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 Globodera of 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 8

4.3.2 .1 G.rostochiensis...... - ...... 78

4.3.2.2 G.pallida...... 8 0

4.3.3 H um id sto ra g e ...... g 2

4.3.3.1 G.rostochiensis...... 8 2

4.3.3.2 G.pallida...... 8 5

4.4 D iscu ssio n ...... 8 7

CHAPTER 5: Behavioural and Physiological Responses of Dormant Cysts...... 9 4

5.1 In tro d u ctio n ...... 9 4

5.2 M aterials and m e th o d s ...... 9 5

5.2.1 Infectivity immediately following periods of emergence from "new" cysts...... 9 5

5 .2 .1.1 A nalysis o f re s u lts ...... 9 5

5.2.2 A ssaying crude hom ogenates o f cysts for hatching activities...... 9 5

5.2.2.1 A nalysis o f re s u lts ...... 9 7

6 5.3 Results .97

5.3.1 Infectivity immediately following periods of emergence from "new" cysts...... 9 7

5.3.2 Assaying crude homogenates of cysts for hatching activities...... 9 7

5.3.2.1 G.rostochiensis...... 9 7

5.3.2.2 G.pallida...... 10 0

5.4 D iscu ssio n ...... 101

CHAPTER 6: General Discussion...... 106

References...... 111 Appendixes...... 129

Appendix A: An example of chi-squared test (using two-by-two table) on the

hatching patterns of "new" and "old" cysts ofG.rostochiensis ...... 129

Appendix B: An example of chi-squared test (using two-by-two table) on the

hatching patterns of "new" and "old" cysts ofG.pallida ...... 1 3 0

Appendix C: An example of one way analysis of variance (AVOVA) on number-of

eggs in "new" and "old" cysts ofG.rostochiensis and G .pallida...... 131

Appendix D: Mean temperature and rainfall at Silwood Park, Ascot, 1986 to 1989__ 1 3 2

Appendix E: An example of two way analysis of variance (ANOVA)G.rosto­ on

chiensis cysts stored dry at 5°C...... 1 3 3

Appendix F: An example of two way analysis of variance (ANOVA)G.pallida on

cysts stored dry at 5 ° C ...... 134

Appendix G: An example of two way analysis of variance (ANOVA)G.rosto­ on

chiensis cysts stored w et at 5 °C ...... 135

Appendix H: An example of two way analysis of variance (ANOVA)G.pallida on

cysts stored wet at 5 ° C ...... 136

Appendix J: An example of two way analysis of variance (ANOVA)G.rosto­ on

chiensis cysts stored humid at 5°C...... 137

7 Appendix K: An example of two way analysis of variance (ANOVA)G.pallida on

cysts stored hum id at 5 °C ...... 138

Appendix L: Mean total hatched and unhatched eggs ofG.rostochiensis stored dry at

various tem peratures for various periods...... 139

Appendix M: Mean total hatched and unhatched eggs ofG.pallida stored dry at

various tem peratures for various periods...... 140

Appendix N: Mean total hatched and unhatched eggs ofG.rostochiensis stored wet at

various tem peratures for various periods...... 141

Appendix P: Mean total hatched and unhatched eggs ofG.pallida stored wet at

various temperatures for various periods...... 142

Appendix Q: Mean total hatched and unhatched eggs ofG.rostochiensis stored

hum id at various tem peratures for various p eriods...... 143

Appendix R: Mean total hatched and unhatched eggs ofG.pallida stored humid at

various temperatures for various periods...... 144

Appendix S: An example of two way analysis of variance (ANOVA)G.rosto­ on

chiensis infectivity assay...... 145

Appendix T: An example of two way analysis of variance (ANOVA)G.pallida on

infectivity a s s a y ...... 146

Appendix U: An example of Chi-Squared test onG.rostochiensis ...... 147

Appendix V: An example of Chi-Squared test onG .pallida ...... 148

A ppendix W: E lectrophoresis...... 149

8 CHAPTER 1: GENERAL INTRODUCTION AND LITERATURE REVIEW.

1.1 Introduction.

Nematodes occur in all natural environments where life is found and their numbers probably exceed the total number of other living animals put together (Stone, 1979). Nematodes are found either living free or as parasites of their respective plant or animal hosts; perhaps 50% of nematodes are marine, 25% free-living, 15% animal parasites and 10% plant parasites (Ayoub, 1980). Plant parasitic nematodes (PPN) are generally distinguished from others, most especial­ ly animal parasitic nematodes, by possession of a stylet (though some entomo- philic nematodes possess stylets, Hominick, personal communication, 1990) which is used to pierce plant cells. Plant nematodes are serious pests of food and cash crops and they occur in all regions of the world, in some places resulting in a devastating effect on agriculture with associated economic and social conse­ quences. Sasser and Freckman (1987) reported that average annual yield loss of the world’s major crops due to damage by plant parasitic nematodes in 1984 was estimated to be 12.3%, of which 10.7% are life sustaining crops while 14% are economically important crops;8 .8% of this damage was sustained in developed countries and 14.6% in developing countries. In monetary terms, $77 billion are lost annually worldwide and about $125 million are spent worldwide for nematology research, teaching and extension. In Europe generally and the United Kingdom (UK) in particular some of the most important plant parasitic nematodes economically areGlobodera rosto- chiensis (Wollenweber, 1923) Mulvey and Stone, 1976 andG.pallida (Stone, 1973) Mulvey and Stone, 1976. There are five pathotypesG.rostochiensis of and three o f G.pallida. Their host ranges are confined to the Solanaceae, of which potato is the most important, hence the name potato cyst nematodes (PCN). Although originally described as a distinct species, PCN’s were for some time regarded as a population ofHeterodera schachtii, until Franklin (1951) recog­ nized the species H.rostochiensis. Stone (1973) described H.pallida as different from H.rostochiensis while Behrens (1975) separated the round cyst nematodes from other species ofHeterodera to form the new genusGlobodera. PCN’s are thought to have originated in the South American Andes (Evans et al., 1975) and were introduced into Europe with the introduction of potato from the Andes. PCN was first noted in Spain in 1570, Britain in 1588 (Salaman, 1926 in Evans et al., 1975) and Germany in 1881 (Evanset al., 1975). Since then PCN

9 has spread to almost all parts of the world where potatoes are intensively grown (Fig. 1.1). Initially they were thought to spread in soil adhering to tubers from infected soils but later it was established that means other than transport of contaminated soil also enhance their spread. Europe became the secondary distribution centre of the PCN probably due to improved varieties of seed tubers developed in Europe and exported around the world either through agricultural trading or other fields of economic cooperations.G.rostochiensis is more widely reported than G.pallida, but this may be because the latter has only recently been described and is, as a result, less well recognized. In crop protection, the ultimate aim is either to eradicate the pest (pest control which is regarded as an effort to kill) or reduce it below an acceptable economic threshold (pest management which is directing population numbers) (Thomason and Caswell, 1987). The former is uneconomical and practically difficult to attain while the latter is economical and possible in practice. This approach is achieved by various means of pest management measures which may include cultural practices, use of resistant varieties and nematicide treatments; however none of these measures alone is successful. Therefore it has now been recognized that the most efficient method of management is achieved through integrated control schemes. To achieve this objective, the life cycle and the ecology of the nematode as it relates to its ability to survive unfavourable conditions need to be investigated and understood. This work is an attempt to shed further light on the phenomenon of dormancy as a strategy in the survival of G.rostochiensis and G.pallida. 1.2 Bionomics of Potato Cyst Nematodes.

G.rostochiensis and G.pallida have become widely distributed over the world (Fig. 1.1). Although the two sibling species have similar ecological requirements and can be found in mixed populations in the fields, they do not interbreed freely (Parrott, 1972). In the UK about 80% of infections are due to G.rostochiensis, while the remaining 20% are either due to G.pallida or a mixture of the two species (Turner, 1985), although this may be changing rapidly (Whitehead, personal communication 1990). There is usually only one generation each year, but a limited second generation occurs in some parts of Europe (Evans, 1969). Cysts of these nematodes are able to persist in the absence of a host crop for ten or more years (Williams, 1978). In the absence of host plants the

10 Fig. 1.1 World dispersal of the potato cyst nematode (PCN) from their primary epicentres in the South American Andes and secondary centres in Europe (updated from Evans and Stone, 1977).

11 population declines at about 30-50% per annum (Huijsman, 1961). However this decline is not a continuous process as most of the annual decline is associated with a short period of spontaneous hatching in the spring (den Ouden, 1960). PCN survives in the soil as cysts, each containing about 200-500 eggs (Fig. 1.2). Some eggs within the cyst will hatch spontaneously in spring, while many can remain dormant for many years or until a suitable host is planted. Dormant cysts are resistant to severely unfavourable environmental conditions. G.rosto-In chiensis and G.pallida, dormancy occurs more prominently in the cyst stage of the life cycle (Fig. 1.2), though it may equally occur in some developmental stages which may be less robust compared to the cysts. Specific chemicals exuded by the growing root system of the host plant stimulate those second stage juveniles (J2 or juveniles) which are prepared for hatching to emerge from the eggs within the cysts in a short space of time in the root zone of the host plant. These juveniles rely on stored nutrients and do not feed until they have invaded the roots of host plants. They enter the host root just behind the growing tip or where a lateral root emerges, and migrate until the head of a J2 reaches the cells of the pericycle, cortex or the endodermis, there to form a large syncytial transfer cell on which the nematode feeds. Further development from J2 to adult occurs within the root of the host plant (Fig. 1.2) and, depending on temperature and other factors, the J2 stage is completed within about seven days after entry. The third stage juvenile (J3) is completed in about another 11 days with a well developed rectum and genital primordium appearing. Sexual differentiation starts in the fourth stage juveniles (J4) (Fig. 1.2) with the female juveniles having paired ovaries while the male has a single testis. Sex is possibly determined by availability of food, but once determined it is irreversible (Trudgill, 1967). The motile vermiform male emerges from the root tissue about 20 days after entry of the J2 and becomes free living in the soil. The female J4 is flask-shaped and the body cavity is filled with developing ovaries. During the final moult to the adult stage the reproductive system opens to the exterior with the formation of the vulva. By about the twenty third day after entry of the J2, most internal organs are obliterated with the exception of the uterus which is by now full of eggs. The adult female is sessile and globose. It ruptures the root cortex and protmdes from the root exposing the vulval region with the head and the neck remaining inserted in the root (Fig. 1.2). It is thought that, when receptive, a female at this stage releases pheromone secretions which attract males. Vermi-

12 DIAPAUSE

Fig. 1.2 Life cycle ofGlobodera species. form males move towards the females and many males may surround each female and multiple mating may result (Greenet al., 1970). The males are generally short-lived, remaining active for only about ten days. After mating, the female is fertilized, dies and then her egg-filled body hardens as the cuticle tans and becomes the new cyst. When infected host-plants are lifted, the cysts can be seen as small bead-like objects attached to the roots. Initially cysts of both species are white but those of G.rostochiensis pass through a golden yellow-coloured phase while those of G.pallida pass through a paler cream-coloured phase. Both species finally become' dark reddish-brown, the final colour change coincides with the tanning of the cysts and death of the female. When crops are lifted many mature cysts remain in the upper 30cm of the soil and are usually the source of future infestations.

1.3 Hatching mechanisms of PCN’s.

Hatching of most plant parasitic nematodes follows a particular pattern in which development is completed within the egg and, with the juvenile completely matured in optimum conditions and with the right stimulus, eclosion proceeds. In cyst nematodes the first process involves biochemical and physiological changes leading to the movement of the juveniles within the egg, followed by the complicated process of eclosion. At the eclosion stage the hatching process may be delayed while waiting for an external stimulus before proceeding (Banyer and Fisher, 1980). InH.avenae and M.naasi, such a stimulus is thought to be exposure to a short period of warm temperature following a longer period of low temperature (Banyer and Fisher, 1980; Ogunfowora and Evans, 1977; and Antoniou, 1983). In PCN it is potato root diffusate (PRD) in combination with favourable conditions such as temperature (Clarke and Perry, 1977), while in som e Meloidogyne species hatching occurs spontaneously in water, but once hatching ceases no further stimulation enhances hatch (de Guiran, 1979b). The ability of nematodes to hatch is controlled by the physical and chemical structure of the eggs. Detailed studies of most plant parasitic nematodes show that the egg is composed of three layers: the outer, median and inner layers (Bird, 1976; Bird and McClure, 1976 and Perryet al., 1982). In PCN the outer layer is thought to be made of quinone-tanned protein, the median layer comprises a matrix of collagen-like unstriated fibres and chitin (Clarke et al., 1967) and the inner layer is thought to be of low melting point lipid (Clarke, 1968 and Perryet al., 1982). The outer layer is of minor importance in the hatching process and acts

14 as a shield, the median layer provides strength and rigidity while the inner layer provides a permeability barrier. When cysts of PCN are soaked in water, the inner layer of the eggs becomes permeable to water and gases in both directions but to few other substances (Ellenby, 1974 and Perry, 1987). It also restricts outward flow of solutes in the egg fluid surrounding the unhatched juveniles (Clarke and Hennessy, 1976). Although the eggshell is permeable to water, the unhatched juvenile remains only partially hydrated. Only when the eggs are treated with PRD does the juvenile start to take up water (Ellenby and Perry, 1976). The water content of the unhatched juvenile increases and reaches a maximum after 48 hours (Ellenby and Perry, 1976). Perry and Feil (1986), working withG.rostochiensis , incorporated fluorescent stain into hatching agent and reported that the percentage of unhatched juveniles fluorescing correlated with the percentage hatched juveniles in routine hatching tests. The uptake of the fluorescent stain by the unhatched juveniles is a further indication that, prior to hatching, eggshell permeability changes and this results in water uptake by the juveniles which is a prerequisite for hatching. However, inH.schachtii there was no increase in water content of the unhatched juvenile whether stimulated by root diffusate or water, and water uptake occurred only after hatch of the juveniles (Perry, 1977).G.rostochien­ In sis, oxygen consumption of the cysts increases within 24 hours when stimulated with PRD (Atkinson and Ballantyne, 1977a) and during this period a fall in adenylate energy charge was also observed (Atkinson and Ballantyne, 1977b). These observed changes all preceded movement of the juveniles within the eggs. Three days after stimulation of cysts with PRD, movements of juveniles were observed within the eggs of G.rostochiensis (Doncaster and Shepherd, 1967). The nucleolus of the nucleus in the pharyngeal glands was observed to increase in size (Atkinson et al., 1987 and Perry et al., 1989) but without emission of gland secretions (Clarke and Perry, 1977). Ogunfowora and Evans (1977) observed a similar phenomenon inM.naasi juveniles. However, Banyer and Fisher (1980) working with H.avenae and Perry et al. (1989) working with G.rostochiensis, reported accumulation of secretory granules in the nucleus of the pharyngeal glands. Whether this contributes in the hatching process of the juvenile is not known. The unhatched juvenile of the PCN emerges from the egg by splitting one end of the eggshell following persistent mechanical thrusts of its stylet, emerging head first (Doncaster and Shepherd, 1967). Ogunfowora and Evans (1977)

15 reported the same phenomenon inM.naasi and further, that during movement of juveniles the eggshell was not distorted except at the point of emergence. However the possession of a stylet is not essential in the hatching process of some nematodes. Laughlin et al. (1974) reported that there was no stylet activity during the hatching of H.iri, the eggshell was ruptured by the tail tip and the juveniles emerged tail first. Whether this is typical of hatching ofH.iri or a chance observation is difficult to say. Hydration of the PCN juvenile before hatching was thought to be preceded by a change in the permeability of the eggshell, which allows leakage of trehalose thus removing the osmotic stress on the unhatched juveniles (Clarke and Perry, 1980 and Wharton, 1980). Trehalose in the egg fluid of the PCN was thought to regulate the water content and the maintenance of dormancy while the juveniles are within the egg (Clarke and Hennessy, 1978). Banyer and Fisher (1980) working with H.avenae, suggested that movement of water into the egg-fluid is not determined by trehalose but by the rigid eggshell which limits the volume of water once an equilibrium is reached. They further suggested that an increase in egg volume of about 5% prior to hatching would allow juvenile mobility and escape of trehalose is therefore not essential. They concluded that the change in osmotic pressure before hatching is one of the automatic steps following stimulation by root diffusate. These opposing views on the role of trehalose in the hatching mechanisms of plant parasitic nematodes are yet to be resolved. Though the explanation on the role of trehalose in the hatching process remained unresolved, various authors have offered alternative points of view on how the juvenile hatch. Clarke and Perry (1977) suggested that when eggs are placed in hypotonic solution such as water and subsequently PRD, hatching is initiated by the diffusion of the egg-fluid solutes from the eggs to the hatching medium. This results in increased hydration of the unhatched juvenile which might cause an increase in its metabolic activity and finally lead to the emergence of the juvenile from the egg. Other views expressed on the permeability of the egg shell was that, the changes in the permeability barrier, are caused by either direct or indirect action of the hatching factor, by causing the release of enzymes from the egg-fluid, eggshell or the unhatched juveniles (Clarke and Perry, 1977; Perry and Clarke, 1981 and Perry, 1987); or that the hatching factor, initiates a neurosecretory response from the stimulated juveniles in the egg (Ellenby and Perry, 1976). Bird (1968), working withM.javanica , reported that hatching stimuli trigger

16 release of an enzyme(s) which hydrolyses the eggshell lipid layer before hatching. Perry (1987) suggested that the lipid layer of the eggsH.schachtii of is probably disrupted by fungal enzymes such as lipases and eggshell digestive enzymes need not necessarily emanate from within the eggs. He further suggested that if enzymes are present in the egg fluid, such enzymes could be kept in an inactive condition by the physical separation of the enzymes and substrates by the lipid membrane or that enzyme activity might be suppressed by the trehalose in the egg fluid. Tefft and Bone (1984 and 1985a) attributed the hatching ofH.glycines to permeability of the eggshell created by enzymatic activities. However root diffusate was not found to increase activities of the enzymes (Tefft and Bone, 1985b). Therefore this suggests the possibility of factor(s) other than the root diffusate that may activate the enzymes in the eggshell anchor the egg-fluid. Other workers (Atkinson and Ballantyne, 1979; Atkinson et al., 1980; Atkinson and Taylor, 1980 and 1983) suggested that in PCN, permeability and hatching involves an active calcium transport system and the calcium binding site was thought to be a glycoprotein on the inner surface of the eggshell. Other workers (Clarke et al., 1967; Bird and McClure, 1976 and Perryet al., 1982) suggested that the inner eggshell is composed of lipoprotein membrane and not a glycoprotein. Recently, Clarke and Perry (1985b) and Perry (1986) suggested that there are three binding sites on the eggshell ofG.rostochiensis. The first site is the outer layer where the more strongly binding calcium ions occur. However they are thought not to be involved in the hatching mechanism. The second site is the lipoprotein layer where calcium ions are displaced by artificial hatching agents and decationised PRD. The third site is also a lipoprotein layer whose calcium is available only when eggshells are treated with PRD. The second and third sites are thought to be involved with changes in the lipoprotein membrane structure of the eggshell during hatching (Perry, 1986). The presence of calcium is not in much dispute, but its possible role in the hatching process, the binding site(s) on the eggshell membrane and the specific effects caused in the eggshell structure are the subject of different opinions (Clarke and Perry, 1985a; Clarke and Hennessy, 1981, 1983 and 1987 and Atkinson and Taylor, 1983). These different opinions led Clarke and Hennessy (1981, 1983 and 1987) to suggest that free calcium ions may not be essential for hatching and that a calcium ion transport mechanism is probably not involved in the permeability changes in the eggshell ofG.rostochiensis. These opposing views on the role of calcium ions in the hatching mechanisms ofG.rostochiensis

17 are yet to be resolved. Hatching process(es) seem to be determined by the permeability changes in the egg shell, therefore any process that would hinder the permeability process would equally interfere with the hatching process(es). This probably explains why D ropkin et al. (1958) reported that NaCl with concentration greater than 0.1M suppresses hatching ofG.rostochiensis. This was suggested to be possible because the internal osmotic pressure of the nematode was equivalent to about 0.1M (Wright and Newall, 1976), although Clarke and Hennessy (1976) reported that it is 0.34M. This phenomenon of differential osmotic pressure suppressing hatching, is a further indication that diffusion of substances from the egg-fluid to the sorrounding hatching environment is essential in the hatching process of the juveniles within the eggs. Though much of the work done on the hatching mechanism is onG.rosto­ chiensis, the same may apply toG.pallida considering that both share the same host and hatch only when stimulated by PRD or similar artificial hatching agents. Generally, for hatching to proceed it is necessary for the juvenile within the egg to be in an appropriate developmental condition for the hatching mechanism to be initiated. Any condition which interferes with the developmental state of the juvenile may lead to dormancy, which may include quiescence anchor diapause. In this state most hatching processes may become inoperative.

