suppression o f the nematode and increase in y ie ld simultaneously. Some treatments are, ho?/ever, b e tter than others. Even though nematicides can be very e ffe c t iv e in reducing nematode population to low le v e ls in s o i l , the costs are often p roh ib itive and cannot be afforded by m ajority o f yam growers in the trop ics . The residue e ffe c ts o f nematicides in s o i l and food and resistance of species or biotypes to nematicides have been l i t t l e studied in the trop ics ; v/hereas cu ltu ra l control is inexpensive and often a valuable adjunct to the chemical control o f plant pests. The resu lts o f th is experiment demonstrate that there is a good deal o f p o ten tia l for t r ia ls with various cu ltu ra l, b io lo g ic a l, and chemical methods of con tro l. 260 The e f fe c t o f gamma radia t io n on the yam nematode. The resu lts o f the irrad ia tion experiments show that to achieve complete suppression o f nematocie population in in fected tubers, a dose o f 30 Krads and above which was found to have an adverse e f fe c t on the internal tissue o f yams was necessary(Adesuyi and MaoKenzie, 1973). These prelim inary investigations also confirm th e ir finding that sprouting o f dormant tubers was suppressed at dosages between 5 and 13 Krads whereas the untreated yams sprouted luxurian tly . There was also no incidence o f rot recorded in a l l the tubers irrad iated a fte r 3 months o f storage. About 60% o f the tubers in the control group rotted com pletely. B arlier reports by Kahan and Gorodeiski (1966) showed that sprouting was prevented in stored potatoes between 10 and 14 Krads. In th is in vestiga tion , lower doses were required than those reported for potatoes. Sparrow and Christensen (1954), Sawyer and Dallyn (1955) found that the lowest dose found to in h ib it the development o f embryonated eggs is the same as the dose found to in h ib it completely the sprouting o f potatoes. But in th is in vestiga tion , th is was not so. At 5 Krads when sprouting 261 o f yams was suppressed, the nematode numbers were not reduced. Although nematode numbers were considerably reduced at high doses, th is condition would be disadvantageous to the yam tubers. Irrad ia tion at high dosages is known to decrease natural resistance o f potatoes to phytopathogenic organisms (M e t lits k ii, 1968). So fa r , no dose has been found to in h ib it sprouting and to suppress nematode population completely. However, doses between 10 and 15 Krads showed some promise. 262 CK PT'SR 5 SUMMARY 1 . D istribution o f nematode paras_ites_ of yars tubers in the Mid-’,Vest. N igeria . A survey of the yam growing areas of Mid-Western N igeria , showed that root-knot nematodes, espec ia lly Meloidogyne incogn ita,, were as important as Scutellonema hradvs in causing yarn losses in the f ie ld and in storage. The root-knot species were found assoc kited with only tubers o f water yam (D. a la ta ) in the Mid-Western State. Reports from other countries g ive growing evidence o f a wider geographical d is tribu tion of nematode genera associated with yam tuber decay. Scutel l onema bradys is therefore not the only nenr.tode capable o f in it ia t in g decay in yam tubers. Other species lik e Pratylenchus coffeae (West Indies) and root-knot nematodes (Guatemala and N igeria ) may be equally important in some countries or in certa in lo c a l i t ie s o f the same country. Reference to ,3. bradys as the ’ yam nematode’ in older lite ra tu re is therefore l ik e ly to assume only lo ca l s ign ificance in future. Yam decay is obviously a serious problem in Mid-Western 263 N igeria . In festa tion o f tubers ranged from 80 - 1 00% in yam barns and markets. A l l ava ilab le evidence shows that nematodes have spread from farm to farm by vegetative propagation through the use of in fected yam setts or seeds and also from tuber to tuber in the yam barn because o f the method o f storage which involves close contact o f tubers. Yam ’ seeds' were found to be fre e r o f nematode in fec tion than yam ’ s e t ts ’ cut from regular tubers. They are therefore recommended for yam propagation in preference to yam 's e t t s ’ . 2. Surface s t e r i l i z a t i on o f nematodes. Four known methods o f surface s te r i l iz a t io n o f nematodes were tested , namely, 0.1 % streptomycin sulphate; 1 : 1 ra tio o f 0.1% streptomycin sulphate and 20 ppm malachite green; 20 ppm o f malachite green; and 0.01% mercuric ch loride. The method involving the use o f 0.1 % streptomycin sulphate alone appears most e f f ic ie n t from th is in vestiga tion . 