DIAPAUSE IN THE NEMATODES Globodera rostochiensis AND G.pallida. by ZANNA MUHAMMAD B.Sc.(Hons)(BUK); M.Sc.; DIC; CBiol, MIBiol. A thesis submitted for the degree of the Doctor of Philosophy of the University of London. Department of Biology, Imperial College at Silwood Park, Ascot, Berkshire. May, 1990. DEDICATION. To Late Maina Mai who believed in me, Alhaji Muhammadu Alkali and Hajiya Faratu M. Alkali who offered me the opportunity. 2 ABSTRACT. Diapause was investigated in populations Globoderaof rostochiensis and G.pallida from Scotland raised at Silwood Park, Ascot. In newly harvested cysts (’’new" cysts) of G.rostochiensis dormancy was shown in October, January, February, April, May and July, and was absent in November, June and August. However in the same cysts stored in an outdoor gravel plunge for 12 months ("old" cysts), dormancy was absent. In "new" cysts ofG.pallida dormancy was shown in October, November, December, February and April, and absent in May, July and September. In "old" cysts dormancy was shown in October, May and July, and absent in November, December, February, April and September. Results showed that G.rostochiensis hatched more freely and faster over short period, whileG.pallida hatched slowly over a prolonged period. In three-year old cysts ofG.rostochiensis and G.pallida populations from Scotland stored dry, humid and wet at 5, 10, 15, 20 and 25°C for 2, 4, 6, 8, 10 and 12 months at Silwood Park, Ascot, both species showed the greatest emergence in cysts stored wet while the least was in cysts stored dry. Under these conditions emergence was influenced by storage conditions rather than storage temperatures or periods, but emergence was generally higherG.rostochiensis in than in G.pallida irrespective of storage conditions, temperatures or periods. G.rostochiensisIn dormancy was shown only in cysts stored dry at 5 and 25°C, whileG.pallida in dormancy was shown in all storage conditions and temperatures except in cysts stored wet at 25°C. Neither infectivity assay nor electrophoresis of crude proteins showed any difference between dormant and nondormant cystsG.rostochiensis of and G.pallida. However electrophoresis showed differences between juveniles and cysts of both species, and also between cysts soaked in sterilized tap water (STW) and those stimulated by potato root diffiisate (PRD). Crude homogenates of cysts in STW and PRD increased emergence in both species, however none of the preparations induced termination of dormancy. The dormancy observed in bothG.rostochiensis and G.pallida suggested that many of the features of diapause may be operating in these particular populations. 3 ACKNOWLEDGEMENTS. I wish to sincerely acknowledge Dr. Adrian A. F. Evans for supervising this work and for creating an atmosphere which made my doubts irrelevant; Dr. William M. Hominick for being my adviser and useful tips in the course of this work; Dr. Jock M. S. Forrest for kindly supplying potato seed tubers and cysts for this work; Dr. Stephen Young for assistance in statistics and data presentation; David Fergusson of The University of Reading for the electrophoresis experiment in his laboratory; Frank Wright for skillfully printing the photographs and all the staff of Silwood Park for their extra kindness beyond the official line. I also wish to express my gratitude to Professor N. M. Gadzama who helped in the development of my career as an academic; the Tenente family for being very wonderful friends throughout the duration of this work - thank you very much; Valerie Walters for her eagerness to help always, even at odd requests and all my friends all over the world. Finally I wish to very sincerely thank Fatima Dabai for her immense understanding and affection. This work was financially supported by a fellowship award from The University of Maiduguri, Nigeria and a scholarship award from the Association of Commonwealth Universities, Which I gratefully acknowledge. 4 CONTENTS. Title page................................................................................................................................................. 1 D e d ica tio n ............................................................................................................................................. 2 A b stra c t................................................................................................................................................... 3 A cknow ledgem ents.............................................................................................................................4 C o n te n ts..................................................................................................................................................5 \ CHAPTER 1: General Introduction and Literature Review...........................................9 1.1 In tro d u ctio n ....................................................................................................................................9 1.2 Bionom ics o f Potato Cyst N em atodes...................................................................................10 1.3 H atching m echanism s o f P C N ’s ........................................................................................ ....14 1.4 D ia p au se ......................................................................................................................................... .18 1.5 Factors influencing d ia p a u se ....................................................................................................2 6 C H A P T E R 2: M ate ria ls a n d M e th o d s ..................................................................................... 3 5 2.1 N em atode population u s e d ........................................................................................................3 5 2.2 Setting up c u ltu re s .......................................................................................................................3 5 2.3 Production of potato root diffusate (PRD........................................................................... ) 3 7 2.4 Setting up hatching s y ste m ............................................................................................... 3 9 2.5 Assessing infectivity of juveniles in different potting media..........................................4 2 2.6 Estimating numbers of unhatched juveniles......................................................................... 4 2 C H A P T E R 3: E m erg en ce P a tte rn s in ’’new " a n d " o ld ” C y sts.......................................4 5 3.1 In tro d u ctio n ....................................................................................................................................:4 5 3.2 M aterials and m e th o d s ............................................................................................................... 4 6 3.2.1 A nalysis o f re s u lts .....................................................................................................................5 0 3.3 R e su lts........................................................................................................... 5 0 3.3.1 G.rostochiensis...........................................................................................................................5 0 3.3.2 G pa llid.............................................................................................................................., a ......53 3.3.3 Eggs in "new" and "old" c y s ts ...............................................................................................5 6 5 3.4 Discussion........................................................... 57 CHAPTER 4: The Effect of Storage Conditions and Temperatures over various P erio d s on S u b seq u en t H a tch in g P a tte rn s o f C y sts...............................................................66 4.1 In tro d u ctio n ...................................................................................................................................... 66 4.2 M aterials and m e th o d s .................................................................................................................6 7 4.2.1 D ry s to ra g e ....................................................................................................................................6 7 4.2.2 W et storage /.................................................................................................................................. 6 9 4.2.3 H um id sto ra g e .............................................................................................................................72 4.2.4 A nalysis o f re s u lts ......................................................................................................................72 4.3 R e s u lts ............................................................................................................................................ 72 4.3.1 D ry sto ra g e ........................................................................................................................... 7 2 4 .3 . 1.1 G.rostochiensis.........................................................................................................................72 4.3.1.2 G.pallida.................................................................................................................................... 7 5 4.3.2 W et s to ra g e .................................................................................................................................7