1.4 Diapause.

Many plant parasitic nematodes, like other animals, develop and grow progressively through the season under normal conditions. However, when conditions become detrimental to their development, some respond in different ways to survive and eventually re-colonize their hostile environment. These unfavourable conditions may extend beyond the nematode’s developmental limits and this may result in senescence, quiescence, diapause anchor cryptobiosis and may eventually lead to the death of the nematodes (Cooper and Van Gundy, 1971), (Fig. 1.3). Plant parasitic nematodes have some exceptional qualities in withstanding environmental pressures. For example,G.rostochiensis was found to contain viable eggs after eight years in soil (Franklin, 1937),Meloidogyne javanica was found after land had been left fallow for over four years (Martin, 1967), an Indian population ofHeterodera avenae survived two years of dry storage and yet hatched freely when placed in water (Williams, 1978) and M.incognita w as found

18 Fig. Fig. fo CopradVa ny 1971). undy, G an V and ooper C (from

1 PHYSIOLOGICAL AGEING .3 A diagram m atic representation o f the life span o f a plant parasitic nem atode atode nem parasitic plant a f o span life the f o representation atic m diagram A .3 19 infecting large numbers of vegetables grown in the coastal and desert areas of Libya and Saudi Arabia where soil temperatures at various times in the season exceed 40°C (Khan and Siddiqui, 1986 and Eissa et al., 1979). Adaptations for survival are even more remarkable in JapaneseH.glycines populations which produce two types of eggs. One type is released into the gelatinous matrix and hatches readily and the other type remains in cysts which have been found capable of surviving up to nine years in the field without hosts (Ishibashiet al., 1973). These abilities of nematodes to survive very unfavourable conditions have interested many plant nematologists who have proposed various terms to describe the phenomena (Tables 1.1 and 1.2). The term diapause originates from its application to arthropods, but it has been equally used in describing phenomena in earthworms (Satchell, 1967) and plant parasitic nematodes (Shepherd and Cox, 1967; Oostenbrink, 1967; Evans and Perry, 1976; Hominicket al., 1985 and Evans, 1987). Lees (1955), referring to arthropods, defined dormancy as characterized by a temporary failure of growth or reproduction by reduced metabolism which often enhances resistance to adverse climatic factors such as cold, heat and drought. The appearance of the resting stage, he suggested, may be evoked by just those conditions which it is adapted to survive. He further defined diapause as restricted to instances where development or activity is arrested ’’spontaneously", as opposed to an interruption of growth directly controlled by unfavourable conditions which he regarded as quiescence. Within the definition of diapause he distinguished two categories, facultative and obligatory diapause. He suggested that facultative diapause does not necessarily occur in every generation, its onset and arrest seem always to be influenced at some stage by the environment and it can be induced or averted by appropriate external conditions. Obligatory diapause, in contrast, is such that when arthropods are reared under different conditions every individual enters diapause in each generation regardless of the environment. Keilin (1959) restricted himself and used the metabolic state of the animal as the fundamental criterion for discussing survival activities in animals. He described four types of dormancy as hibernation, aestivation, diapause and quiescence and suggested that factors that influence dormancy include loss of water, cooling, lack of oxygen, high salt concentration and a combination of any of these factors. Mansingh (1971) regarded any response in insects away from the normal path of development as dormancy and he defined it as an evolved physiological

20 Table 1.1 Terms used to describe survival of plant parasitic nematodes under unfavourable conditions.

— Term s Description R eferences

Anhydrobiosis. Tolerance to dehydration. 39, 42, 43, 86, 87

Osmobiosis. Tolerance to high salt concen­ 31,46, 149, 191 tration.

Anoxybiosis. Tolerance to lack of oxygen. 39, 40, 89

Cryobiosis. Tolerance to low temperature. 3 8 ,1 3 8 ,1 8 6

Thermobiosis. Tolerance to high temperatures.1,20

Quiescence. Reaction to one or a combina­ 39, 62, 186 tion of the above factors and easily reversible.

Cryptobiosis. "Hidden life". No visible sign 39, 62, 186 of life and metabolic activities become hardly measureable.

Diapause. Endogenous or exogenous fac­1, 13, 14,61,62, 87, 88,91, tors which might be influenced 92, 129, 136, 157, 168 by environmental factors acting on the nematodes for minimum period of time leading to delay or arrest in development.

21 Table 1.2 Reported incidences of diapause in plant parasitic nematodes.

Nematodes References

G. rostochiensis 61,62, 96,136,168 H. avenae 13,14,157 M.naasi 1 ,1 2 9 M.incognita \ M.arenaria 1 87, 88,91,92 M.javanica l M.hapla I

22 adaptation to overcome adverse environmental conditions of a particular climatic zone. Thus, ecological stimuli, characteristic of the climatic zone set in motion complex physiological and biochemical processes which immediately or event­ ually bring about the arrest of development. He suggested that there are basically three types of dormancy: quiescence, oligopause and diapause. He defined quiescence as a response to a sudden, unanticipated, noncyclic and usually short duration deviation of one or more environmental factors from the optimum. The reaction of the individual is spontaneous and results in growth retardation only within its natural tolerance limits and it can occur at any time of the year and may be experienced by any or all of the developmental stages of the insects. Diapause was defined as the most highly evolved system of dormancy for overcoming cyclic, long term and extreme environmental conditions. It is induced well before the adversity and maintained for some time irrespective of the environmental factors and it is fixed at a particular developmental stage of a given species. The third category was oligopause which he regarded as an intermediate between quiescence and diapause. It is closer to diapause in character except that it is activated by mild and moderate environmental conditions and operates slightly before the adversity. Laudien (1973) also grouped all resting stages under dormancy and regarded the cause of the arrest in development as the fundamental criterion for separating categories within dormancy. He attributed such causes to the environment, which he suggested could induce as well as reverse dormancy. This, he suggested, differed in diapause, which he characterised as the condition in which develop­ ment has been arrested and cannot be resumed until specific requirements have been satisfied even if favourable conditions return. While most of the reviews cited above dealt with the phenomena as experienced in arthropods, Evans and Perry (1976) extensively reviewed the phenomenon of dormancy in nematodes. They suggested that inhibited develop­ ment in animal parasitic nematodes is an expression of quiescence in which development has been interrupted under the influence of some factor(s) other than the external environment, that is factors usually associated with, or acting through, the host. Arrested development was considered as an expression of diapause, which they described as a phenomenon in the nematode’s life cycle which is induced by, or has arisen through selection as, a developmental adaptation to seasonal changes in the environment of the infective juveniles. They therefore regarded quiescence and diapause as types of dormancy, quiescence

23 being induced in response to unfavourable environmental conditions and ended by the return of favourable conditions, while diapause is the condition in which development has been arrested and cannot be resumed until specific requirements have been satisfied (Table 1.3a). Wharton (1986), in agreement with the earlier classifications of Lees (1955), suggested a classification which recognizes the role of "metabolism" and "development" in the expression of dormancy in nematodes. He considered metabolic dormancy to comprise quiescence and anabiosis, while developmental dormancy includes obligate dormancy and diapause. Evans (1987) however criticized this concept and the earlier categories of Evans and Perry (1976) by suggesting that they both suffer the disadvantage of not carrying the analysis of the causes of dormancy far enough. He therefore suggested a classification which considered all the factors so far raised and included the developmental potential of the nematodes as well (Table 1.3b). In this classification he regarded normal life to be when ontogenetic development is unaffected and somatic development is proceeding. Facultative quiescence occurred when environmental conditions change and as a consequence affected only somatic development. Obligate quiescence or diapause occurred when ontogenetic and somatic development are both affected by endogenous factor(s). The final category, regarded as a prediapause, has only ontogenetic development affected while somatic develop­ ment continues. In discussing a subject as diverse and complex as diapause, various classification schemes and definitions are appropriately offered as highlighted above. However, there are basically two schools of thought governing the various concepts, which are "metabolic" and "developmental" types of dormancy. Thus, there are those who advocated: a) "metabolic" dormancy (Lees, 1955 and Keilin, 1959); b) "developmental" dormancy (Mansingh, 1971; Laudien, 1973 and Evans and Perry, 1976); and, c) both "metabolic" and "developmental" dormancy (Wharton, 1986 and Evans, 1987). Each school has its advantages. However, as discussed by Evans and Perry (1976), the "metabolic" dormancy school of thought suffers some shortcomings (such as the view on the primacy of metabolic activity in controlling diapause) highlighted by evidence deduced from work on the control of development, especially in the field of diapause in arthropods. Therefore, I have regarded diapause as a form of dormancy and the cause of arrest in development is considered to be the fundamental criterion for separating categories within dormancy. This would agree with some of the proposals of

24 Table 1.3 (A) A classification of types of dormancy in nematodes (from Evans and Perry, 1976). (B) States of nematode dormancy arranged according to observed effects on somatic and ontogenetic development (from Evans, 1987).

(A)

Dormancy

Quiescence Diapause

Facultative Obligate Facultative Obligate 1 4 4 Initiated by Environmental factors becoming Stage in the life cycle Environmental factors Endogenous factors unfavourable acting as signals to receptive stage E nded by Return of favourable conditions Environmental signals, Spontaneous endogenous Environmental conditions (readily reversible) indicating favourable factors acting after a acting on the receptive conditions, which minimum period of time stage for a minimum “trigger" further period of time development Examples W idely shared by nem atodes; Resistant stages, e.g. Arrested development of Larvae in eggs of Anoxybiosis, dauer larvae of some trichostrongyle parasites H eterodera spp. and Cryobiosis, rhabditids; of animals, e.g. M eloidogyne naasi Osmobiosis, Third stage infective Obelescoides ostertagi, Unfavourably high temperature, larvae of trichostrongyles;Haemonchus contortus, Anhydrobiosis, Cysts ofH eterodera sp p .; O stertagia cuniculi Cryptobiosis Eggs of many species

(B)

Somatic Ontogenetic development development Unaffected Affected

Unaffected I. Normal life IV. Prediapause (like oligopause [46]) Active growth. Ontogeny stopped by token stimuli Active metabolism. from environment. Metabolism largely unaffected until diapause proper ensues. Affected II. Facultative Quiescence III. Quiescence and Diapause Reduced activity. Growth a) Obligate quiescence. Stage in life much reduced or stopped. cycle. Readily overcome in Metabolism reduced. favorable conditions. b) Diapause. Delayed resumption of development. (i) Facultative-induced bv token stimuli in environment. (ii) Obligate-stage in life cycle.

25 Evans and Perry (1976). However, I disagree with their classifications of quiescence and diapause into facultative and obligate categories. Use of these categories excluded most plant parasitic nematodes from their scheme and the categories are difficult to recognize in practice (see Chapter 6). The recent review of Evans (1987) also suffers similar shortcomings.

1.5 Factors influencing diapause.

Various factors, abiotic and biotic, combine together to elicit complex behavioural adaptations in plant parasitic nematodes. The most basic factors which bring about changes in nematodes are the abiotic factors. All animals are governed by their genes interacting with the environment. The most successful animals survive and continue their generations while the less adaptable ones perish. Shepherd and Clarke (1971) suggested that the abiotic factors affecting hatching and subsequent survival of the PCN included temperature, soil moisture, aeration and pH, with temperature and moisture having the major role. This part of the literature review will look at the important biotic and abiotic factors and relate them where possible to the overall survival of the PCN. Most workers treat each of the factors highlighted above separately in discussing nematode survival. Such an approach in discussing diapause in the PCN will limit the appreciation of the ecosystem as an ecological unit. Therefore, in this review, all the factors considered above and others which may influence diapause in the PCN will be discussed in an integrated approach. Where necessary, examples other than from the PCN will be discussed to shed further light on the phenomenon of diapause. Also most early work failed to appreciate the presence of diapause, and most of the experimental designs were not planned towards such studies. In the few cases where dormancy was studied, fundamental information was frequently lacking on some or all of the following: a) history of cysts, b) species involved G.rostochiensis( or G.pallida), c) storage conditions of cysts, d) hatching conditions, e) hatching medium used and how it was produced, f) host plants on which cysts were raised. Absence of these details makes assessment of results from work done prior to 1973 difficult, most especially in ascertaining which species of the PCN was involved (see Section 1.1). Neverthe­ less, it is important to review earlier works (with their shortcomings in mind) so

26 that a historical perspective of studies of dormancy in plant parasitic nematodes can be appreciated. It is anticipated that with such a background, the contribution of the present work will become clear and add to our understanding of dormancy in the PCN. Gemmell (1940) and Ellenby (1946a) observed that when G.rostochiensis was hatched in PRD, daily emergence of juveniles reached a peak of numbers then declined. They further reported that when hatching ceased, some eggs still remained in the cysts and failed to hatch even when fresh PRD was added. However, when cysts were dried and later stimulated with PRD, further hatching was achieved. Ellenby (1946a) explained this phenomenon by suggesting that metabolic products produced by the hatching juveniles accumulated in the cysts and inhibited further hatch. He investigated this problem by comparing punctured and unpunctured single cysts and concluded that emergence was greater in the punctured cysts (Ellenby, 1946b). Shepherd (1962) and Shepherd and Cox (1967), however, reported that with replicated batches of 50 cysts they found no significant differences between punctured and unpunctured cysts in either water-soaked or air-dried cysts. Onions (1955) and Hesling (1959) working withG.rostochiensis and Wallace (1959) working withH.schachtii suggested that hatching is delayed or stopped because of an oxygen deficit and increased acidity of the hatching medium caused by the activities of the hatched juveniles. Wallace (1959) reported that changing the hatching medium at various intervals removed the inhibitory substances and allowed hatching to resume. Kaul (1962) suggested that a hydrolysable catechin- tannin in the cyst wall of G.rostochiensis might be responsible for inhibiting hatching during autumn and winter since he found more of this compound during these seasons than during spring when hatching was prominent. Shepherd and Cox (1967), working withG.rostochiensis , reported that more eggs hatched when freed from new cysts than in intact cysts. However, they apparently contradicted Kaul (1962) when they reported that hatching was further increased when fragments of tanned cyst wall were left among the freed eggs. Dropkin (1976) suggested that a stimulus for delay or cessation of hatching in plant parasitic nematodes may be derived from some metabolites of soil biota rather than from the physical factors of the environment.

27 In H.glycines, Okada (1972a) reported that washed cyst walls added to a suspension of eggs did not affect emergence of juveniles, whereas aqueous extracts of crushed whole cysts did inhibit hatching. Paradoxically, Okada (1974) also reported that crushed whole cysts ofH.glycines, G.rostochiensis and H.oryzae induced emergence of juveniles. Clarke and Perry (1977) suggested that the method of extraction of the hatching factor(s) from the cysts determines the results of the hatching tests. This probably explains why Okada (1972b) reported obtaining hatching stimulation by ultrasonic disruption of a suspension of eggs and juveniles ofH.glycines. Okada (1977) also reported that when root diffiisates are combined with aqueous cyst extracts the rate of hatching H.in glycines w as found to be significantly greater than either of the two alone. This suggests that the two stimulants possibly have different sites of stimulation. He suggested inhibiting and stimulating substances to be antagonistic in in vitro hatchings. He further reported that hatching inhibitors are found only in old cysts but are absent in young cysts and adult females. InM.naasi, failure of hatching was considered to be due to inhibitory substances in the eggshell (Watson and Lownsbery, 1970; and Gooris and d’Herde, 1972). Watson and Lownsbery (1970) reported that there were no cytological or morphological differences between chilled and unchilled eggs ofM.naasi, even though the chilled eggs gave a higher percentage hatch. They therefore suggested that the chilling effect produces a chemical rather than a structural effect which inactivates the hatching inhibitor(s). In H.glycines and G.rostochiensis, the hatching factors produced by host plants were found to be unstable (Okada, 1977 and Shepherd, 1962). Perry (1986) reported that potato root diffusate is inactivated at pH greater than 8 and contains 4-6 "hatching factors" which are moderately strong organic acids. Therefore, substances "derived" from nematodes which promote or inhibit hatching could be complex, as diffiisates from host plants are. It is also difficult to establish whether such substances are from the nematodes or from other organisms associated with the nematodes. For these reasons, most in vitro experiments to show the effects of such substances are difficult to evaluate and results should be treated with caution. Triffitt (1930), Calam et al., (1949) and Lownsbery (1951) suggested that the observed phenomena of suppression of hatching inG.rostochiensis and H.schachtii are an expression of dormancy which is inherent in the nematodes due to seasonal effects. They reported that winter juveniles failed to emerge even under optimum conditions in the laboratory and suggested that neither tempera­

28 ture nor fresh root diffiisates can stimulate further hatching in winter. Lownsbery (1951) suggested that exposure to winter conditions is not necessary to induce dormancy because dormancy is part of the nematode’s normal physiology. Fenwick and Reid (1953b) and Ellenby (1955), while recognizing dormancy in G. rostochiensis, reported that temperature and seasonal fluctuations do not induce dormancy. Winslow (1956), working withG.rostochiensis , H.schachtii and H. cruciferae, reported that dormancy in the winter season was expressed only by G.rostochiensis. Fenwick and Reid (1953a), Winslow (1956) and Cunningham (1960) accepted the view thatG.rostochiensis exhibits dormancy, but thought that it might have been induced by the conditions to which cysts are exposed before or during storage. Cunningham (1960) concluded that winter hatch depression or dormancy is not inherent in the seasonal hatch cycle of G.rostochiensis but is induced by the soil conditions in the previous autumn and late summer. The contradictory findings highlighted above were clarified by Shepherd and Cox (1967) when they interpreted periodicity in the hatching G.rostochien­of sis as facultative diapause. They argued that the term dormancy is not accurate enough because all second stage juveniles in eggs Heterodera of species are quiescent until shortly before they are about to hatch. They further stated that in the field, loss of the ability to hatch starts long before temperature conditions become unfavourable for hatching. Once this happens, no treatment is known which stops it. However, removing cysts from the field early in their formation seems to keep some in a state in which they can hatch. The inability to hatch seems therefore not to be obligatory. The stimulus the eggs receive may be one that does not take effect immediately but sets off diapause at a later stage. They could not identify the exact nature of the environmental conditions that set off diapause in their work, but they did suggest that removal of cysts from the field before August reduced diapause onset. Fushtey and Johnson (1966), working with a Canadian population ofH.avenae, were more specific when they reported that a period of at least eight weeks at 0-7°C is required before the eggs hatch. Whether this period is characteristic ofH.avenae cysts in the field or only a laboratory-in­ duced condition is not clear. Therefore, it is difficult to relate this behaviour to termination of diapause. Shepherd and Cox (1967) reported that inG.rostochiensis , temperature of storage prior to hatching in PRD had no appreciable effect on hatching at any time of the year except for eggs stored at 30°C for six weeks which showed an accelerated onset of hatching. Oostenbrink (1967) observed the same phenom­