3. Studies on population bui l d-up o f £>. bradjvs . Studies were undertaken on the population dynamics o f £5. bradys at ambient temperatures and at cool temperatures ranging between (16 - 18°C). Results showed that there 264 were increases in nematode populations during storage at ambient temperatures. These increases occurred throughout the f iv e month period of storage whereas at lower temperatures nematode populations only increased for the f i r s t 3 months, but therea fter the populations remained low. Sprouting was a lso completely inh ib ited at low temperatures, whereas yams stored at ambient conditions sprouted luxurian tly . The resu lts obtained demonstrated that there are d ifferences in the depth of penetration amongst the f iv e cu ltivars of D, rotundata. The nematodes (j3. bradys) produce severe damage in the white yam, colon ising mostly the f i r s t k mm o f the periderm, even though the nematode population could be found in the periderm to a depth o f between 0 - 1,5 cm. The oldest portion , adjacent to the stems (top ) contained the highest population, while fewer nematodes were recovered from the centra l (m iddle) and posterior (bottom) portions of the tuber. 5. Histopathology stud ies. The h istopathologica l studies indicate that: The early stage of yam decay is the dry rot stage 265 caused by the yam nematode Scutellonema hr ad.vs in the Western State of N ig e r ia . So fa r , there has been no anatomical evidence of fungi in dry ro t sections. The nematodes are prim arily responsible for providing in fec tion s ites for fungi and bacteria . The 'wet ro t ' stage is essen tia lly associated with fungal and bacteria], a c t iv it ie s . Whereas nematodes employ a slow k i l l at the dry rot stage, the d is in tegration of c e lls at the wet rot stage is fa s t . Fungi are found in the wet rot sections only, espec ia lly the chlamydospores and mycelia o f Fusarium species. Yam decay involves fungi and nematodes each playing a v i t a l ro le at one stage or another in the process. Longitudinal sections through nematode in fected roots of yam tubers revealed the cortex as the favourite feeding s it e . Rapid and re lia b le methods for the determination of carbohydrates are described. The resu lts o f th is study agreed with e a r lie r observations that there is conversion o f starch to soluble 266 simple sugars during storage, and these interconversions were influenced by the presence of decay organisms lik e nema t od e s and fu ng i . The conversion of starch to simple sugars aided hy the presence o f nematodes helps to explain how nematodes may predispose yam to in fec tion by other secondary organisms, e .g . fungi and bacteria . 7.. Amino aci ds. Detection of free amino acids in 3 species of healthy and nematode-infected tubers of Dioscorea was carried out by means o f paper chromatography. About 13 ninhydrin-positive amino acids were extracted from the uninfected white yam (D. rotundata ) wereas 10 were detected in the in fected . Eleven free amino acids were detected in the uninfected yellow yam (D. cayenensis) whereas only 9 were id en tified in the in fected yam. Only 10 and 8 free amino acids were extracted in the healthy and nematode-in fee ted tubers o f D. ala ta resp ective ly . In the nematode in fected yam tubers fewer essen tia l amino acids were detected than in the healthy tubers. Eighteen ninhydrin-positive m aterials were detected in the protein hydrolysate. The in fected tubers of 267 5* Q¥ensis and D. a la ta for the most part hacl more protein amino acids than did the corresponding healthy tubers. There was no corre la tion between the absolute number of free amino acids in the yam tubers and the protein amino acids. The absolute amount o f each protein amino acids from both the healthy and in fected tubers varied considerably within each o f the yam species. However, the r e la t iv e amounts varied w ithin rather narrow lim its between the healthy and the in fected tubers of each species. Except in the case o f white yam (D. r otundata) „ percentage protein also increased in the nematode-infected tubers. 8. Weight loss in healthy and nematode-infected tubers Considerable varia tion in water loss was found to \s f i occur between the in fected and uninfected yam tubers o f yellow yam (D. cayenensis ) and white yam (D. rotundata ) . The nematode-infected yam tubers of both species were found to lose more weight; than the uninfected. This loss was found to be s ta t is t ic a l ly s ign ifican t at 268 9. Measurement of edib le portion o f the nemntode- in fe c ted tubejrs. Percentage peeling losses due to dry ro t recorded were 28.1 ; 26.2$ and 26.3 for species o f D. rotundata. D. cayenensis and D. a lata resp ec tive ly . The highest peeling loss recorded for the in fected tuber was The d ifferen ce in peeling losses between the healthy tubers and nematode in fected tubers was s ta t is t ic a l ly s ig n if ic a n t. 1 0. Substances discharged^ by Scutellonema brady_s . Amino acids emitted and extracted from surface- s te r il iz e d larvae and adults of S. bradys were id en tified by paper chromatography. T ive amino acids id en tified ■were aspartic acid, phenylalanine, isoleucine, leucine and hydroxyinol ace tic acid. In addition to the amino acids discharged by J3. bradys, the presence o f p ec to ly tic and amylase enzymes was demonstrated in aqueous extracts of a population o f mono-axenic cultures of £5. bradys iso la la ted from nematode-infected yam. In viscometric te s ts , pectin was degraded at pH 6, and amylase was detected spectrophoto- m etrica lly at pH 6.9. The p ecto ly tic enzymes are also produced in v it r o by the nematode, and amylase a c t iv it ie s 269 were not detected in healthy yam extracts and sodium ch loride. However, the d i f f ic u lt y in obtaining su ffic ien t quantity of nematodes for enzyme experiments can severely l im it the type and number o f enzymes detected. Yams showing symptoms o f dry rot and wet rot were examined with N.M.R. Spectrometer for other classes of compounds. The conversion o f stero id compounds in the healthy yam to aromatic compounds ( i . e . substances having fragrant odours) in both the dry and wet rot yams was revealed.. This conversion is probably disease re la ted . 