29 enon in G.rostochiensis. Bishop (1953 and 1955) reported that alternating temperatures at 25°C and 15°C for five days in every seven resulted in greater hatch o f G.rostochiensis cysts than continuous incubation at 25°C. Wallace (1955) also demonstrated that incubation under a continuous regime of eight hours at 24°C followed by 16 hours at 15°C gave the highest hatch H.schachtii.in Hatching behaviour in field and in vitro tests often differs. Shepherd (1963) demonstrated this difficulty when she reported that H.goettingiana enters what seems to be a permanent state of diapause when brought into the laboratory at whatever age dr time of the year. However, eggs in the field hatch readily and infect pea plants during spring and summer in response to the stimulus from pea roots. It is interesting to note that Perryet al. (1980) and Beane and Perry (1983) obtained 40-74% emergence in vitro and showed that the hatching response in H.goettingiana is related to the age of the plant (4 and 6 weeks) producing the diffusate and the storage temperature of the cyst infested soil (a low temperature of about 2°C) prior to hatching at a higher temperature (15°C). Perry and Beane (1983) also showed that duration of stimulation for 18 and 24 hours\week is of major importance in the hatching of free eggs ofH.goettingiana. Whether this supports the presence of diapause as reported by Shepherd (1963) is difficult to say because hatching may only have marked the end of a quiescent period. These studies suggest that induction or termination of diapause is associated with seasonal cycles, which in turn are a function of temperature. The combined effects of temperature changes and root diffiisates tend to be seasonal markers which might provide signals for the PCN to synchronize its life cycle with that of its host. Jones (1975a and b) suggested that for nematodes living near the soil surface, diurnal temperature fluctuations may be large and may have a direct or indirect effect on their life cycle, while for those living deeper in the soil the small diurnal temperature fluctuations may not be important. Since cysts of G.rostochiensis and G.pallida are produced and generally remain in the root zone, most will be found within 30cm of the soil surface, where temperature fluctu­ ations in the soil will have an effect on their behaviour. Further insights into the effects of temperature fluctuations in the field followed by laboratory hatching at constant temperature were provided by Banyer and Fisher (1971a). They used cysts ofH.avenae brought directly from the field and set to hatch at 20°C, over periods of up to 20 weeks, without ever recording complete hatch. However, they observed differences in the hatching rates within this period. Based on these observations, they suggested that eggs (within a cyst)

30 which hatched within a short time were those in which dormancy had been overcome in the field, whereas those eggs which hatched after a longer period (when hatching rate had declined) were dormant when brought from the field, their dormancy having been broken during the incubation period in the labora­ tory. They explained this phenomenon by suggesting that high soil temperature (>20°C) and high soil moisture were not essential for overcoming dormancy in the field. However, after termination of dormancy, both temperature and soil moisture would become important in controlling continuation of hatching. In the laboratory, dormancy was overcome at 20°C, while in the field it was overcome when temperatures were low in mid-autumn-winter periods. They therefore concluded that breakage of dormancy is not an inherent seasonal rhythm, but is due to the action of environmental factors which in this instance is the onset of low temperature. Although storage at low temperature for specified periods was found to increase the rate at which dormancy was broken, they suggested that it may not be an essential single factor. Dormancy could not be broken by a period of eight weeks at 7°C followed by hatching at 20°C, but a period of eight days did break the dormancy. This therefore suggested that the duration of the low temperature is an equally important factor in dormancy termination, perhaps by a diapause mechanism. In another work, Banyer and Fisher (1971b) suggested that hatching in H.avenae involves two processes, the first regarded as a period of juvenile "development" with an optimum temperature of about 10°C which must be completed before a second phase begins. Phase-two was regarded as an eclosion period with an optimum temperature of about 20°C. Regardless of the temperature at which each phase was completed, the optimum temperature for each phase remained the same. They explained the phenomenon of dormancy by suggesting that when soil temperature remains high (>20°C) dormancy is not broken because the rate of phase-one development will be slow at such a temperature, and as a result, the hatching rate remains low regardless of soil moisture. The stage at which dormancy is induced, they suggested, was dependent on the magnitude of temperature rise. The remarkable effect of seasonal rhythm on plant parasitic nematodes, and on H.avenae in particular, was demonstrated by Rivoal (1978 and 1979). He reported that H.avenae which originated from Northern France has an optimum hatching temperature of 10°C and that transfer to 15°C is followed by a fast and short emergence of juveniles. In contrast those populations which originated from

31 Southern France hatched best at 5°C and a further rise in temperature suppressed their hatching ability. When both populations were raised in an intermediate climate in central France for three years or were transferred reciprocally from their geographic origins to that of the other race, in both instances they retained their individual hatching characteristics, although dates of initiation of hatch and peak juvenile emergence followed local temperature regimes as expected (Rivoal, 1986). Kerry and Jenkinson (1976) and Williams and Beane (1979) reported that in the UK, H.avenae hatches in seasonal cycles, with low hatch in winter and maximum hatching attained when the soil temperature in summer reaches about 20°C. Other workers (Watson and Lownsbery, 1970 and Ogunfowora and Evans, 1977) reported the same phenomenon in the hatching behaviourM.naasi of w here eggs hatch at 20°C only after a chilling period of 7-11 weeks at 5-10°C. Antoniou (1983) reported the same with populations ofM.naasi from Belgium, California, France, Germany, New Zealand and Wales (UK). H om inick et al. (1985) suggested that inG.rostochiensis , dormancy appears to be inherent in seasonal cycles with maximum emergence in spring and early summer, while from July until next spring emergence was observed to be suppressed; neither PRD nor distilled water stimulated hatching. They suggested that this dormancy had characteristics of a diapause which may be a consequence of signals received by the developing females and their eggs from the plant during its growing season. Oostenbrink (1967) was cautious in attributing diapause in G.rostochiensis to a single factor. He suggested thatG.rostochiensis show ed a distinct diapause, which may be influenced, but not controlled by, age of cysts, storage conditions, temperature, root diffusate or seasonal cycle. He went on to suggest that mechanisms which control diapause might be fully understood by studying the role of fluctuating temperatures, genetics and biochemistry of unhatched eggs. Evans (1974 and 1987) suggested that factors from the host plants may have effects on the eggs of nematodes contributing to the induction of diapause in plant parasitic nematodes. Hominick (1986) tested this hypothesis by hatching two batches of G.rostochiensis cysts, one from field-grown potatoes where light was not a limiting factor, and the other batch from potatoes grown in canisters in an incubator in the dark with light as a limiting factor. He reported that cysts from the field had pronounced diapause while cysts from the canisters had no evidence of extended diapause. This implies that daylength may be a factor in inducing diapause in PCN through the host plant. Evans (1982) reported that photoperiod-

32 ism had an effect on host plants and their exudates, which in turn affected the hatching behaviour ofG.rostochiensis. However, such an effect was found to be due not only to photoperiodism but also to the cultivar of the host plant (Farrer and Phillips, 1983 and Forrest and Phillips, 1984). So far, it has been possible to suggest the phenomenon of diapause as a form of dormancy in temperate nematodes, de Guiran (1979b) reported that a M.incognita population from Ivory Coast in West Africa had some viable unhatched juveniles remaining after being hatched at 28°C for 28 days. He suggested that- this dormancy could be considered as a diapause, de Guiran (1979a) further suggested that stress due to environmental conditions led to quiescence in the eggs ofM.incognita and that the longer the period of the stress, the more the quiescence tended to resemble diapause (de Guiran, 1980; de Guiran and Germani, 1980; de Guiran and Villemin, 1980a and b). However de Guiran (1979b) reported that egg masses in diapause could not be stimulated to hatch either in the presence of host roots or their diffiisates, or after dissociation by sodium hypochlorite (NaCIO). He showed that such eggs were alive by using a vital staining technique. He further reported that 10-20% of the eggs in a population tend to exhibit diapause characteristics which were transmitted from one generation to the other irrespective of the percentage in the original egg m asses. Evans and Perry (1976) suggested that in some nematodes diapause may be obligatory for most individuals in a population. However, they cautioned that it is often difficult in nature to separate the state of quiescence from diapause. Quiescence may precede diapause and follow on after the termination of diapause. Therefore there is no clear cut behavioural boundary which separates various degrees of dormancy in plant parasitic nematodes. Environmental and endogenous factors acting on the nematodes tend to complement each other. Dormancy of various kinds, but especially diapause, is seen as one of the processes which, if present, may need to be overcome to allow the chain of events leading to hatching to be completed. In the first part of this work, an opportunity was taken, while attempting to establish the nature of the dormancy inG.pallida, of making parallel experiments onG.rostochiensis to confirm work on the same population as used by Hominicket al. (1985). The second part examined, in more detail than previously, some of the various combinations of environmental influences that may act on potato cyst nematodes surviving in soil for several years in the absence of a host crop. Various temperature treatments ranging from

33 5-25°C and storage periods of up to 12 months in different conditions (dry, wet and humid) were used and their effects were tested by assessing hatching. In the third part attempts were made to detect changes other than hatching in cysts during and while ending dormancy, in the hope that this would give some indication of mechanisms involved in expressing a type of dormancy that would lead to our understanding of diapause. Finally, the evidence from this and other work is considered in the light of the ecological pressures acting on the two potato cyst nematodes,G.rostochiensis and G.pallida selecting them for the kinds of dormancy they have.

34 CHAPTER 2: MATERIALS AND METHODS.

2.1 Nematode Population Used.

Cysts o f Globodera rostochiensis and G.pallida used in these experiments were isolates that had been continuously grown outdoors for a number of years at the Scottish Crop Research Institute (SCRI) at Invergowrie, Dundee. On 27:2:87, in response to a written request, Dr. J.M.S. Forrest of SCRI sent two vials, one w ith G.rostochiensis pathotype Rol (Rol) and the other withG.pallida pathotype Pa 2\3 (P2\3) (both species were raised on potato plants cv. Pentland Crown), by post. Both species had been harvested from the 1985 season, extracted from soil, and stored as cysts at SCRI at room temperatures in the dark. On arrival at Silwood Park, Ascot on 29:2:87, cysts were counted into batches of 100 and placed in glass vials for storage in the dark at 20°C. During storage, a constant temperature of 20°C was used with minimal exposure to light by keeping all vials in light proof plastic containers until required for experimentation.

2.2 Setting up Cultures.

On 2:3:87 certified potato seed tubers of cultivar Pentland Crown, obtained from commercial sources, were spread out in a plastic tray in a greenhouse at 5-8°C until sprouts were observed on the eyes. Sixty 18cm plastic pots with the drainage hole covered with fine muslin cloth were filled with sterilized loam: sand (2:1). A sprouting tuber of potato was planted into each pot and kept for three days in a greenhouse at 18-23°C. On 28:4:87, thirty batches of 100 cysts of each species,G.rostochiensis and G.pallida, were soaked in sterilized tap water (STW) for seven days in the dark at 20°C. On 5:5:87, each pot was inoculated with the pre-soaked cysts ofG.rosto- chiensis and G.pallida respectively. Pots were maintained outdoors in a gravel plunge (Fig.2.1) and watered when necessary through the growing season. About three months later as the potato foliage was senescing, the 30 pots of each population were removed from the plunge. The soil and the roots were carefully emptied into a large tray, spread out thinly and dried slowly in a warm air chamber in the dark. After five days, the dry contents of 15 pots of each population was extracted using a Fenwick can. The float containing the cysts and debris was wrapped in a muslin cloth and again dried in the dark in a warm air chamber. When the debris and the cysts were completely dry after three days,

35 Fig. 2.1 Potato plants in plastic pots in an outdoor gravel plunge.

36 cysts were handpicked from the debris and randomly counted into batches of 50 and stored in glass vials at 20°C in the dark inside a light-proof plastic container until required for experimentation. The soil from the remaining 15 pots of each population was sieved (a 60 mesh sieve placed over a 20 mesh sieve) to remove all traces of living potato roots or tubers. Soil was then divided into sets of 18cm plastic pots according to nematode speciesG.rostochiensis ( or G.pallida) and returned to the gravel plunge outdoors (at the same time as the other half began their storage at 20°C in the dark, Table 2.1). Here they were left for 365 days, after which the soil from each pot was extracted with a Fenwick can on 4:9:88. Cysts were dried, handpicked and counted into batches of 50, for storage in glass vials at 20°C in the dark on 8:9:88. Precautions were taken as previously to limit exposure to light and cysts were stored under these conditions until required for experimentation. The cysts extracted soon after maturity on host roots are referred to as "new" cysts, while those cysts returned to the outdoor gravel plunge for one year are referred to as "old" cysts. In the absence of any living potato tissue to allow reproduction, the "old" cysts are presumed to be identical to the "new" cysts, the only difference being one of age.

2.3 Production of Potato Root Diffusate (PRD).

Sprouting potato tubers produced as described above were planted in a plastic tray filled with sterilized loam: sand (2:1) and kept in a greenhouse at 5-10°C until shoots developed. When the first sets of shoots were observed, the tubers were transferred to a controlled temperature room (CT room) maintained at 20-22°C with a 16 hours light and 8 hours dark regime (Hussey and Stacey, 1981). Plants were watered when necessary. About one month later, when shoots developed to a height of about 3cm, 40 sprouts were separated at their base from the tubers and planted into trays of sterilized sand and grown in a CT room at 20°C with a 16 hours light and 8 hours dark regime. Plants were watered with "Phostrogen" plant food supplement according to the manufacturers recommendation whenever necessary. After two weeks of growth, roots were washed and immersed in STW, with five sets of roots per 125ml of STW in a glass beaker in the dark for two hours (Hominicket al.t 1985). The diffusate produced was pooled and filtered through Whatman Nol filter paper into several one litre polythene bottles. The bottles were individually

37 Table 2.1 Calendar of production of culture G.rostochiensis of and G.pallida at Silwood Park, Ascot.

Date T reatmentsNRemarks

27:2:87 Cysts of G.rostochiensis and G.pallida were obtained by post from SCRI.

29:2:87 Cysts were divided into batches of 100 and stored in glass vials at 20°C in the dark.

28:4:87 Cys.ts were soaked in STW for seven days at 20°C in the dark.

5:5:87 60 pots containing sprouting potato seed tubers were each inocu­ lated with 100 presoaked cystsG.rostochiensis of and G.pallida respectively. Pots were maintained outdoor in a gravel plunge.

28:8:87 Pots were removed from gravel plunge and their contents air dried in the dark.

4:9:87 Cysts were extracted from 30 pots using Fenwick can. 15 pots containedG.rostochiensis while the other halfG.pallida.

8:9:87 Cysts from the 30 pots were handpicked and counted into batches of 50 ("new" cysts) and stored in glass vials at 20°C in the dark. The contents of the other half of the pots (30) withG.rostochiensis and G.pallida (similar to above) were sieved free of all traces of living potato roots and tubers; the soil and the cysts were then returned to the gravel plunge outdoors and kept there for one calendar year.

5:10:87 Hatching started on batches of "new" cysts.

28:8:88 Pots maintained outdoors in a gravel plunge for one calendar year were removed and air dried in the dark.

4:9:88 Cysts were extracted from the 30 pots using Fenwick can, one half (15 pots) containedG.rostochiensis and the other halfG.pallida.

8:9:88 Cysts were handpicked and counted into batches of 50 ("old" cysts) and stored in glass vials at 20°C in the dark.

5:10:88 Hatching started on batches of "old" cysts.

38 covered with double wrap of black plastic sheet and placed into a black plastic storage jar and stored in the dark at 3-4°C (Fenwick, 1949 and Hominicket al., 1985). When PRD was required for hatching, the required quantity was drawn from the stock into a light proof glass flask. Transfer of PRD into the hatching plates was done with light proof automatic pipette tips. These precautions were to minimize exposure of PRD to light. Fenwick (1949) and Oostenbrink (1967) reported that storage of PRD at 3-4°C does not diminish the activity of the diffusate. This' batch of PRD was used throughout these experiments unless otherwise stated.

2.4 Setting up Hatching System.

Hatching tests were made in incubators, at different temperatures, according to the requirements of each experiment, using batches of 50 cysts replicated four times. In each experiment, cysts were stored under defined conditions (details are provided under individual experiments). Cysts were first soaked in STW for seven days in the dark at chosen hatching temperatures, rinsed in STW, and then hatched in PRD. During the experiments, each batch of cysts was kept on a small (7mm diameter)45 m nylon hatching sieve (Fig. 2.2d), in 1ml of PRD in a well of 24-well Linbro culture plates (Flow Laboratories, UK. Fig. 2.2b and c). The plates were individually wrapped in a black plastic sheet (Fig 2.2a) to limit exposure to light. While changing the hatching medium care was taken to expose the cysts to minimal light. Before changing PRD or rinsing cysts with STW, both media were removed from their storage temperatures and equilibrated at the respective hatching temperatures for about 15-20 minutes. This protocol was applied for all the hatching experiments, because sudden changes in temperatures during incuba­ tions might trigger effects on subsequent juvenile emergence (Oostenbrink, 1967). Such a phenomenon results in either unduly large or small juvenile emergence (Oostenbrink, 1967). Cysts on sieves were transferred to fresh PRD weekly and the emerged juveniles counted. The concentration of juveniles in suspension were adjusted before sampling so that 100-200 juveniles were counted (Peters, 1952). Fungal growth appearing during incubation of cysts was removed with a soft brush when hatching medium was changed.

39 Fig. 2.2 Hatching system apparatus a) black plastic cover b) 24-well Linbro multiwell culture plate c) culture plate with cover and d) hatching sieves.

4 0 41 2.5 Assessing Infectivity of Juveniles in Different Potting Media.

To assess the best growing medium in which to check nematode infectivity, three-week old tomato plants, cv. "Moneymaker", were planted into 9cm plastic pots with the drainage holes covered with fine muslin cloth. Pots were filled with either a) 40-100 mesh acid washed sand (BDH), b) sterilized river bed sand or c) sterilized loam:sand mixture (1:1). Plants were watered lightly when necessary with "Phostrogen" plant food supplement and kept in a CT room at 20°C with a 16 hours light and 8 hours dark regime for two days. During this period, the tomato plants were allowed to get established in the potting medium before inoculation. On the third day, four pots of each medium were inoculated with approximately 1000 juveniles (J2)G.rostochiensis of or G.pallida. Pots were then arranged randomly in a CT room at 20°C with a 16 hours light and 8 hours dark regime and watered lightly with "Phostrogen" solution when necessary. 14 days after inoculation, each plant was gently lifted, the roots washed under slow running tap water and stained in 0.05% acid fuchsin in lacto-glycerol (Bridgeet al., 1982). Stained nematodes were counted under a dissecting microscope and the mean percentage infectivity determined for each potting medium. The lowest invasion occurred on plants potted in river bed sand, while the highest invasion was recorded on plants potted in BDH sand. However, the loam:sand mixture (1:1) was chosen as the best compromise between maximal invasion and cost of materials.

2.6 Estimating Numbers of Unhatched Juveniles.

After the completion of each experiment, numbers of viable eggs (i.e. those eggs containing unhatched coiled second stage juveniles) (Fig. 2.3c) were estimated in each batch of cysts. Cysts were individually broken open under a dissecting microscope using fine mounted needles. The contents of the broken cysts were diluted as previously described (Section 2.4) and then thoroughly mixed using an MSE homogeniser to ensure even dispersion of eggs. Three aliquots were taken from the dilution of each batch and counted as previously described (Section 2.4). Juveniles which were freed in the process of breaking the cysts were counted as viable unhatched eggs, while empty eggs (Fig. 2.3d) were disregarded. Emergence was calculated as a percentage of the total hatched and unhatched viable eggs from each replicate.

42 Fig. 2.3 Potato cyst nematode a) cysts b) second stage juvenile c) egg containing second stage juvenile and d) empty egg.

43 X100

X400 X400 CHAPTER 3: EMERGENCE PATTERNS IN "NEW" AND "OLD" CYSTS.

3.1 Introduction The classical method of controlling pests in agricultural soils relies on cultural practices. Such practices include one or more methods such as fallow farming, shifting cultivation or crop rotation. These practices in general terms achieve some success, however soil inhabiting pests re-emerge and re-colonize most agricultural lands once host crops are introduced.G.rostochiensis was found to contain viable eggs after eight years in soil (Franklin, 1937),M.javanica was found after land had been left fallow for over four years (Martin, 1967)H.schachtii and cysts survived the summer period on fallow soil where soil temperature frequently exceeded 40°C (Thomason and Fife, 1962); also the same nematode survived a winter period during which the soil freezes (Rrusberg and Sardanelli, 1989). The phenomenon of dormancy, including delayed emergence, quiescence or diapause in plant parasitic nematodes, has long been a subject of contradictory findings among nematologists. Gemmell (1940) and Ellenby (1946b) observed an incomplete hatch byG.rostochiensis in PRD, daily emergence of juveniles reached a maximum rate, then declined. Ellenby (1946b) attributed such failure by the nematodes to hatch completely to inhibiting substances produced as a result of metabolic activities of the juveniles within the eggs as they are stimulated by the potato root diffusate (PRD). Hesling (1959) and Wallace (1959) suggested it was caused by oxygen deficiency. Triffit (1930) and Calamet al. (1949) suggested it was a seasonal expression, while El-Shatoury (1978) suggested it was a genetically inherited character of selective advantage to the nematode population. Shepherd and Cox (1967) and Oostenbrink (1967) produced evidence that G.rostochiensis eggs which failed to hatch were in diapause and they compared the phenomenon with that found in insects. Even with this indication there was confusion in the usage of terminology and definition of the term diapause in plant parasitic nematodes. This led to an extensive review on the subject in an attempt to resolve the contradictions (Evans and Perry, 1976 and Evans, 1987). Hominicket al. (1985) demonstrated unequivocally that one populationG.rostochiensis of exhibited diapause which aids it in surviving unfavourable conditions. Other workers have demonstrated the phenomenon of diapauseH.avenae in (Banyer and Fisher, 1971 a,b; Rivoal, 1979, 1983 and 1986);M.naasi (Ogunfowora and Evans, 1977 and Antoniou, 1983) andM.incognita (de Guiran and Demeure, 1978, de Guiran and Villemin, 1980a,b and de Guiran 1979a and 1980).

45 In this chapter, the pattern of emergence of second stage juveniles(32) from eggs of one population each ofG.rostochiensis and G.pallida were compared according to a "nematode-response" hatching protocol of "new" and "old" cysts. "New" cysts (cysts extracted soon after maturity on host roots) are hypothesized to be those exhibiting diapause, while "old" cysts ("new" cysts that were stored for one calendar year outdoors in a gravel plunge) are hypothesized to be those that have overcome their diapause (Hominicket al. 1985). The aim was by observing the hatching behaviour in these experiments to establish the presence or absence of diapause inG .pallida, and if present, to compare for the first time its occurrence with that ofG.rostochiensis.

3.2 Materials and methods

Hatching was done according to a "nematode-response" protocol (Fig. 3.1). "New" and "old" cysts were both stored in batches of 50 cysts at 20°C in the dark (SI). Four batches were hatched at 20°C in potato root diffusate (PRD) whenever required. The first "new" cysts were set to hatch in October 1987 by soaking in sterilized tap water (STW) for two weeks (H I) then hatching in PRD (H2) as described in Chapter 2 Section 2.4. Emerging juveniles were counted weekly until less than 100 juvenilesVeplicate\week emerged. Hatching was then concluded for these cysts which were rinsed in STW, dried, and stored on their hatching sieves at 20°C in the dark for a period of 12 months (S3). A new batch of cysts was set to hatch immediately. This protocol was continued for one calendar year (Table 3.1) "New" cysts had a second hatching after each replicate had had 12 months of dry storage at 20°C (Fig. 3.1). Cysts were first soaked in STW for one week (H3) and then hatched in PRD (H4) as previously described. Emerging juveniles were counted weekly until less than 10 juvenilesVeplicate\week emerged. These protocols were performed on each replicate according to a strict calendar (Table 3.1). Cysts were then broken open and the number of viable unhatched eggs determined as described previously (Chapter 2, Section 2.6). The same protocol (Fig. 3.1) and strict calendar (Table 3.1) was followed to hatch "old" cysts, the only exception being that in this case cysts were first stored for 12 months outdoors and then for various periods at 20°C in the dark (S2). When emergence was less than 100 juvenilesVeplicate\week cysts were broken open and the number of viable unhatched eggs determined. Cysts were not stored and hatched the second time as in "new" cysts due to lack of time.

46 Fig. 3.1 "Nematode-response" hatching protocols for "new" and "old" cysts of G.rostochiensis and G.pallida incubated dry at 20°C and hatched at 20°C in PRD over a period of one and two calendar year(s).

Key: ------Storage period...... Soaking in STW. ------Hatching in PRD. 51 First storage period of "new" cysts at 20°C. 52 First (and second) storage of "old" cysts outdoor (and 20°C). 53 Second storage period (dry) of "new" cysts for 12 months at 20°C. H I First soaking ("new" and "old" cysts) in STW. H2 First hatching ("new" and "old" cysts) in PRD until < 100 J2 emerged\week. H3 Second soaking ("new" cysts) in STW. H4 Second hatching ("new" cysts) in PRD after 12 months storage until < 10 J2 emerged\week.

47 "New "cysts "Old'bysts

StorageNHatching Storage temp Hatching temp Storage temp StoNHat period 20C 20C 20C period

0-12 SI 12 Months Months (outdoors)

S2 1I 0-12 2 Weeks H i j Months

i i 1 Variable H 2 ! i h i ! | 2 Weeks T * 1----- ! 1 12 Months S3 i i i j 1 W eek H3 ; j H 2 i i j i Variable H4 1 1 Variable i i t i Cysts broken

4 8 Table 3.1 Calendar of hatching of "new" and "old" cystsG.rostochiensis of and G.pallida. "New" cysts had second hatching after dry storage for one calendar year at 20°C in the dark. All hatching as in PRD at 20°C in the dark.

Hatching period "New'' cysts "Old" cysts

First hatch Second hatch First hatch

G.rostochiensis

October 5:10:87 5:10:88 5:10:88

November 23:11:87 23:11:88 23:11:88

January 11:1:88 11:1:89 11:1:89

February 15:2:88 15:2:89 15:2:89

April 4:4:88 4:4:89 4:4:89

May 21:5:88 21:5:89 21:5:89

June 6:6:88 6:6:89 6:6:89

July 4:7:88 4:7:89 4:7:89

August 22:8:88 22:8:89 22:8:89

G.pallida

October 5:10:87 5:10:88 5:10:88

November 16:11:87 16:11:88 16:11:88

December 21:12:87 21:12:88 21:12:88

February 15:2:88 15:2:89 15:2:89

April 4:4:88 4:4:89 4:4:89

May 9:5:88 9:5:89 9:5:89

July 4:7:88 4:7:89 4:7:89

September 12:9:88 12:9:89 12:9:89

49 As earlier stated in Chapter 2, Section 2.1, cysts in this work originated from SCRI where they were raised on the potato cv. Pentland Crown. For the purpose of the experiments in this Chapter, both "new" and "old" cysts were raised on the potato cv. Pentland Crown at Silwood Park, Ascot as also detailed in Chapter 2, Section 2.2. Also all PRD used in this experiments was from the potato cv. Pentland Crown as detailed in Chapter 2, Section 2.3. Therefore, maximum care was taken to reduce the possibility of differences in hatching patterns due to host influence. At the end of the experiments, the number of viable eggs in "new" and "old" cysts at each hatching period was determined, to see whether differences in hatching patterns were as a result of differences in egg contents or not. Also the numbers of eggs in "old" cysts were compared with those in "new" cysts to assess whether spontaneous hatching had occurred during storage in the soil outdoors.

3.2.1 Analysis of results.

The statistical package "Statistix" (NH analytical software, St. Paul MN 55117 USA) was used to perform the Chi-squared test to assess whether the hatching curves in "new" and "old" cysts differed significantly from one another in any hatching period; using the hatching curve of "old" cysts as "expected" (see Appendixes A and B). The numbers of eggs in "new" and "old" cysts were tested with one way analysis of variance (ANOVA) to assess whether there were significant differences between their contents during any hatching period (see Appendix C).

3.3 Results.

3.3.1G.rostochiensis. Cumulative percentage emergenceG.rostochiensis of juveniles from "new" cysts (first year following their production) and from "old" cysts (stored 12 months in the field) is shown in Fig. 3.2. Nine hatching cycles were achieved in 12 months by following the "nematode-response" protocol. Cumulative percentage emergence in "new" cysts can be grouped as low emergence (<50%), medium emergence (50-60%) and high emergence (>70%). Cysts hatched in January and April had about 40% emergence; those hatched in October, February, May, June, July and August had 50-60% emergence while those hatched in November had the highest emergence of over 80%. In all hatches the peak rate of emergence was noted in the second and third week of hatching in PRD, except in the November hatch where there were two peaks of emergence, one in the

50 October 1987/88 November Cumulative Percentage Hatch Cumulative Percentage Hatch Cumulative Percentage Hatch Cumulative

Time (weeks) Time (weeks) Cumulative Percentage Hatch Cumulative Percentage Hatch line indicates resumption of hatching (H3 and H4) after storage period 83- period storage after andH4) (H3 resumption hatching of indicates line y End of first hatching period in "new" cysts (HI and H2). Continuing H2). and (HI cysts "new" hatchinginfirst of period End y i. . Cmltv ecnaehtho nw ad"l" yt of cysts "old" and "new" of hatch percentage Cumulative 3.2 Fig. hatching in PRD at 20C over a hatching cycle of one and two and one of cycle hatching a over 20C at PRD in before hatching periods various for 20C at dry stored G.rostochiensis calendar year(s). calendar August June 52 "Newcysts" "Old cysts" July

third week and another in the fifth week. The shortest total emergence period was six weeks during the April hatch, while the longest was 13 weeks during the October hatch. Most emergence was completed between seven and nine weeks. Dry storage of "new" cysts at 20°C for 12 months after first hatch (Fig. 3.1 S3) and subsequent second hatching in PRD (Fig. 3.1 H3 and H4) gave a negligible additional increase to the cumulative hatch in the first year (Fig. 3.1 HI and H2) on all hatching, except October hatch where there was an increase in cumulative emergence of 2%. When "old" cysts were hatched, emergence was about 90% in all except the last period (August hatch) which had about 75% emergence. The maximum period of emergence was 10 weeks in October, the minimum was five weeks in the November, January, February, April and June hatches, while the May, July and August hatches lasted six, seven and eight weeks respectively. As in "new" cysts most nematodes emerged during the second and third week. Emergence in "new" and "old" cysts ofG.rostochiensis was significantly different in October, January, February, April, May and July (P<0.05 or P <0.005, see Fig. 3.2) hatches; suggesting the presence of dormancy in these periods. However hatches in November, June and August were not significantly different (Fig. 3.2), suggesting the absence of dormancy in these periods. 3.3.2G.pallida. Results of hatching "new" and "old" cysts ofG.pallida following the same protocol as in G.rostochiensis are shown in Fig. 3.3 and produced only eight hatching cycles. Cumulative percentage emergence could be grouped asG.rostochiensis in , except that in this case there was no medium emergence. Hatches of "new" cysts in October, November, December, February and April produced less than 50% emergence, while hatches in May, July and September gave about 80% emergence. In all hatches, peak emergence was in the third or fourth week of hatching, except in May, where there were two peaks- the first peak starting in the second week and the second peak in the fifth week. The shortest emergence period was eight weeks seen in October, February and April, while the longest was 12 weeks in July. As in G.rostochiensis, dry storage at 20°C for 12 months after first hatch and subsequent second hatching in PRD produced no further significant emergence in all hatches.

53 Fig. 3.3 Cumulative percentage hatch of "new" and "old" cystsG.pallida of stored dry at 20°C for various periods before hatching in PRD at 20°C over a hatching cycle of one and two calendar year(s).

Key: I End of first hatching period in "new" cysts (HI and H2). Continuing line indicates resumption of hatch (H3 and H4) after storage period S3.

54 October 1987/88 November

55 However, "Old" cysts showed differences in their emergence according to the period of hatching. October hatch was lowest (less than 20% ), April, May and July hatches had 50-60% emergence, and the highest emergence of about 80% was in November, December, February and September. All peaks of emergence were in the second and third week except for November hatch which had various peaks. The longest emergence period of 10 weeks was in November hatch, all other hatches had an emergence period of four to five weeks. Emergence in "new" and "old" cysts ofG.pallida was significantly different (P<0.05 or P<0.005, see Fig. 3.3) in November, December, February, April, May and July hatches; suggesting the presence of dormancy in these periods. However, hatches in October and September were not significantly different suggesting absence of dormancy in these periods, even though October hatching had less than 50% emergence and September hatching had more than 80% emergence (Fig. 3.3).

3.3.3 Eggs in "new" and "old" cysts.

The numbers of eggs in "new" and "old" cysts ofG.rostochiensis were significantly different (P<0.05) in October, January, February and May hatchings (Table 3.2). However, G.pallida in only cysts in November hatching were signifi­ cantly different (P<0.05) (Table 3.2). Generally, there were more eggs in "old" cysts of G.rostochiensis than in "new" cysts; while inG.pallida there were more eggs in "new" cysts than in "old" cysts. The observation inG.rostochiensis was unexpected and cannot be explained.

3.4 Discussion.

The main problem associated with describing and comparing reported phenom­ enon of diapause in cyst nematodes is principally due to lack of information on: a) history of cysts; b) species involved prior to 1973G.rostochiensis ( or G.pallida); c) storage conditions of cysts; d) hatching conditions; e) hatching medium used; f) host plant on which cysts were raised. It was with these problems in mind that Hominick et al. (1985) described detailed protocols in their experiments in determining diapause G.rostochiensis.in In this present work, similar protocols were strictly observed. In Hominicket al. (1985) hatching was set at particular predeter-

56 Table 3.2 Total number of eggs in batches of 50 "new" and "old" cysts (means and standard deviation (SD) of four replicates)G.rostochiensis of and G.pallida.

Hatching period Total number of eggs in batches of 50 cysts (means and SD)

"New" cysts "Old" cysts

G.rostochiensis

October 14949 ±840 27733 ± 2309 *

November 20178 + 1577 24619 ±3688

January 16623 ± 1396 32678 ±2417*

February 21872 ±1782 35048 ± 3096 *

April 22291 ± 4 0 0 2 30445 ± 2574

May 17082 ±591 22317 ±1346*

June 19457 ±2810 23549 ± 1498

July 17903 ±2187 21419 ±3800

August 21263 ± 1272 18633 ± 4 3 1

G.pallida

October 20998 ± 892 18987 ±1699

November 29497 ± 2413 18875 ±1590*

December 24034 ± 2672 23672 ± 3483

February 25940 ± 852 28902 ±3171

April 24128 ±1661 22443 ±1925

May 27818 ±2542 23645 ± 2981

July 24307 ±1177 23244 ± 2667

September 26334 ±1468 31919 ±4174

* Significantly different at P<0.05

57 mined months as representative of the seasons in the U.K. In this present work, hatching was done continuously according to a "nematode-response" protocol. In this method, nematodes were set to hatch at the beginning of Autumn (October 1987) and then each species was left to indicate when its hatching ability was exhausted (when less than 100 juveniles\replicate\week emerged) before another batch of cysts was set to hatch. The advantage of this approach is that, at any time of the year for the period this experiment was done (12 months) there were cysts hatching. At the end, it was the cysts that determined their own behaviour according to how they responded to the experimental conditions. To obtain a better comparison of emergence in both "new" and "old" cysts, it would have been desirable to have identical treatments on both sets of cysts. However, as mentioned earlier, due to lack of time "old" cysts could not be stored for 12 months after the first hatch. As a result they had no opportunity of having a second hatch. Even with this shortcoming in the design of the experiments, results of emergence in "old" cysts showed up to 90% emergence in most cases. As a result there would have been very few extra J2 to emerge at a second hatching. Variability in cysts was controlled by replication, while efficacy of PRD was achieved by storage of stock solution below 4°C (Fenwick, 1949). In this condition, PRD was shown to retain its hatching activity for well over two years (Oostenbrink, 1967). Temperatures in respective incubators were set according to the manufacturers instructions and checked regularly to ensure they were working properly. However, in the course of this work slight fluctuations were encountered occasionally and corrected. G.rostochiensis, according to the "nematode-response" hatching protocol, had nine hatching cycles in 12 months compared to eightG.pallida. in This agrees with findings of Parrott and Berry (1976) and McKenna and Winslow (1972) that G.pallida hatches less freely thanG.rostochiensis. Parrott et al. (1976) suggested that this makesG.pallida more persistent in soils. Ellis and Hesling (1975) reported that any differences in hatching betweenG.rostochiensis and G.pallida may be more apparent than real. Clarke and Perry (1977), suggested that a more valid comparison needed to include the total percentage emergence and to ensure identical past histories of the cysts before comparison of the hatching of the two species could be made. This work has met the conditions that Clarke and Perry (1977) suggested and the results confirmed findings of other workers thatG.pallida hatches less freely than G.rostochiensis.

58 The prevalence of diapause as recorded by some earlier workers (Shepherd and Cox, 1967; Oostenbrink, 1967; Cunningham, 1960; Winslow, 1956 and Shepherd and Clarke, 1971), and its absence recorded by other workers (Fenwick and Reid, 1953b; Ellenby, 1955 and Rode, 1971), are very difficult to resolve. This difficulty arises because much of the earlier work was done without realizing that there are two species of the then knownH.rostochiensis. It was in 1973 that Stone (1973) describedH.rostochiensis as different fromH.pallida and Behrens (1975) separated the PCN from Heterodera to form a new genus Globodera. One problem of interpreting the results of workers prior to 1973 is the possibility that they may have been working with one or other species or with a mixed populationG.rostochien- of sis and G.pallida which may have had completely different hatching patterns. Due to these differences, different workers at different times, despite using similar experi­ mental conditions, reported either the presence or absence of diapause in PCN. In both G.rostochiensis and G.pallida the form of the emergence curve in "new" and "old" cysts is an asymmetric sigmoid curve. This begins with a rapid emergence of many J2 and later, a slow but continuing progress. Hominicket al. (1985) suggested that the rate of initial hatch may be more relevant in assessing evidence for a diapause rather than the final cumulative hatch. In this work, the rate of initial hatch and cumulative percentage emergence of "old" cysts was considered in assessing evidence for diapause. In G.rostochiensis, hatching of "new" cysts in October, January, February, April, May and July showed low emergence which was overcome in "old" cysts during the same period in the subsequent year. Statistically, when hatching curves in "new" cysts in these months were compared with "old" cysts they are significantly different (P<0.05 or P<0.005, see Fig. 3.2), indicating the presence of dormancy in "new" cysts. Hatching in November, June, and August was not significantly different from hatching in "old" cysts in the same period; indicating the absence of dormancy in "new" cysts. In all the nine hatching cycles G.rostochiensis in the exceptional emergence pattern was in November hatch where in both "new" and "old" cysts emergence was over 80%. Dormancy in G.rostochiensis in this population is shown in October, January, February, April, May and July which seasonally corresponds to early Autumn, Winter, Spring and mid Summer. However, absence of dormancy was shown in November, June and August corresponding to late Autumn and Summer. These results differ remarkably with those of Hominicket al. (1985) who reported diapause to be present in Autumn and early Winter and absent in Spring and Summer. These

59 differences may be due to differences in hatching periods when hatching tests were conducted. They did their hatching tests at prefixed periods at intervals of two months for 12 months. Though this may give a good representation of the various seasons, the reality is that seasons in the U.K. are not fixed in a rigid chronological monthly timetable. Another factor in differences in seasons is how each particular worker determines when a particular "season" ends and another begins. There is always an extension of one season over another and also overlapping of seasons. The Summer of 1987 in which cysts in this work were raised, was wet and warmer compared to the Summer of 1988 in which cysts were hatched (Appendix D). This difference may have influenced the emergence patterns in both populations of G.rostochiensis and G.pallida. On closer examination of individual hatching periods, there are striking similarities between the hatching curves in this present work and those of Hominick et al. (1985) in hatching tests done in June. The other similarities were in hatching tests done at Scottish Crop Research Institute at Invergowrie, Dundee (SCRI) in February, April and October on cysts grown either at Imperial College, Silwood Park, Ascot (IC) or SCRI. However, the hatching behaviour they reported differed when the same cysts were hatched at IC. Differences between the present experi­ ments and those of Hominicket al (1985) agree with the comments made by Hominick et al. (1985) when they suggested that "even when identical cysts, hatching agents and environment are used, significantly different hatching curves were obtained by different laboratories". In this present work, cysts originated from SCRI, though they were raised at Silwood Park, Ascot (in a similar environment and identical location with cysts used in the work of Hominicket al., 1985). Neverthe­ less, they still maintained their SCRI characteristics. Hominick (1979) showed that cysts of G.rostochiensis from two farms in Scotland, one where early potatoes are cultivated and the other where potatoes are cultivated in a conventional manner, showed differences in their hatching patterns when they were raised and hatched in identical laboratory conditions (at IC). Each population showed hatching patterns characteristic of it’s cultivation conditions in Scotland. Also Rivoal (1978 and 1986) reported that whenH.avenae from northern France was transferred and reared in southern France and vice versa their hatching rhythm remain basically unaltered, the same thing happened when both northern and southern populations were reared and hatched at an intermediate climate. Maybe cultivation practices in different areas are selecting for a population of nematodes adapted genetically to those areas.