11. Host range stud ies. "From green house experiment invo lving 30 tes t plants, 20 were found to be ’ hosts ’ to Scu tellonema bradys. Beniseed and cowpea were regarded as good hosts. Eupatorium. Synedrella . yam bean ( Snhenostylis stenocorpn) are among the hosts. Tobacco, maize and cotton are non-hosts. 1 2. Fungi associated with the dry-rot of yam JjUbers. The d irec t p lating methods of diseased portions o f yam tissues in agar (P .D ,.:,) supported the growth o f two fungi, namely, Fusarium oxysporum and Ehizopus n ig r ic ans. even though plenty o f m ycelial growth was observed on most p la tes . The keeping of yams in humidity chambers before p la ting them on agar showed the growth o f more Pan ic i l iu m s c le ro t ig e n u m . Trichoderm a v i r i d e . and a theobromae 13 Results from th is investigation showed that S. hradys obviously has a profound influence on the development o f ’ dry rot* disease of yams. I t is, in fa c t, the primary pathogen in th is complex. Although fungi may be associated with ’ dry r o t ’ disease, they are not responsible for i t s formation. Association of Aspergillus niger and nematode (S . brad.vs) did not produce any aggravated condition in the disease complex because the fungus inh ib ited nematode reproduction. The presence o f the fungus A. n iger seemed to have some anti-nematode e f fe c t and to have some adverse e ffe c ts on the numbers of nematodes that invaded the roots and tubers and subsequently on nematode development. 12+. Control o f £>. bradys by hot_ water trea tment. Observations on the use o f hot water treatment as a means o f con tro llin g yam nematodes revealed that dipping nematode-infected tubers of water yam D. a lata and 271 D. cavenensis in hot water at 50 - 55°C fo r 1+0 minutes completely suppressed the nematode. Germination, growth, storage l i f e , y ie ld , and p a la ta h ility o f tubers of D. alata so treated were not adversely a ffec ted . The best time to use hot water starts as from two months a fte r harvest, that is during the post-dormancy period. 15. Some cu ltu ra l and chemical methods o f con tro l of S. bradvs. F ive d iffe ren t methods o f nematode control were employed. Treatment involving organic manuring gave the highest y ie ld of yam tubers, and considerably reduced nematode population. Application of nemagon was e f fe c t iv e in con tro llin g J3. bradvs but i t reduced the y ie ld o f water yam probably due to some degree o f phytox ic ity . Application o f D-D Y/as in e ffe c t iv e as i t neither increased y ie ld nor reduced nematode population. Application o f wood ash increased the y ie ld o f water yam while HPK f e r t i l i z e r reduced nematode population. 16. Control o f S. bradys by gamma irrad ia tion . About 70 - 8Op reduction o f nematode population Y/as achieved at 15 Krads and above. In order to achieve 272 complete suppression o f nematode population in in fected tubers, a dose o f 30 Krads and above was required, but th is led to ro ttin g o f tubers. Complete suppression o f sprouting was achieved as from 5 Krads. 273 REFERENCES Acosta, N, (1974). Depth of penetration o f phytoparas i t ic nematodes in yam tubers. Nemntropica ^(1 ) p.7 - 11 . A d en iji, M.O. (1970). Fungi associated with storage decay o f yam in N igeria . Phytopathology 6o(4) p.590 - 592, Adesiyan, 3 .0 .s R.A. Odihirin and M.O. Aden iji (1974). Histopathology studies o f the yam tuber (21 oscorea rotundata)in fected by Scutellonema bradys Steiner and LeHew. In t . B iodetn. B u ll. ( in p ress ). - ............ - (1975). Economic losses caused by the yam nematode Scutellonema bradys in N igeria , P I. P is . Reptr. ^ ( 6 ) p.477 - 480. _ _ (1975). Changes in carbohydrate constituents induced in the yam tuber ( Dioscorea rotundata P o ir ) by a plant p a ra s itic nematode, Scutellonema bradys. In t . Biodetn. B u ll. (In p ress ). —— ( 1975) . D istribu tion o f nematode parasites o f yam tubers in Mid-West State, N igeria . N iger. P I. Protect . J, ( in p ress ). 274 Adesuyi, S.A. and J.A. MacKenzie (1973). The inh ib ition o f sprouting; in stored yams ( Dioscorea rot undata) by gamma rad iation and chemicals. I .A .A. A . S .M. -166/35 p .1 27 - 1 36. A is t, 3. and R.D. Riggs (1969). Amino acids from Heterodera g lyc ines. J. Nematol. 1,(3) p.25