60 Hatching in "new" cysts of G.pallida was remarkably different from that obtained in "new" cysts ofG.rostochiensis. Hatching started slowly in October with about 40% emergence. In the following months of November, December, February and April hatching slowed down much further with less than 20% emergence. However, in May, July and September, emergence dramatically increased to about 80%. Comparing this pattern of emergence with "old" cysts showed hatchings in November, December, February, April, May and July to be significantly different (P<0.05 or P<0.005, Fig. 3.3); indicating the presence of dormancy in "new" cysts. However, hatchings in October and September were not significantly different, indicating absence of dormancy in "new" cysts. These results suggest the presence of dormancy in "new" cysts in Autumn, Winter and early Spring which was overcome in late Spring and Summer. In these results, hatchings of great interest were in October and September; hatchings in October showed the presence of dormancy in both "new" and "old" cysts while it was completely absent in September hatching. These results suggest synchronization between the hatching patterns of "new" cysts of G.pallida and the development of its potato host plants. Dormancy sets-in during Autumn and Winter, corresponding to the post harvest plant dormancy period, while it is overcome in Spring and Summer corresponding to periods when potato plants start growing and are liable to infection by PCN. Winslow (1956) suggested that dormancy exists inHeterodera species but, it varies between species with some being slight or negligible and in others more pronounced. Gemmell (1943) and Ellenby (1946c) showed that the number of juveniles that emerged from cysts of the same species are influenced by the variety of host on which they were grown. The work of Hominick (1979) supported this suggestion when he showed that there are significant differences between sizes of cysts (and by inference their content) from two farms in the same localities. This variance among cysts of the same species may possibly be among the reasons for the sharp differences between emergence in "new" cystsG.pallida of and G.rostochiensis, although McKenna and Winslow (1972), reported that emergence was similar between pathotypes of the same species. Differences in dormancy within species also exist inMeloidogyne species, M.hapla overwinters successfully whileM.incognita and M.javanica were unable to overwinter in either bare soil, under-cover crops or roots of perennial host plants (Johnson and Potter, 1980).

61 A second hatching of "new" cysts in PRD did not increase emergence in all hatching periods in bothG.rostochiensis and G.pallida. In some of the first hatching periods where emergence had reached over 90% obviously there are few juveniles left in the cysts for further emergence. However, in hatching periods where emergence was less than 50% it was expected that restimulation after one year storage would elicit further emergence. Diapause in the caseG.rostochiensis of was mainly present in "new" cysts and since the "new" cysts were stored for one year after their first hatch, it was anticipated that their diapause would have been overcome by the time of their second hatching. Hominicket al. (1985) did a similar experiment but, they exposed their cysts to distilled water, dried them and then exposed them to PRD one year later. Their results differed in emergence patterns. Cysts hatched at IC, whether grown at IC or SCRI, had low emergence in February, June, August and April. While in similar cysts which were hatched at SCRI emergence was almost 100%. In this present work as well as in Hominicket al. (1985) the cysts used originated from SCRI but were grown at Silwood Park, Ascot. Probably the differences observed in both works maybe due to differences in climate available in the north (SCRI), where daylength is short in winter and the south (IC) of the U.K. where daylength is longer. Rivoal (1978) reported the same phenomenon in H.avenae in the north and south of France. Oostenbrink (1967) temporarily exposed one year old cysts G.rostochiensisof to PRD and repeated restimulation for four successive years and in each year he recorded decreased emergence. He suggested that this is not uncommon in nature, and attributed the phenomenon to diapause which acts to protect the species from hatching under unfavourable conditions. Another possibility for lack of emergence during the second hatching period may be because cysts were exposed to PRD before they were stored dry for one year. Perry (1989) reported that such a treatment removes or alters the protection afforded by the eggshell and trehalose, and as a result the unhatched juveniles become susceptible to desiccation. In G.pallida where dormancy is seen in both "new" and "old" cysts, occurring in a less defined pattern comparedG.rostochiensis to , it is difficult to offer plausible explanations. However, Janssenet al. (1987) reported that cutting cysts G.rosto­ of chiensis in halves bypasses diapause. In this present work, both "new" cysts of G.rostochiensis and G.pallida which exhibited dormancy during their hatching periods were cut into halves at the end of the second hatching. However, there was no increase in emergence after two weeks of hatching in PRD. The only juveniles observed were those released as cysts were cut. Janssenet al. (1987) obtained

62 emergence in their experiments probably because the cysts they used were grown on agar medium. As a result, such cysts have little relationship with a host plant experiencing a seasonal environment which was suggested to influence the develop­ ment of the female and the induction of diapauseG.rostochiensis in cysts (Hominick et a l, 1985 and Hominick, 1986). In "old" cysts of G.rostochiensis, emergence was about 90% in all hatching periods except August where emergence was about 75% . This result indicates absence of dormancy in cysts ofG.rostochiensis when stored outdoors for one year. Cunningham (1960) stored cyst-infested soil in an outdoor gravel plunge and at room temperature; in the laboratory stored cysts he reported that level of hatching depends on when cysts were extracted from soil and suggested that any effect on hatching is induced by the soil conditions. Oostenbrink (1967) demonstrated that cysts taken from the field before August showed high emergence in Autumn and Winter when such cysts are supposed to be in diapause. Ellenby (1955) hatched his cysts one year after harvest and he reported that cysts stored at room conditions showed a higher emergence than cysts stored at constant temperature. He attributed the daily temperature fluctuations in the room as the reason for the high emergence. Since temperature fluctuations have been demonstrated to trigger the successful emergence of one year post harvest cysts (Ellenby, 1955 and Oostenbrink, 1967), and similar temperature fluctuations occur outdoors, it may have contributed to the emergence of "old" cysts of G.rostochiensis in this present work. However, Jones (1975a) reported that nematodes living near the soil surface experience larger diurnal temperature fluctuations than those deeper in the soil where diurnal temperature fluctuations are not very great. Population of cysts ofG.rostochiensis and G.pallida in this present work were distributed randomly in 16cm long plastic pots in a gravel plunge during outdoors storage, some cysts may have been at the soil surface while others may have been deeper at the bottom of the pots. As a result, they might have been exposed to different diurnal temperature fluctuations. From the literature available, it is very difficult to determine the relationship between hatching pattern of cysts and the influence of storage in soils outdoors. This difficulty arises from the complexity of soil as an ecosystem (Jones, 1975a). Also from climatic factors imposed on nematodes via host plants independent of the soil conditions (Evans, 1974). Considering all these difficulties in interpreting results in this son of work, it is nevertheless clear that storage of cystsG.rostochiensis of in the soil outdoors overcomes dormancy which was present in "new" cysts. Whether

63 this is attributed to maturity of cysts (Ellenby and Smith, 1967a), soil conditions (Cunningham, 1960) or cyclic and temperature (Oostenbrink, 1967) influences it is difficult to explain. Contrary to behaviours in "old" cysts ofG.rostochiensis, "old" cysts of G.pallida responded completely differently. Emergence reached 70-90% in No­ vember, December, February, April and September; declined to about 50% in May and July and declined further to less than 20% in October. This trend showed high emergence in Autumn and Winter then a slowing down in Spring and Summer and an almost complete suppression in mid Summer. These results suggest that storage of cysts of G.pallida for one year in the field does not overcome the strong dormancy observed in "new" cysts. Probably because dormancy is very strong in the "new" cysts it may require a much longer storage period before it overcomes such a dormancy. Hatching patterns ofG.pallida in both "new" and "old" cysts were complex, regardless of this complexity, emergence from "old" cysts was far below that of "new" cysts in October, May and July. It appears as if a new dormancy has set in, suggesting the possible presence of multiple dormancyG.pallida. in Oostenbrink (1967) suggested the induction of a second diapause in cystsG.rostochiensis of which was independent of cyclic effects but influenced by physiological maturation. Perhaps this is what is happening in the "old" cysts G.pallida. of The complexity of offering possible explanations in this type of work was summarised by Shepherd and Cox (1967) when they reported that; "an interesting feature of the behaviour of G.rostochiensis in the field is that diapause seems not restricted to new generation eggs but can recur, and eggs that would have hatched given the right stimulus enter diapause a second time and periods of quiescence may alternate with diapause". Contrary to expectations, "old" cysts ofG.rostochiensis and G.pallida were found to be full and their egg contents were almost similar to those of "new" cysts. From this observation neither the theory of spontaneous hatching nor that of micro-organism induced hatching can be supported. Perryet al. (1981) reported that evidence exists to show persistence of hatching factors in soils because solution drained from soil 100 days after removal of potato roots still caused some hatching. However Fenwick (1956) reported that PRD activities are lost in four days in soil and suggested that the breakdown was caused by micro-organisms. Stelter and Sager (1987) reported that leachings from potato grown soil immediately after harvest caused more than 50% emergenceG.rostochiensis. in However, as the season progressed, hatching due to leachings from a potato plot and a non-host plot were very low and not significantly different. They suggested that hatching was due to

64 hatch-stimulating substances produced by soil micro-organisms. Ellenby (1963) and Ellenby and Smith (1967b) also suggested that hatchingG.rostochiensis of in the absence of host crops is due to effects of micro-organisms in the soil. In this present work, cyst-infested soil was sieved to exclude potato tubers and roots before the soil was stored outdoors. Probably this significantly reduced hatching in the soil during the storage period. Another possible reason may be because the soil in this work was not subjected to continuous potato cultivation. Consequently there was no accumulation of hatching factors in the soil to cause further hatching during cyst storage. The reason why soil micro-organisms and their activities have not caused hatching during the storage period cannot be explained. Considering the definitions of dormancy and their categorisation by Evans and Perry (1976) and Evans (1987), and from evidence advanced by Hominicket al. (1985) in establishing the phenomenon of diapauseG.rostochiensis in , the dormancy observed in both populations ofG.rostochiensis and G.pallida in this work, suggested that a physiological state similar to diapause was operating in both species.

65 CHAPTER 4: THE EFFECT OF STORAGE CONDITIONS AND TEM­ PERATURES OVER VARIOUS PERIODS ON SUBSEQUENT HATCHING PATTERNS OF CYSTS.

4.1 Introduction.

Nematodes are poikilothermic organisms and lack the capacity to change their microenvironment in soil or move away completely from it. The habitat of the PCN is very variable ranging from low temperatures in winter to high temperatures in summer. Though humidity in soil is always relatively high, in spring when rain is abundant some soils remain waterlogged for considerable periods, while in summer when temperatures are high and no rain falls, top layers of soil tend to become dry. Waterlogged and dry soils both influence the osmotic pressure experienced by nematodes. These experiences may result from either dilution or concentration of organic or inorganic chemicals available in the soil, which may be from the soil itself, plant organs, soil inhabiting organisms or introduced through agricultural practices. Banyer and Fisher (1971a) suggested that dormancyH.avenae in is broken by the onset of low temperature and enhanced by the duration of the cold temperature. However a rise in temperature after incubation at low temperature induced dormancy which is dependent on the magnitude of the temperature rise (Banyer and Fisher, 1971b). Antoniou (1983) reported thatM.naasi in , chilling interrupted by short periods of warmth stimulated hatching in diapausing eggs, de Guiran (1979b and 1980) reported a diapauseMeloidogyne in spp. and found that neither temperature nor humidity had an influence in ending diapause.G.rostochiensis, In Lewis and Mai (1957 and 1960) reported that storage of cysts at up to 24°C or alternating between 0°C and 24°C in dry soil had no effect on emergence. However, storage in moist soil under the same conditions resulted in more emergence. Shepherd and Cox (1967) reported that cysts stored moist hatched better than those stored dry, and cysts exposed to 30°C for 6 weeks had their diapause broken. Dunn (1954) reported that there was no emergence from cysts preheated at 29°C, while Ellenby (1955) showed that cysts stored dry at room temperature, 5°C or 23°C had no marked differences in the form of their emergence curve. Bishop (1955) and Oostenbrink (1967) showed that alternation of temperatures resulted in higher emergence compared to constant temperature storage. Because of these contradictory results on the effects of temperatures and moisture on emergence in PCN, Dunn (1962) suggested that previous temperature and moisture states on PCN are very important factors when

66 assessing hatching rates. In this work, these two factors (storage temperatures and moisture) were studied in detail to determine their influence on the hatching patterns of three-year old post harvest cysts ofG.rostochiensis and G.pallida from SCRI (Chapter 2, Section 2.1). Recent studies have examined the influence of soil moisture and storage periods on emerged juveniles ofG.rostochiensis and G.pallida (Robinson et al., 1987a) and influence of temperature over a short period on hatching (Robinsonet a l., 1987b). However little is known about the combined effects of different moisture conditions and storage temperatures over a long storage period on the hatching of G.rostochiensis and G.pallida. It was decided to investigate the pre-conditioning effects of storage temperatures on juvenile emergence from batches of cysts stored under dry, humid and wet conditions for various periods. It was anticipated that these experiments would show a) whether cysts stored at temperatures other than constant 20°C vary in their hatching response; b) whether different humidity regimes during various storage periods could modify hatching behaviour; c) whether combinations of a) and b) above influence the degree of dormancy in three-year old post harvest cysts. Hominick et al. (1985) showed that diapauseG.rostochiensis in occurs only in new cysts and storage of such cysts for one year was shown to overcome the diapause. No work has been done to investigate whether cysts older than one year and not exposed to hatching conditions can be induced to enter a state of diapause. This is the first systematic work to study diapause in cysts G.rostochiensis of and G.pallida older than two years.

4.2 Materials and Methods.

4.2.1 Dry storage

Batches of 50 cysts ofG.rostochiensis and G.pallida were counted from stocks supplied by Scottish Crop Research Institute (SCRI) from their 1985 harvest onto hatching sieves. Cysts were three years old after harvest at the time of these experiments (Chapter 2, Section 2.1). From the time of harvest at SCRI to when cysts arrived at Silwood Park, Ascot (IC), they were stored at room temperature in the dark at SCRI. When the cysts arrived at IC on 29.2.1987, they were stored at constant 20°C in the dark until required for further experimenta­ tion. Each sieve was then placed into a well of a 24-well Linbro culture plate; each plate therefore contained 24 sets of hatching sieves each with 50 cysts of eitherG.rostochiensis or G.pallida (Fig. 4.1). One set of plates of each species

67 Fig. 4.1 A 24-well Linbro culture plate containing hatching sieves. Each hatching sieve contains 50 cysts of eitherG.rostochiensis or G.pallida.

68 was stored dry at 5, 10, 15, 20 and 25°C respectively, with each plate wrapped individually in a black plastic sheet, as described in previous chapters, prior to storage. Cysts were stored at their respective temperatures and, at two month intervals for 12 months, were set to hatch (Fig. 4.2, SI). At the end of each storage period, four replicates of 50 cystsG.rostochiensis of and G.pallida were soaked in 1ml of sterilized tap water (STW) for two weeks (Fig. 4.2, H I); in the third week cysts were hatched in 1ml of potato root diffusate (PRD) (Fig. 4.2, H2) as described in previous chapters. Hatching medium was changed weekly and the emerged juveniles counted. Cysts stored at 5, 10, 15 and 25°C were hatched at 20°C, while cysts stored at 20°C were hatched at 25°C. When emergence was less than 100 juveniles\replicate\week, further hatch­ ing was stopped and cysts were stored dry on their sieves at their respective hatching temperatures (20 and 25°C) for one month (Fig. 4.2, S2). At the end of the one month storage period, cysts were again soaked in 1ml of STW at their respective hatching temperatures for one week (Fig. 4.2, H3) and then hatched in lml of PRD (Fig. 4.2, H4). Emerging juveniles were counted weekly while changing the hatching medium, and hatching continued until emergence was less than 50 juveniles\replicate\week. At this point further hatching was again stopped and cysts were stored dry on their hatching sieves at their initial storage temperatures (5, 10, 15, 20 and 25°C) for six months (Fig. 4.2, S3). At the end of this storage period, cysts were soaked in lml of STW for one week (Fig. 4.2, H5) and then hatched in lml of PRD (Fig. 4.2, H6) with emergence counted weekly while changing the hatching medium. Hatching continued until emergence was less than 10 juvenilesVeplicate\week; cysts were then broken open, and aliquots counted to record viable nematode eggs from which the percentage hatch was determined as described in previous chapters.

4.2.2 Wet storage.

Similar protocols as described above were followed for wet stored cysts, the exception here being that cysts were stored wet in 2ml of STW during first storage (Fig. 4.2, SI) and second storage (Fig. 4.2, S2). During wet storage cysts were closely monitored to ensure that there was enough STW to keep them wet. Whenever the STW level became low, more water was added. Fungal growth appearing during storage was removed with a soft brush whenever necessary.

69 Fig. 4.2 Hatching protocols forG.rostochiensis and G.pallida stored dry, wet and humid for various periods at various temperatures and hatched at 20 and 25°C in PRD.

Key: ------Storage periods...... Soaking in STW. ------Hatching in PRD. 51 First storage period (dry, wet or humid at various temperatures). 52 Second storage period (dry, wet or humid at hatching temperatures). 53 Third storage period (all dry at various temperatures). HI First soaking in STW. H2 First hatching in PRD until < 100 J2 emerged\week. H3 Second soaking in STW. H4 Second hatching in PRD until < 50 J2 emerged\week. H5 Third soaking in STW. H6 Third hatcing in PRD until < 10 J2 emerged\week.

Cysts stored at 5, 10, 15 and 25°C were hatched at 20°C while those stored at 20°C were hatched at 25°C.

70 StorageXHatching Storage temp.C Hatching temp.C

period 10 15 20 25 20 25

2,4,6,8,10,12 Months s: Dry,Wet or Humid

i . JLJL 1 ______•' H I 2 Weeks

Variable i H 2 ..... *____j 1 Month

Dry,Wet or Humid T T ______1 W eek H 3

Variable H 4

■ 1 , t _!

6 Months all dry S3 .1.___ T H 5 1 Week

Variable H 6 f Cysts broken

71 4.2.3 Humid storage.

Similar protocols to those described in dry storage were followed for humid-stored cysts, the exception here being that cysts were stored humid during first storage (Fig. 4.2, SI) and second storage (Fig. 4.2, S2). Humid chambers (Fig. 4.3) were created by lining plastic sandwich boxes with tight-fitting lids with rolls of Kimwipe tissue paper wetted with STW. Culture plates containing batches of cysts as described in Section 4.2.1 were placed into the humid box and closed tightly with the lid. Humid chambers were closely monitored to ensure that the Kimwipe remained wet and occasionally lids were opened to allow circulation of fresh air. Fungal growth was checked and controlled as in wet storage.

4.2.4 Analysis of results.

The statistical package "Statistix" (NH analytical software, St. Paul MN 55117 USA) was used to perform a two way analysis of variance (ANOVA) on angularly transformed percentages, to assess whether the hatching patterns in each of the storage periods (e.g. in first hatching) differed significantly with one another at a given storage temperature (e.g. at 10°C) in a particular storage condition (e.g. humid storage) (see Appendixes E, F, G, H, J and K). The alternative hypotheses in this work are: Is emergence influenced by the "clock mechanism" of the juveniles in the eggs, signalling that diapause period is over? For example, in cysts stored at 10°C and hatched at 20°C in PRD (both hatching temperature and PRD are seasonal markers) after specified storage periods (e.g. four months, since winter conditions in the soil rarely exceed four months). Or, do the juveniles not recognize the passage of a season and as a result emergence is influenced by the presentation of optimum hatching conditions at any given time?

4.3 Results.

4.3.1 Dry storage.

4.3.1.1G.rostochiensis. The results of hatching the dry-stored cysts ofG.rostochiensis are shown in Fig. 4.4. Cysts hatched at all storage temperatures and periods during the first hatching period (Fig. 4.2 HI and H2). Emergence from cysts previously stored at 5,10 and 15°C were not significantly different in percentages of J2 emerging after

72 Fig. 4.3 Humid chamber a) sandwitch box and lid lined with wetted rolls of Kimwipe tissue paper b) a tightly closed humid chamber containing cysts.

73 Cumulative Percentage Hatch Cumulative Percentage Hatch Cumulative Percentage Hatch i. . Cmltv pretg hth jvnls ad ecnae nac (gs of (eggs) unhatch percentage and (juveniles) hatch percentage Cumulative 4.4 Fig. G.rostochiensis Lines above bars indicate mean standard errors (see Appendix L for mean total hatch total mean for L Appendix (see errors standard mean indicate bars above Lines and unhatch eggs). unhatch and 5 n 2° wr hthd t 0C n toe trd t 0C ee ace a 25°C). at hatched were 20°C at stored those and 20°C at hatched were 25°C and 15 0 1, 0ad2°. yt eehthdi R a 2 ad2° css trda , 10, 5, at stored (cysts 25°C and 20 at PRD in hatched were Cysts 25°C. and 20 15, 10, yt soe dy vr pro o 2 4 6 8 1 ad 2 ots t 5, at months 12 and 10 8, 6, 4, 2, of period a over dry stored cysts 5C 74 First hatch in PRD (H2) PRD in hatch First eodhthi R fe 1 month 1 after PRD in hatch Second Third hatch in PRD after 6 months 6 after PRD in hatch Third hatching temperatures (H4) temperatures hatching at respective storage dry of storage temperatures (H6) temperatures storage respective previous at storage dry of Unhatched eggs Unhatched percentage 20C

IOC

the various storage periods, while those previously stored at 20 and 25°C were significantly different (P<0.05). Cysts stored at 5°C had no pattern in emergence. However, there were two peaks in the fourth and tenth month, with about 70% emergence. At 10°C storage, emergence increased with time reaching a peak in the sixth and eighth months with about 80% emergence before declining in the subsequent months. Peak emergence was in the first eight months of storage at 15°C storage and thereafter it declined. The same pattern occurred in cysts stored at 25°C except that peak emergence was about 50% in the first six months of storage followed by a sharp decline in the subsequent months to about 5% in the twelfth month. Emergence in cysts stored at 20°C after the first two months was near 90% then it dropped in the fourth month followed by a steady increase reaching a peak in the tenth month with about 95% emergence before once more declining in the twelfth month. A common feature in all the storage temperatures is the decline in emergence in the twelfth month of storage. Second (Fig. 4.2 H3 and H4) and third hatchings (H5 and H6) had little effect on emergence even though there was a large percentage of unhatched eggs (Fig. 4.4). The percentage of unhatched eggs was not significantly different between the different storage periods in cysts stored at 5, 10 and 15°C, although there was a higher percentage at 5°C. In cysts stored at 20 and 25°C, the percentage unhatched eggs was significantly different at the various times (P<0.05), with no clear pattern at 20°C. However, at 25°C the percentage unhatched eggs remained the same in the first six months. Thereafter it increased with an increase in storage period reaching a peak of about 95% in the twelfth month. The general emergence pattern in this result supports the second hypothesis (Section 4.2.4) which suggests that emergence from cysts was influenced by optimum conditions at any given time. However, emergence from cysts stored at 25°C decreased with storage period. This may suggest some sort of control mechanism in the juveniles which is difficult to explain in relation to seasonal changes.

4.3.1.2G.pallida. The results of hatching of dry-stored cysts ofG.pallida are shown in Fig. 4.5. As in G.rostochiensis, hatching occurred at all storage temperatures and periods during the first hatching period (Fig. 4.2 HI and H2). However, in this case significant differences (P<0.05) in emergence during first hatching was

75 Cumulative Percentage Hatch Cumulative Percentage Hatch Cumulative Percentage Hatch 5C ee ace a 2° ad hs soe a 2° wr hthd t 5C. Lines 25°C). at hatched were 20°C at stored those and 20°C at hatched were 25°C i. . Cmltv pretg hth jvnls ad ecnae nac (gs of (eggs) unhatch percentage and (juveniles) hatch G.pallida percentage Cumulative 4.5 Fig. unhatch eggs). unhatch 0ad2°. yt eehthdi R t2 ad2° (yt soe t5 1, 5 and 15 10, 5, at stored (cysts 25°C and 20 at inPRD hatched were Cysts 25°C. and 20 bv as niae en tnad ros se pedx frma ttl ac and hatch total mean for M Appendix (see errors standard mean indicate bars above css trd r oe apro o , , , , 0 n 1 mnh a 5 1. 15, 10. 5. at months 12 and 10 8, 6, 4, 2, of period a over dry stored cysts 5C 76 110 100 90 40 50 60 70 80 20 30 10 4 8 0 12 10 8 6 4 2 First hatch in PRD (H2) PRD in hatch First Third hatch in PRD after 6 months 6 after PRD in hatch Third storage temperatures (H6) temperatures storage respective previous at storage dry of of dry storage at respective at storage dry of hatching temperatures (H4) temperatures hatching eodhthi R fe 1 month 1 after PRD in hatch Second Unhatched eggs Unhatched Storage Period (months) Period Storage percentage 20C IOC

found at all storage temperatures after the various storage periods. Emergence patterns were similar at 5, 10 and 15°C storage, with high emergence in the first two months followed by a decline in the fourth and sixth months before reaching a peak of about 70% in the eighth month. Thereafter emergence declined in the subsequent months to about 10% in the twelfth month. In cysts stored at 20°C, emergence started at about 40% after two months then declined for several intervals before again rising to 40% in the tenth month and thereafter it declined to less than 5% in the twelfth month. After 25°C storage, emergence decreased with increase in storage period up to the sixth month. In the eighth month a peak of about 55% was reached followed by a sharp decline in the subsequent months to about 1% in the twelfth month. A common feature during the first hatching was that at all storage temperatures, emergence declined in the twelfth month. At 5, 15 and 25°C storage, peak emergence was in the eighth month while at 10 and 20°C storage there were two peaks, with the former in the second and the eighth months and the latter in the second and the tenth months of storage. Emergence patterns were significantly different (P<0.05) between the different storage periods at all storage temperatures during the second hatching period (Fig. 4.2 H3 and H4). These differences occurred in the first six months when peak emergence reached about 15% in the sixth month of storage, except for cysts stored at 20°C where emergence was less than 5% at all storage periods. Emergence ceased almost completely in the last six months of storage at all storage temperatures. A third hatching (H5 and H6) following a third period of dry storage, had no effect on emergence at all storage temperatures and periods except at 15 and 25°C storage. Here, there was 5-10% emergence in the tenth and twelfth months, even though there were high percentages of unhatched eggs at all storage temperatures and periods. Percentage unhatched eggs was significantly different (P<0.05) between the different storage periods at all storage temperatures. At 5, 10, 15 and 25°C storage, the percentage unhatched eggs ranged from 35% to 60% in the first ten months. However, in the twelfth month there was a massive increase to about 90% . In cysts stored at 20°C there was an increase in percentage unhatched eggs with an increase in storage period up to the sixth month, then it declined in the eighth and tenth months before reaching a peak in the twelfth month with about 95% . The common feature is the high percentage of unhatched eggs in the twelfth

77 month at all storage temperatures and similarities in patterns except at 20°C storage, which had the highest percentage of unhatched eggs at all storage periods. The general emergence patternG.pallida, for like that forG.rostochiensis, supports the second hypothesis (Section 4.2.4) suggesting emergence to be influenced by optimum hatching conditions at any given time.

4.3.2 Wet storage.

4.3.2.1G.rostochiensis. Results of hatching cysts ofG.rostochiensis in the wet-storage treatment are shown in Fig. 4.6. Cysts at all storage temperatures and periods hatched during the first hatching period (Fig. 4.2 HI and H2). After storage at 15, 20 and 25°C emergence between the different storage periods was not significantly different, ranging from about 70% to almost 100%. However, emergence after 5 and 10°C storage was significantly different (P<0.05) between the different storage periods. In cysts stored at 5°C, emergence started at about 60% after the first two months of storage, then it declined to about 30% by the fourth month. Thereafter, it increased with storage period reaching peaks of about 90% following the last six months of storage. After 10°C storage, emergence was similar to 5°C storage except that it started slowly after the first four months of storage. Then, in the sixth month, emergence increased as length of storage period increased reaching a peak of about 80% in the last six months. Emergence during the second hatching ( Fig. 4.2 H3 and H4) was significantly different (P<0.05) after the various storage periods at all storage temperatures. After storage at 5 and 10°C, emergence reached peaks of about 45% and 65% respectively in the fourth month of storage before it declined in the subsequent months. After storage at the other temperatures, peak emergence was about 25% in the sixth month at 15°C, 10% in the fourth month at 20°C and about 5% in the eighth month at 25°C. A third hatching (H5 and H6) had no effect on emergence at 20 and 25°C storage after the various storage periods. However, emergence increased in the first two months of storage with 5% at 5°C storage, 25% at 10°C storage and 15% at 15°C storage. The percentage unhatched eggs was not significantly different between the different storage periods at 15, 20 and 25°C storage, with unhatched eggs ranging from less than 5% to a maximum of 15%. However, at 5 and 10°C storage, the percentage unhatched eggs was significantly different (P<0.05) between the

78 Cumulative Percentage Hatch Cumulative Percentage Hatch Cumulative Percentage Hatch i. . Cmltv pretg hth jvnls ad ecnae nae tgs of teggs) unhaten percentage and (juveniles) hatch percentage Cumulative 4.6 Fig. G.rostochiensis Lines above bars indicate mean standard errors (see Appendix N for mean total hatch total mean for N Appendix (see errors standard mean indicate bars above Lines and unhatch eggs). unhatch and 0 1,2 n 5C Csswr ace nPDa 0ad2° css trd t5 10, 5, at stored (cysts 25°C and 20 at PRD in hatched were Cysts 25°C. and 20 15, 10, 5 n 2° wr hthd t 0C n toe trd t 0C ee ace a 25°C). at hatched were 20°C at stored those and 20°C at hatched were 25°C and 15 yt soe wt vr pro o 2 4 6 8 1 ad 2 ots t 5, at months 12 and 10 8, 6, 4, 2, of period a over wet stored cysts C IOC 5C 79

different storage periods. After 5°C storage, percentage unhatched eggs rose from 15% in the first two months of storage to about 20% in the fourth month, while after 10°C storage, the percentage unhatched eggs declined from 40% in the first two months to about 25% in the fourth month. However, at both these storage temperatures, percentage unhatched eggs declined after the fourth month to less than 10% in the remaining storage periods. The general emergence pattern documented by these results supports the second hypothesis (Section 4.2.4) except for those cysts stored at 10°C and hatched at 20°C in PRD during the first hatching period. For these, emergence was low in the first four months of hatching and thereafter increased with storage periods. This suggests that the first four months of low emergence (possibly a response to a winter period simulated by the previous storage at 10°C) may be because the cysts are in diapause. After the passage of the perceived winter period, emergence resumed (i.e. diapause had been overcome) in the subsequent storage periods. This result is very interesting in the sense that it occurred at 10°C in wet stored cysts, which is similar to winter conditions in the field when emergence is low. Therefore, the hatching behaviour of cysts stored wet at 10°C supports the first hypothesis in Section 4.2.4.

4.3.2.2G.pallida . Results of hatching of wet-stored cysts ofG.pallida are shown in Fig. 4.7. Emergence at all storage temperatures during the first hatching (Fig. 4.2 HI and H2) was significantly different (P<0.05) after the various storage periods. The highest emergence occurred in cysts stored at 25°C, where emergence increased with an increase in storage period reaching a peak of about 90% in the twelfth month of storage. After storage at 5, 10 and 15°C, emergence was very low during the first six months of storage. In cysts stored at 5 and 10°C, emergence increased from the eighth month and continued with increase in storage period reaching a peak of about 70-80% in the twelfth month, while at 15°C, storage emergence peaked at about 60% in the eighth and twelfth months with a decline to about 25% in the tenth month. Emergence after 20°C storage was second highest after 25°C. Though it started slowly from 20 to 30% in the first four months, by the sixth month it had reached a peak of about 80% , then declined in the eighth and tenth months before rising to about 70% in the twelfth month.

80 Cumulative Percentage Match Cumulative Percentage Hatch G.pallida unhatch eggs). unhatch i. . Cmltv pretg hth jvnls ad ecnae nac (gs of (eggs) unhatch percentage and (juveniles) hatch percentage Cumulative 4.7 Fig. bv br idct ma sadr err (e Apni P o en oa hth and hatch Lines total mean 25°C). for at P hatched Appendix were (see 20°C errors standard at mean stored indicate bars those andabove 20°C at hatched were 25°C 0ad2°. yt ee ace nPD t 0 n 5C(yt soe t , 0 1 and 15 10, 5, at stored (cysts 25°C and 20 at PRD in hatched were Cysts 25°C. and 20 yt soe wt vr eido , , , , 0 n 1 ots t , 0 15, 10, 5, at months 12 and 10 8, 6, 4, 2, of period a over wet stored cysts 5C 81 IOC

Emergence was significantly different (P<0.05) between the different storage periods at all storage temperatures during the second hatching period (Fig. 4.2 H3 and H4). In cysts stored at 5, 10 and 15°C, emergence started slowly and by the sixth month peaks of 40-50% were reached, followed by declines in the subsequent months to less than 10% in the twelfth month. After 20°C storage, peak emergence was about 40% in the fourth month followed by less than 10% in the other months, while after 25°C storage, peak emergence was about 10% in the sixth, eighth and tenth month of storage. During the third hatching (H5 and H6), there was an exceptionally large increase in emergence after 5, 10 and 15°C storage during the first two months of storage with about 70% at 5°C, 75% at 10°C and 80% at 15°C. In the fourth month, emergence decreased to about 10% and thereafter it ceased completely. After storage at 20°C, emergence was about 10% and was limited to only the first two months, while after 25°C storage, there was no emergence at all. The percentage unhatched eggs was significantly different (P<0.05) at all storage temperatures after the various storage periods. In cysts stored at 5 and 10°C, there were low percentages in the first two months then in the fourth month they peaked to about 70% . Thereafter, the percentage unhatched eggs declined with an increase in storage period. After 15 and 20°C storage, there was no pattern, so that at 15°C there were peaks in the fourth and the tenth months of storage, while at 20°C there were peaks in the second and tenth months of storage. However, after 25°C storage, the percentage unhatched eggs remained at about 40% in the first four months of storage, followed by a decline with an increase in storage period in the subsequent months. The emergence patterns G.pallidafor stored wet, generally supports the first hypothesis (Section 4.2.4) except that for cysts stored at 25°C, which supports the second hypothesis.G.pallida cysts stored wet showed diapause in cysts stored at 5 ,1 0 , 15 and 20°C, during either the first two or four months of storage in the first hatching period. Contrary to the results forG.rostochiensis , where diapause was induced only at 10°C, those forG.pallida suggest induction of diapause in almost all seasons as represented by storage at 5 ,1 0 ,1 5 and 20°C.

4.3.3 Humid storage.

4.3.3.1G.rostochiensis.

82 Results of hatching of humid-stored cysts ofG.rostochiensis are shown in Fig. 4.8. Emergence occurred after all storage temperatures and periods during the first hatching (Fig. 4.2 HI and H2). With cysts stored at 5, 10, 15 and 20°C, emergence was significantly different (P<0.05) between the different storage periods; while those stored at 25°C were not significantly different. The highest emergence occurred after 25°C storage, where it started at about 60% and increased with an increase in storage period to a peak of about 80% in the tenth month before a slight drop to about 70% in the twelfth month. Emergence from cysts stored at 5, 10 and 15°C had similar patterns, starting with high emergence in the first two months and then dropping in the fourth month before rising with an increase in storage period to a peak. This was followed by a decline in emergence in the subsequent storage periods. Cysts stored at 5 and 10°C had their peaks in the eighth month while those stored at 15°C peaked in the tenth month. At these three storage temperatures, peak emergence was about 90%. Emergence after 20°C storage had no pattern, except that there were two peaks in the fourth and eighth months. Emergence was significantly different (P<0.05) between the different storage periods at all storage temperatures during the second hatching period (Fig. 4.2 H3 and H4). In cysts stored at 5, 10 and 15°C, emergence continued during all storage periods except during the tenth months, with peaks of about 30% at 5°C, 50% at 10°C and 60% at 15°C all in the fourth month. After 20°C storage, emergence had no pattern except that it occurred at all storage periods with a peak of about 35% in the twelfth month. However, in cysts stored at 25°C, emergence started with about 10% in the first two months. Thereafter, it declined with an increase in storage period to about 1% in the twelfth month. As with other storage temperatures, there was no emergence in the tenth month of storage. A third hatching (H5 and H6) had no effect on emergence at all storage temperatures and periods, even though there were unhatched eggs at all storage periods. The percentage unhatched eggs was significantly different (P<0.05) between the different storage periods after 5 and 10°C storage. The highest percentage of about 25% was in the sixth and twelfth months at 5°C and about 35% after the twelfth month at 10°C. However, after 15, 20 and 25°C storage, the percentage unhatched eggs was not significantly different at the various storage periods, with unhatched eggs ranging from 20% to 30%.

83 G.rostochiensis i. . Cmltv pretg hth jvnls ad ecnae nac (gs of (eggs) unhatch percentage and (juveniles) hatch percentage Cumulative 4.8 Fig. Lines above bars indicate mean standard errors (see Appendix Q for mean total hatch total mean for Q Appendix (see errors standard mean indicate bars above Lines and unhatch eggs). unhatch and 5 n 2° wr hthd t 0C n toe trd t 0C ee ace a 25°C). at hatched were 20°C at stored those and 20°C at hatched were 25°C and 15 0 1, 0ad2°. yt eehthdi R t2 n 5C(yt soe t , 10, 5, at stored (cysts 25°C and 20 at PRD in hatched were Cysts 25°C. and 20 15, 10, Cumulative Percentage Hatch Cumulative Percentage Hatch Cumulative Percentage Hatch cysts stored humid over a period of 2, 4, 6, 8, 10 and 12 months at 5, at months 12 and 10 8, 6, 4, 2, of period a over humid stored cysts 2 5C SC 84 First hatch in PRD (H2) PRD in hatch First eodhthi R fe 1 month 1 after PRD in hatch Second Third hatch in PRD after 6 months 6 after PRD in hatch Third hatching temperatures (H4) temperatures hatching respective at storage humid of storage temperatures (H6) temperatures storage respective previous at storage dry of Unhatched eggs Unhatched Percentage Storage Period (months) Period Storage IOC

The general emergence pattern from these results supports the second hypothesis (Section 4.2.4) suggesting that emergence is influenced by the presence of optimum conditions at any given time.

4.3.3.2G.pallida. The results of hatching of humid-stored cysts ofG.pallida are shown in Fig. 4.9. Emergence occurred at all storage temperatures, but not after all storage periods, during the first hatching periods (Fig. 4.2 HI and H2). However, emergence was significantly different (P<0.05) between the different storage periods at all storage temperatures. The highest emergence occurred after 25°C storage, where it started slowly after the first two months of storage and then it increased in the subsequent months reaching peaks of about 80% in the sixth, eighth and tenth months before declining to about 50% in the twelfth month. In cysts stored at 5, 10 and 15°C, emergence started very slowly in the first four months of storage. This was followed by an increase in or after the sixth month, with peaks of about 70% at 5°C, 60% at 10°C and 90% at 15°C. Thereafter, emergence declined to about 10% in the twelfth month of storage. However emergence was completely different after 20°C storage, starting at about 30% and then declining with duration of storage period to complete cessation in the twelfth month. Emergence was significantly different (P<0.05) between the different storage periods at all storage temperatures during the second hatching period (Fig. 4.2 H3 and H4). In cysts stored at 5, 10 and 15°C, emergence patterns were similar, with peaks of about 30% at 5 and 15°C and about 20% at 10°C, all in the sixth month of storage. At these three storage temperatures, emergence decreased with an increase in storage period after the sixth month. After 20°C storage, peaks of about 10% were seen in the first six months of storage and further emergence ceased thereafter. After 25°C storage, peak emergence was about 20% in the first two months of storage and thereafter it declined to less than 5% in the twelfth month. The third hatching (H5 and H6) had no significant effect on emergence at all storage temperatures and periods. However, there was about 10% emergence at 5, 10 and 15°C at different storage periods. The percentage unhatched eggs was significantly different (P<0.05) at all storage temperatures after the various storage periods. The highest percentage unhatched eggs was after 20°C storage, where it started with about 60% in the first two months of storage and continued to increase with storage period to about

85 i. . Cmltv pretg hth jvnls ad ecnae nac (gs of (eggs) unhatch percentage and (juveniles) hatch G.pailida percentage Cumulative 4.9 Fig. unhatch eggs). unhatch Lines 25°C). at hatched were 20°C at stored those and 20°C at hatched were 25°C and bv br idct ma sadr err (e Apni R o en oa hth and hatch total mean for R Appendix (see errors standard mean indicate bars above 5 2 ad 5C Csswr ace nPD t 0 n 5C(yt soe a 5 1, 15 10, 5, at stored (cysts 25°C and 20 at PRD in hatched were Cysts 25°C. and 20 15, Cumulative Percentage Hatch Cumulative Percentage Hatch Cumulative Percentage Hatch 100 110 90 40 60 70 50 80 20 30 10 4 8 0 12 10 8 6 4 2 yt soe ui oe apro o , , , , 0 n 1 mnh a 5 10, 5, at months 12 and 10 8, 6, 4, 2, of period a over humid stored cysts 25C 15C 5C 86 First hatch in PRD (H2) PRD in hatch First eodhthi R fe 1 month 1 after PRD in hatch Second Third hatch in PRD after 6 months 6 after inPRD hatch Third hatching temperatures (H4) temperatures hatching respective at storage humid of storage temperatures (H6) temperatures storage respective previous at storage dry of Unhatched eggs Unhatched Percentage IOC

100% in the twelfth month. In cysts stored at 5, 10, 15 and 25°C, the percentage unhatched eggs started at 60-75% in the first two months and then declined with storage period up to the tenth month, before rising in the twelfth month. After 5 and 10°C storage, peaks were in the twelfth month at about 80% ; while after 15 and 25°C storage, peaks were in the second month at about 75% and 60% unhatched eggs respectively. These results are like those forG.pallida cysts stored wet (Section 43.2.2) in that emergence patterns support the first hypothesis (Section 4.2.4). Diapause was shown at all storage temperatures and ranged from two months after 25°C storage, to four months after 5 and 15°C storage, to six months after 10°C storage, to an exceptional 12 months after 20°C storage.

4.4 Discussion

Cysts of G.rostochiensis and G.pallida, as inhabitants of British soils, are continuously exposed to different moisture levels and fluctuating temperatures in agricultural fields. In this work, the choice of storage temperatures (5-25°C) and conditions (dry, humid and wet) was an attempt to provide as wide a range of conditions as the cysts might be exposed to during their development in British agricultural soils. Both storage temperatures and moisture levels were an attempt to simulate conditions which cysts might experience in the field during different seasons. Therefore, the experiments in this Chapter addresses the questions: a) Is emergence influenced by the "clock mechanism" of the juveniles that diapause period is over (as a result of hatching at 20°C in PRD which are both seasonal markers) and it is time for emergence to proceed after specified storage periods and temperatures? b) are the juveniles unable to recognize the passage of a season, so that emergence is influenced by the presentation of optimum hatching conditions at any given time? Hatching of cysts at constant 20 or 25°C after their respective storage periods is not realistic and does not reflect the hatching temperatures in natural agricultural soils, where temperature may vary over daily and longer periods. However, 20°C had been found to be the optimum hatching temperaturesin invitro hatching experiments (Oostenbrink, 1967 and Hominicket a l., 1985). Thus, 20°C was chosen to provide optimum conditions for emergence. Any lack of emergence, was assumed not to be due to absence of optimum conditions. On the other hand, hatching at 25°C, after storage at 20°C, was done to see whether a temperature shock will elicit higher emergence in cysts stored at constant 20°C. The effect of constant storage and

87 hatching at 20°C had already been tested in Chapter 3. Thus, storage temperatures in this work represent temperatures in the soil during one calendar year and storage conditions represent soil conditions during one calendar year. In both cases, conditions were limited by experimental design to suit these experiments. The choice of three-year old post harvest cysts for these experiments was deliberate, because in many agricultural fields some of the nematode inoculum is from the many cysts that are carried over for several seasons. The carry-over capacity of PCN is estimated to be about 35-40% of viable cyst contents from year to year (Jones and Parrott, 1969). In Chapter 3, dormancy similar to a physiological state which could be regarded as diapause was observed in a populationG.rostochiensis of and G.pallida. This observation was based on hatching tests with freshly harvested cysts and one-year old post harvest cysts, both stored dry and hatched at a constant temperature (20°C). In this chapter, three-year old post harvest cysts were stored under different humidity regimes, at storage temperatures ranging from 5°C to 25°C, and for periods ranging from two to twelve months. This was to investigate whether these variables affect the type of dormancy observed in Chapter 3. The results suggest that storage in wet and humid conditions at high temperatures provide optimum conditions for the increased physiological activities which are essential for emergence to occur (Dropkin and Johnson, 1958, Wallace, 1961, Doncaster and Shepherd, 1967, Wallace, 1968b, Bird, 1968 and Bhatt and Rohde, 1970). In contrast, in cysts stored dry, emergence was low and ceased with prolonged storage at higher temperatures. Diapause was observed inG.rostochiensis both and G.pallida cysts stored either wet or humid. However, diapause was not observed in cysts stored dry. During storage of cysts under either dry, humid or wet conditions, storage chambers were regularly opened and the cysts exposed to fresh air. Cysts stored wet were not deprived of oxygen by the use of 2ml of STW per well (well capacity is 2.5ml) and the tissue culture plate cover is specifically designed to allow free diffusion of air. Generally the hatching system used was simple and reliable allowing cysts to be in contact with the hatching medium (PRD) while being well exposed to air, thus simulating soil conditions during hatching. Covering the hatching plates with black plastic sheets limited exposure to light which may have had an effect on the cysts and the PRD, and it also replicated conditions existing in soils.

88 The hatch resulting fromG.rostochiensis stored under either wet, humid or dry conditions showed that emergence was high following wet and humid storage, while the lowest emergence was in cysts stored dry. In wet-stored cysts, prior to the first hatching, cysts were stored in water at different temperatures. After cessation of emergence during first hatching, they were again maintained in water, but at higher storage temperatures before being hatched the second time. The availability of water and cumulative effects of higher temperatures during first hatching and second storage may have increased the physiological activities of the juveniles within the eggs, which resulted in the high emergence observed. Differences in emergence between wet and humid-stored cysts may be due to lack of free water in humid-stored cysts to enhance the physiological activities of the juveniles. Robinson et al. (1987a) reported that hatched juvenilesG.rostochiensis of stored at various moisture levels used their lipid reserves rapidly at soil moistures corresponding to the point of inflection of the soil moisture curve. Though juveniles in this present experiments were within eggs stored wet. Nevertheless, water would have permeated the eggshell and hydrated the juveniles resulting in greater metabolic activities than those cysts stored humid, hence the resultant high emergence in wet-stored cysts. The effects of storage temperatures on wet-stored cysts during second hatching were greatest at 5 and 10°C storage and this resulted in further emergence. In humid-stored cysts, there was further emergence at all storage temperatures though with variable percentages depending on storage periods. This contrasts sharply with the absence of emergence during the third hatching except in wet-stored cysts where there was emergence in the second month of storage at 5, 10 and 15°C. At low temperatures, physiological activities are generally low, including the movements of juveniles which are necessary for hatching (Doncaster and Shepherd, 1967). Probably these factors contributed to the low initial emergence in wet and humid-stored cysts, and were overcome during second hatching due to storage at higher temperatures for up to one month. Banyer and Fisher (1971a) suggested that in H.avenae it is the onset of a suitable temperature, and its duration, that affects hatching behaviour. WithG.rostochiensis stored at low temperatures, a minimum period may be required before the cysts are able to hatch completely, as cysts in wet storage needed four months of storage at 10°C to achieve this. The same reasons may explain behaviour in humid-stored cysts.

89 In contrast to wet and humid-stored cysts, in dry-stored cysts, emergence occurred only during first hatching; second and third hatching had no influence on emergence of juveniles at all storage temperatures and storage periods. The highest emergence occurred from cysts stored at 10 and 15°C, while in cysts stored at 5, 20 and 25°C, emergence was comparatively low after various storage periods. The exception was cysts stored at 25°C, where emergence decreased with an increase in storage period. These results suggest that neither accumulated temperatures during first hatching and second storage nor fluctuating temperatures have provided the trigger effects observed in the hatching of wet and humid-stored cysts. The lack of moisture may have inhibited the physiological activities of the juveniles during the first and second dry storage periods of the cysts. Robinsonet al. (1987a) showed that G.rostochiensis juveniles utilized more of its lipid reserves at different moisture levels, which suggests that level of activity is correlated with degrees of moisture juveniles are exposed to. In general, these results agree with those of Shepherd and Cox (1967), who showed that when cysts are stored dry or moist at various temperatures for various periods, the moist-stored cysts showed higher emergence than dry-stored cysts. However, inM.incognita, de Guiran (1979a) reported that the hatching of eggs was delayed and diminished proportionally to the time of their previous stay in water-saturated soil. These differences in response to levels of moisture by G.rostochiensis and M.incognita may be due to the hard and strong nature of the cyst wall in PCN, which may prevent the disintegration of the cysts due to microbial activities, compared to the much weaker and fragile egg-mass matrixM.incognita. of It also points to the adaptive capacity of the two species.G.rostochiensis , being an inhabitant of wetter regions, is not greatly inhibited by excessive moisture levels in its hatching ability whereasM.incognita, being an inhabitant of drier regions, is deterred by excessive moisture levels in its hatching ability. Oostenbrink (1967) reported that storage of cysts ofG.rostochiensis under wet conditions (ranging from 17 days at 23°C to one month at 5°C) is noxious to the hatchability of the cysts and hatching of such cysts at 23°C after incubation at 5°C results in low emergence. The results presented here do not support these observations. Although Oostenbrink attributed noxiousness of wet storage to microbial activities and fungal disintegration of the cyst wall, in natural soil these conditions are an integral part of the soil system to which the cysts are continuously exposed. Therefore, the observation of Oosten-

90 brink cannot be due to the factors he associated with wet storage. In any case, there was no evidence in his work to show disintegration of the cyst wall as a result of fungal activities. Hatching behaviour inG.pallida was different fromG.rostochiensis but the general trend was similar with high emergence in wet and humid-stored cysts. The highest emergence inG.pallida occurred in cysts stored wet and the lowest in cysts stored dry. Similarities between wet and humid-stored cysts were seen only in cysts stored at 25°C where emergence was highest. At this storage temperature, emergence during first hatch increased with an increase in storage periods, with the exception of a drop in humid-stored cysts in the twelfth month. This high emergence at high storage temperature contradicts reports thatG.pallida favours low temperatures for its development and hatching (Foot, 1978, Mugniery, 1978, Franco, 1979, Magnus- son, 1986 and Robinsonet al., 1987b); most of these conclusions were reached with dry-stored cysts. In cysts stored at 20°C, the highest emergence was from those stored wet, while low emergence was seen in cysts stored humid and dry, with the lowest in humid-stored cysts. This result was surprising when compared with wet-stored cysts, as both wet and humid-stored cysts had adequate moisture and temperature, yet humid-stored cysts failed to hatch except during the first two months of storage. The higher emergence from dry-stored cysts when compared to humid-stored cysts cannot be explained. Emergence from dry-stored cysts ofG.pallida during the first hatching period after storage at 5, 10 and 15°C showed similar trends. The similarities between dry and humid-stored cysts are that both showed moderate emergence in the first six months of storage, then a peak in the eighth month and the lowest emergence in the twelfth month. The failure of these cysts to hatch in greater numbers in this work (when low storage temperatures were followed by hatching at higher temperatures, 20 and 25°C) may support the suggestion thatG.pallida favours lower temperatures for hatching compared toG.rostochiensis (Robinson et al., 1987b; Franco, 1979; Ellis and Hesling, 1975 and McKenna and Winslow, 1972). Unlike in G.rostochiensis, second hatching inG.pallida resulted in emergence at all storage temperatures and storage conditions. Emergence was high from wet and humid-stored cysts, with the highest in wet-stored cysts and the lowest in dry-stored cysts. The highest emergence was from cysts stored at 5, 10 and 15°C. The result of this second hatching was a consequence of keeping the cysts at their respective hatching temperatures (20 and 25°C) for one month, which was higher than their initial storage temperatures. As was suggested in the caseG.rostochiensis, of the

91 probable explanation is that the accumulated heat units during the second storage period enhanced the physiological activities of the juveniles which may be a prerequisite for the observed emergence. Equally, the importance of moisture in emergence was demonstrated by the high emergence from wet-stored cysts. Dunn (1962), working withG.rostochiensis , and Banyer and Fisher (1971a), working with H.avenae, both reported that high preconditioning temperatures caused an increase in juvenile emergence when cysts were kept in moist conditions. Wallace (1966 and 1968a) suggested that hatching corresponded to levels of moisture available to the juveniles within the eggs. Emergence during the third hatching was sporadic and without a pattern, except for cysts stored wet at 5, 10 and 15°C where there was very high emergence in the second month of storage. This emergence may have been due to a temperature shock when cysts were transferred from their storage temperatures to higher hatching temperatures. It could also have been due to some sort of metabolic adaptation which would take time to overcome. Why this did not occur in other cysts at other storage and hatching temperatures cannot be adequately explained. Watson and Lownsbery (1970) observed that there were no cytological differences in temperature-treated and untreated eggs ofMeloidogyne spp.. However, they reported that temperature treatment may produce a chemical rather than a structural effect, which perhaps inactivates a hatching inhibitor and promotes hatching. Since most chemical reactions have an optimum condition within which they operate, the limitation of third hatch to only the first two months of storage at 5, 10 and 15°C may be due to such a constraint. The rate of most biological processes increases with an increase in accumulated heat units, reaching a maximum beyond which the rate of activity tends to decline (Tyler, 1933 and Wallace, 1961). In the results so far discussed, the various peaks probably indicate the threshold of accumulated heat units necessary for emergence, while in periods where emergence was either moderate or low, the threshold may not have been reached (resulting in moderate emergence) or was exceeded (resulting in low emergence). Magnusson (1986) reportedG.rosto­ that chiensis under Nordic conditions exhibits delayed hatch when temperatures become very low and they hatch only when about 150 day degrees are attained. Potato cyst nematodes normally begin to infect their hosts in spring during the growing season, while from late summer to autumn and winter they remain dormant in the field. InG.rostochiensis , emergence occurred to a greater extent at all storage temperatures and storage conditions, with the highest emergence in wet-stored cysts stored at 15°C and above. This suggests the preferenceG.rostochiensis of for wet

92 conditions with high temperatures, which is near conditions available in nature during spring and early summer. On the other hand,G.pallida had a slower initial emergence pattern at all storage temperatures and storage conditions. However, high emergence did occur from wet-stored cysts, with the highest emergence following storage at 25°C. This preference for a high temperature was also reflected in humid-stored cysts. The erratic and incomplete pattern of emergenceG.pallida in at all storage temperatures and storage conditions probably contributes to its persist­ ence in soils as suggested by Parrott and Berry (1976b) and Robinsonet al. (1987a and b). It has also been suggested that in mixed populationsG.pallida tends to replaceG.rostochiensis (Lane and Holliday, 1974, Parrottet al., 1976a and Foot, 1978). In general, work reported here confirms observations by other workers (McKenna and Winslow, 1972, Parrottet al., 1976a, Parrott and Berry, 1976b and Robinson et al., 1987b) thatG.rostochiensis initially hatches more freely than G.pallida. However, in this work, it has been shown that this is limited to only the first hatching period. In the second and third hatchings,G.pallida hatched much better thanG.rostochiensis, even compared to those cysts ofG.rostochiensis where there was a high percentage of unhatched viable eggs. These results suggest a greater initial juvenile emergenceG.rostochiensis in while in G.pallida emergence occurred over a prolonged period as demonstrated in second and third hatchings. This attribute of G.pallida suggests a reason for poor control by oxamyl in the field (Whiteheadet al., 1984), where juveniles, as a result of erratic hatching behaviour, may emerge later and remain infective for longer than the effective life of the nematicide. Generally, emergence from three-yearG.rostochiensis old cysts is strongly influenced by storage conditions and to a lesser extent by storage temperatures. However, inG.pallida, delayed emergence is inherent in its developmental process and neither storage conditions nor storage temperatures have a profound influence in controlling it. Results in this work suggest that if there is any factor influencing the time and the rate of emergence, it is more likely to be storage conditions rather than storage temperatures.

93 CHAPTER 5: BEHAVIOURAL AND PHYSIOLOGICAL RESPONSES OF DORMANT CYSTS.

5.1 Introduction.

Diapause has been reported in some plant parasitic nematodes (Chapter 1) and most of the work done to explain this phenomenon was from hatching tests (Chapters 3 and 4). Although a substantial body of literature is available on hatching processes (Clarke and Perry, 1977; Perry and Clarke, 1981; Perry, 1987 and 1989), there is little information on cysts that failed to conform to the observed hatching process. Janssenet al. (1987) reported that diapause can be overcome by cutting cysts in half. Though this appears to be very artificial and unrelated to practical situations, there was no explanation on the mechanism involved in overcoming diapause when cysts are cut in half. Other workers (Bishop, 1955; Lewis and Mai, 1957 and 1960; Oostenbrink, 1967; Shepherd and Cox, 1967; Juhl, 1968; Banyer and Fisher, 1971a and b; Rode, 1971 and Antoniou, 1983) suggested temperature changes (a specified period of storage at low or high temperature followed by hatching at low or high temperature depending on the initial storage temperature) as a means of overcoming diapause. While this worked on some nematodes, in PCN the results were inconclusive. Hatching stimulants such as PRD and picrolonic acid are known to be effective hatching agents, but alone they do not overcome hatching inhibition in PCN (Hominick et al., 1985). It has been suggested that hatching factor(s) occur within the cysts themselves and various workers (Shepherd and Cox, 1967 and Okada, 1972a,b and 1977) have used various methods to show the presence and activity of such hatching factor. However, the results were either contradictory or inconclusive and in most cases cysts used were of unspecified history. In this present work, the behavioural and physiological responses of cysts of G.rostochiensis and G.pallida were investigated by: a) studying the infectivity of juveniles according to their hatching periods over 12 months, b) studying the effects of various crude homogenates of cysts on hatching of other cysts, c) using electrophoresis to determine differences between hatched juveniles, sterilized tap water (STW) soaked cysts, potato root diffiisate (PRD) stimulated cysts and dormant cysts (Appendix W). The aim in this work is to determine other responses (other than hatching tests) that may indicate the presence of diapause in cystsG.rostochiensis of and G.pallida.

94 5.2 Materials and methods.

5.2.1 Infectivity immediately following periods of emergence from "new" cysts.

Juveniles ofG.rostochiensis and G.pallida were obtained during the peak of emergence from "new" cysts (those hatched in Chapter 3). As a result of this protocol, there are nine infectivity assays forG.rostochiensis and eight for G.pallida. Three-week old tomato plants cv. "Moneymaker" were planted into 9cm plastic pots filled with loamisand mixture (1:1). After three days when plants were established in the potting medium, four replicates of the plants were each inoculated with approximately 1000 one week old juveniles ofG either .rosto­ chiensis or G.pallida. Pots were kept at 20°C in a CT room with a 16 hours light and 8 hours dark regime for two weeks, and plants were watered lightly when necessary with "Phostrogen" solution. At the end of the two week period, plants were gently lifted, washed, stained and the number of stained nematodes recorded as described in Chapter 2, Section 2.6.

5.2.1.1 Analysis of results.

Using the statistical package "Statistix" (SX) percentages were angularly transformed and two way analysis of variance (ANOVA) performed on the transformed data, to assess whether there were significant differences between periods of infectivity in each species (Appendixes S and T).

5.2.2 Assaying crude homogenates of cysts for hatching activities.

From results in Chapter 4, cysts ofG.rostochiensis and G.pallida were selected according to their hatching response to either give slow and incomplete hatching (SIH) or rapid and complete hatching (RCH). Cysts G.rostochiensis of and G.pallida which had been stored wet for four months at 10°C gave slow and incomplete hatching. While cysts ofG.rostochiensis after being stored wet for four months at 15°C gave rapid and complete hatching, andG.pallida, for following dry storage for four months at 5°C. Ten hatching media were prepared as follows: a) sterilized tap water (STW); b) potato root diffusate (PRD); c) crude homogenates of cysts ofG.rostochiensis, which showed slow and

95 incomplete hatching (RD), extracted in STW (STW+RD); d) crude homogenates of cysts ofG.rostochiensis , which showed slow and incomplete hatching (RD), extracted in PRD (PRD+RD); e) crude homogenates of cysts ofG.rostochiensis, which showed rapid and complete hatching (RA), extracted in STW (STW+RA); f) crude homogenates of cysts ofG.rostochiensis, which showed rapid and complete hatching (RA), extracted in PRD (PRD+RA); g) cmde homogenates of cysts ofG.pallida, which showed slow and incomplete hatching (PD), extracted in STW (STW+PD); h) crude homogenates of cysts ofG.pallida, which showed slow and incomplete hatching (PD), extracted in PRD (PRD+PD); j) crude homogenates of cysts ofG.pallida, which showed rapid and complete hatching (PA), extracted in STW (STW+PA); k) crude homogenates of cysts ofG.pallida, which showed rapid and complete hatching (PA), extracted in PRD (PRD+PA). The various crude homogenates mentioned above were prepared by first cleaning debris and any fungal growth from presoaked cysts, and then the cysts were crushed in a known volume of either STW or PRD in a glass homogeniser over ice. During homogenisation, samples were monitored under a low power microscope to ensure all cysts, eggs and any freed juveniles were completely broken. Homogenates were then stored in dark storage bottles at 3-4°C. Each cmde homogenate was made up of two different strengths a) single strength with 35 cystsVnl of either STW or PRD, b) double strength with 70 cystsNml of either STW or PRD. Four replicates of batches of 50 cysts G.rostochiensis of which were previously stored to induce either slow and incomplete hatching (SIH), or rapid and complete hatching (RCH), were hatched in media a, b, c, d, e and f above. The same protocol was used to hatch cysts ofG.pallida showing slow and incomplete hatching (SIH) and rapid and complete hatching (RCH) in media a, b, g, h, j and k above. Cysts were first soaked in STW for two weeks and then hatched in 1ml of the various hatching media at 20°C for five weeks at single strength and for further five weeks at double strength. Juveniles were counted weekly when changing the hatching medium. At the end of the ten week hatching period, cysts were broken open and percentage hatch assessed as described in Chapter 2, Section 2.6.

96 5.2.2.1 Analysis of results. The statistical package "Statistix" (SX) was used to perform the Chi-squared test on numbers of juveniles emerging from the controls (STW or PRD) and from numbers of juveniles emerging in any of the treatments, to assess whether there were significant differences in emergence due to the different hatching media used. Emergence from the controls was used as "expected". (Appendixes U and V).

5.3 Results.

5.3.1 Infectivity immediately following periods of emergence from "new" cysts.

In the nine infectivity assays forG.rostochiensis and eight for G.pallida (Fig. 5.1), results for both species showed significant differences (P<0.05) between the two species. In both species infectivity was less than 10%, with G.rostochiensis having the maximum of about 7% in August and the lowest of about 2% in January.G.pallida had a maximum of 5% in July and September and the lowest of about 3% in November.G.rostochiensis had no particular pattern except that infectivity was less than 4% from October to April, it then increased to 6-7% from May to August. InG.pallida infectivity increased with hatching periods and there were three small but distinct patterns. The first with about 3% from October to December, the second with about 4% from April to May and the third with about 5% from July to September.

5.3.2 Assaying crude homogenate of cysts for hatching activities.

5.3.2.1G.rostochiensis. The results of the assay ofG.rostochiensis are shown in Table 5.1. Hatching in STW for both slowly (SIH) and rapidly hatching cysts (RCH) resulted in no emergence over the ten weeks hatching period. However, hatching in PRD of slowly hatching cysts (SIH) resulted in 3% emergence over ten weeks, with emergence occurring in the first two weeks of the first five weeks hatching period. Rapidly hatching cysts (RCH) had 50% emergence in PRD over the ten weeks hatching period, with 19% in the first five weeks and 31% in the second five weeks.

97 10

' I I T I I I I------1------1------1 I------Oct/87Nov Dec Jan Feb Mar Apr May Jun Jul AugSep/88 Time(months)

Fig. 5.1 Infectivity ofG.rostochiensis and G.pallida on tomato plant cv. "Money­ maker over a hatching cycle of 12 months. Juveniles were from "new" cysts in Chapter 3 and inoculation was done with about 1000 one week old juvenilesNplant with four replicates. Lines above bars indicate mean standard error.

98 Table 5.1 Cumulative percentage hatch of cystsG.rostochiensis of showing slow and incomplete hatching (SIH) and rapid and complete hatching (RCH) in: a) sterilized tap water (STW); b) potato root diffusate (PRD); c) homogenates of cysts ofG.rostochiensis, which showed slow and incomplete hatching (RD), extracted in STW (STW+RD); d) homogenates of cysts ofG.rostochiensis, which showed slow and incomplete hatching (RD), extracted in PRD (PRD+RD); e) homogenates • of cysts ofG.rostochiensis, which showed rapid and complete hatching (RA), extracted in STW (STW+RA); f) homogenates of cysts ofG.rostochiensis, which showed rapid and complete hatching (RA), extracted in PRD (PRD+RA). Hatching medium at single strength had 35 cystsNml of STW or PRD and double strength had 70 cystsVnl of STW or PRD.

Hatching me­ Cumulative percentage hatch. Total cumulative dium. percentage hatch.

At single strength for At double strength for At 10 weeks. 5 weeks. a further 5 weeks.

SIH RCH SIH RCH SIH RCH

(a) STW + 0 0 0 0 0 0

(b) PRD+ 3 19 0 31 3 50

(c)STW +RD 3 4 0 I 3 5

(d)PRD+RD 6 21 0 21 6 42

(e)STW +RA 10 10 0 2 10* 12*

(f)PRD+RA 17 33 0 7 17* 40

+ Hatching in STW and PRD were all done at the same strength during the 10 week hatching period, the table shows cumulative percentage hatch in each of the 5 weeks and the total 10 week hatching periods. * Results are significantly different at P <0.05 between controls (a or b) and treatments (c, d, e or f).

99 Emergence was different at single and double strengths of the various crude homogenate hatching media, except in the hatching medium PRD+RD, where there was the same percentage emergence in rapidly hatching cysts (RCH). In slowly hatching cysts (SIH), all emergence occurred in the single strength hatching media; while in rapidly hatching cysts (RCH) emergence occurred in both strengths with the highest in the single strength hatching media. In slowly hatching cysts (SIH), all preparations of crude homogenates (STW based or PRD based) at single strength increased emergence, compared to emergence in any of the two controls (STW or PRD). The highest emergence was in the hatching medium PRD+RA. The trend in rapidly hatching cysts (RCH) at single strength also shows that STW prepared homogenates had higher percen­ tage emergence compared to the STW control. The same trend follows for PRD prepared homogenates, With the highest emergence in the hatching medium PRD+RA. The general trend shows that homogenates of rapidly hatching cysts in either STW or PRD (STW+RA and PRD+RA) caused higher emergence than homogenates from slowly hatching cysts in either STW or PRD (STW+RD and PRD+RD). Also homogenates in PRD showed higher emergence than homoge­ nates in STW. At double strength, emergence declined in rapidly hatching cysts (RCH) hatched in PRD prepared hatching media (PRD+RD or PRD+RA) compared to the PRD control. In STW prepared hatching media (STW+RD or STW+RA), there was a small increase in emergence compared to the STW control. However, in slowly hatching cysts (SIH) emergence completely ceased at double strength in all hatching media. In slowly hatching cysts (SIH), the cumulative percentage hatch over the ten week hatching period in STW+RA was significantly different (P<0.05) with emergence in the control STW (Table 5.1). Also emergence in PRD+RA was significantly different (P<0.05) with emergence in the PRD control (Table 5.1). In rapidly hatching cysts (RCH), emergence was not significantly different in all PRD prepared hatching media compared to the PRD control. However, emerg­ ence was significantly different (P<0.05) in STW+RA compared to the STW control (Table 5.1). 5.3.2.2 G.pallida.

100 The results of the assay of G.pallida are shown in Table 5.2. As in G.rostochiensis (Section 5.3.2.1), hatching in STW for both slowly (SIH) and rapidly hatching cysts (RCH) resulted in no emergence over the ten weeks hatching period. Hatching in PRD for both slowly (SIH) and rapidly hatching cysts (RCH) also resulted in no emergence in the first five weeks of hatching at single strength. However, emergence occurred in the second five weeks of hatching in PRD at double strength, with 2% in the slowly hatching cysts and 8% in the rapidly hatching cysts. Emergence was different at single and double strengths in all hatching media containing crude homogenates, except in the hatching medium PRD+PD. Where percentage emergence was the same at single and double strengths for both slowly (SIH) and rapidly (RCH) hatching cysts. However, in both slowly (SIH) and rapidly hatching cysts (RCH), emergence in all crude homogenates hatching media (STW or PRD based) at single strength was higher than in any of the two controls (STW or PRD). In slowly hatching cysts (SIH), the highest emergence at single strength was 3% in the hatching media STW+PD and PRD+PA, and the lowest was in STW+PA with 1%. While in rapidly hatching cysts (RCH), the highest emergence at single strength was 5% in the hatching media STW+PD, and the lowest in STW+PA with 1%. At double strength, in both slowly (SIH) and rapidly hatching cysts (RCH), emergence was low in all PRD prepared crude homogenates hatching media (PRD+PD and PRD+PA) compared to the PRD control. Except in PRD+PD, where there was the same percentage emergence with the control in slowly hatching cysts (SIH). However, in STW control and STW prepared hatching media (STW+PD and STW+PA), emergence completely ceased at double strength preparations. The cumulative percentage emergence over the ten week hatching period in both slowly (SIH) and rapidly hatching cysts (RCH) was significantly different (P<0.05) in STW+PD compared to emergence in the STW control (Table 5.1). There was no significant difference between emergence in PRD prepared crude homogenates (PRD+PD and PRD+PA) and the PRD control in both slowly (SIH) and rapidly hatching cysts (RCH). 5.4 Discussion. Infectivity assessment to determine differences due to period of emergence in Chapter 3 showed no differences between juveniles from dormant and nondormant cysts in both G.rostochiensis and G.pallida (dormancy in these cysts was earlier seen

101 Table 5.2 Cumulative percentage hatch of cysts of G.pallida showing slow and incomplete hatching (SIH) and rapid and complete hatching (RCH) in: a) sterilized tap water; b) potato root diffusate (PRD); c) homogenates of cysts of G.pallida, which showed slow and incomplete hatching (PD), extracted in STW (STW+PD); d) homogenates of cysts of G.pallida, which showed slow and incomplete hatching (PD), extracted in PRD (PRD+PD); e) homogenates of cysts of G.pallida, which showed rapid and complete hatching (PA), extracted in STW (STW+PA); f) homogenates of cysts of G.pallida, which showed rapid and complete hatching (PA), extracted in PRD (PRD+PA). Hatching medium at single strength had 35 cysts\ml of STW or PRD and double strength had 70 cystsNml of STW or PRD.

Hatching me­ Cumulative percentage hatch. Total cumulative dium. percentage hatch. At single strength for At double strength for At 10 weeks. 5 weeks. a further 5 weeks. SIH RCH SIH RCH SIH RCH (a) STW+ 0 0 0 0 0 0 (b) PRD+ 0 0 2 8 2 8 (c)STW+PD 3 5 0 0 3* 5* (d)PRD+PD 2 4 2 4 4 8 - (e)STW+PA 1 1 0 0 1 1 (f)PRD+PA 3 4 1 2 4 6 + Hatching in STW and PRD were all done at the same strength during the 10 weeks hatching period, the table shows cumulative percentage hatch in each of the 5 weeks and the total 10 week hatching period. * Results are significantly different at P<0.05 between controls (a or b) and treatments (c, d, e or f).

102 in the hatching tests in Chapter 3). Infectivity of G.rostochiensis in January was about 2% and in July about 6%, even though these cysts were showing some degree of dormancy during these two periods (Chapter 3). Also inG.pallida infectivity of juveniles from dormant cysts in February was about 4% compared to ones from nondormant cysts in May which also had about 4% infectivity. These results suggest infectivity to be influenced by season of hatching rather than the physiological state of the juveniles in a particular season. In both species, infectivity was low in Autumn and Winter, but in Spring there was an increase in infectivity which continued to increase through Summer. These results are very revealing towards the near perfect synchronization of both species to the growing phase of the potato plants as obtained in the field. This observation is strongly reinforced considering that throughout the period of this experiment (12 months), tomato plants, prior to inoculation and after, were maintained at constant 20°C with a 16 hours light and 8 hours dark regime. Nevertheless, the degree of infection followed a seasonal pattern. Results in this present work, do not agree with Robinson et al. (1985) who reported that the time of hatch in G.rostochiensis had no significant effect on the level of invasion. However, it agrees with Storey (1984) who reported that there was no difference in infectivity between cysts which were dormant for one, four or seven years. In plant parasitic nematodes, survival and infectivity of second stage juveniles depend on food reserves accumulated in the egg. Juveniles do not feed from the time they hatch until they have penetrated a host plant (Van Gundy et al., 1967). Storey (1983 and 1984) and Robinson et al. (1985, 1987a and b) showed that one of the most important components of the food reserves is the neutral lipid whose levels significantly influence host invasion. However, Storey (1984) reported that a decrease in neutral lipid reserves of G.pallida over a dormant period of seven years had no significant effect on invasion. Juveniles used for inoculating tomato plants in this present work were collected after one week of hatching in PRD at 20°C, and it has been shown that storage for such a period had no effect on infectivity (Reversat, 1980; Storey, 1984 and Robinson et al., 1987a). Therefore, the seasonal preference showed by both G.rostochiensis and G.pallida may not be due to adverse experi­ mental conditions, but rather a reflection of their adaptation to increased infectivity during the period of maximum host availability. In the assay to determine the influence of crude homogenate of cysts on hatching, it is difficult to establish that nematodes were actually responding to only the material in the cyst homogenates. Some microorganisms such as bacteria and their metabolic by-products may also have either enhanced or inhibited hatching.

103 However, even with this shortcoming, hatching in both G.rostochiensis and G. pallida was higher at single strength of various crude homogenates. While at double strength, it was negligible. This suggests that there was an optimum concentration at which some of the hatching promoting substances present may be active. Possibly maximum activity may be present at a concentration lower than that used here as a single strength, considering that PRD and picrolonic acid are also optimally active at very low concentrations. The negligible emergence at double strength may also suggest that the hatching promoting substances may need to be used soon after preparation. In this work, they were stored at 3-4°C for five weeks before used in hatching, but greatest emergence with the single strength occurred in the first two weeks of hatching and thereafter emergence was low. Both G.rostochiensis and G.pallida failed to hatch in STW alone, but cyst homogenates prepared in STW did induce emergence in both species. This strongly suggests the presence of substances that induce hatching. When similar cysts were homogenised in PRD they again showed a response but with higher emergence compared to STW homogenates. Okada (1977) reported that inhibiting and stimulat­ ing substances could be competing for the same site and, as a result, they could be antagonistic in vivo. Probably the same phenomena operates between such sub­ stances and PRD during in vitro hatching. In G.rostochiensis, the highest emergence in slowly hatching cysts was in the hatching medium PRD+RA and in rapidly hatching cysts in PRD+RD and PRD+RA. In G.pallida, the highest emergence in slowly hatching cysts was in PRD+PA and in rapidly hatching cysts in PRD+PD. This suggests that substances capable of promoting hatching are not exclusive to a particular physiological state of the cysts, but may be present in all cysts. The most curious result was the absence of inhibitory substances in any of the homogenates. These results differed from those of Ellenby (1946b), Kaul (1962), Ellenby and Smith (1967a) and Gooris and D’Herde (1972) who suggested that failure of eggs of plant parasitic nematodes to hatch was not due to diapause but to inhibitory substances in the eggs or cysts. However, the results in this work support the reports by Okada (1972b and 1974) that crushed whole cysts of H. glycines, G.rostochiensis and H.oryzae or ultrasonicly disrupted suspension of eggs and juveniles of H.glycines contained substances which induced emergence of larvae. How such substances may act is difficult to explain. Watson and Lownsbery (1970) thought that hatching is achieved by a chemical activation rather than a structural effect. Perry (1989), further suggested that such chemicals are likely to be either hormone based or enzymes or the combination of the two. The substances

104 promoting hatching in the various crude homogenates used in this present work have some shortcomings. Their ability to increase the rate of emergence in all cysts are generally low, compared to the rate of emergence in actively hatching cysts (Chapters 3 and 4). However, the interesting point is the ability of the substances in the STW prepared crude homogenates to increase emergence, compared to STW control. ^ While it is clear that dormancy which exhibit! the characteristics of diapause was observed in hatching tests (Chapters 3 and 4), there were no behavioural or physiological responses (with the exception of hatching) in this work to indicate the presence of this phenomenon in eitherG.rostochiensis or G.pallida.

105 CHAPTER 6: GENERAL DISCUSSION. The work reported in this thesis sheds some new light on several aspects of the hatching processes of the potato cyst nematodes, Globodera rostochiensis and G.pallida, and has implications for our understanding of the synchronization of seasonal activity in host and parasites of which at least two aspects of dormancy (diapause and quiescence) are possible components.G.rostochiensis hatches more freely and faster over a short period, while G.pallida hatches slowly over a prolonged period. As regards the storage temperatures after which hatching occurs most rapidly in PRD at either 20 or 25°C, there is no clear cut preference in the range of 5-25°C storage. Storage temperature preference is strongly influenced by storage conditions and duration of storage. In both species, age of cysts has a profound influence on dormancy. In G.rostochiensis, newly harvested cysts showed dormancy which tended to be overcome in older cysts. However in G.pallida dormancy is exhibited irrespective of the age of the cysts. Various workers (Chapter 1, Section 1.5) have suggested the presence of different degrees of dormancy in G.rostochien­ sis, which Hominick et al. (1985) confirmed to be diapause, but this is the first recorded and most persuasive evidence of a dormancy showing the characteristics of diapause in G.pallida. Dormancy in both species cannot be regarded as a simple case of diapause (Hominick et al., 1985), neither can it be described as facultative diapause (Shepherd and Cox, 1967). The phenomenon of diapause in G.rostochiensis and G.pallida is more complicated than in several other nematode species, and there is little evidence to indicate the process(es) involved in diapause initiation anchor termination. All that can be confirmed is evidence of its presence indicated by results from hatching tests. A comprehensive range of possible combinations of optimum conditions known in an agricultural ecosystem have been presented to the cysts of these two species, and regardless of this, some cysts remained unhatched. Hatching processes in plant parasitic nematodes are well discussed and documented (Shepherd and Clarke, 1971, Clarke and Perry, 1977, Perry and Clarke, 1981, Perry, 1987 and 1989), but none of them adequately explained what is involved in dormancy. The hard fact of this kind of work is that evidence is difficult to collect for fitting into an account of growth and development of these species. The induction of diapause inG.rostochiensis and G.pallida in this present work can best be described, within the limits of available information, as "obligate" diapause (Evans and Perry, 1976 and Evans, 1987). While the termination of diapause in G.rostochiensis can also be regarded in the general

106 framework of "obligate" diapause. In G.pallida, termination of diapause cannot be described or classified with certainty, for the simple reason that there is no evidence in this work or elsewhere to show that diapause has been overcome. The recent review by Evans (1987), suggesting the incorporation of ontogenetic development in describing and classifying diapause in plant parasitic nematodes opens new frontiers for further theoretical explanations. In other plant parasitic nematodes such as M.naasi (Ogunfowora, 1973 and Antoniou, 1983) and H.avenae (Banyer and Fisher, 1971a and b and Rivoal, 1978, 1979, 1983 and 1986) where diapause is more responsive to or influenced by temperatures, it is possible to describe and define processes of both induction and termination of diapause with some certainty. It may appear from this discussion so far that difficulties associated with termination of diapause are intrinsic only to cyst forming nematodes. This is not the case, as de Guiran (1979a,b and 1980) and de Guiran and Villemin (1980a and b) showed the presence of what was apparently a diapause in the non cyst forming nematode M.incognita. However, they could not find how such diapause can be terminated, even though they demonstrated that those eggs in diapause are viable. In explaining the phenomenon of diapause inG.rostochiensis , Hominick et al., (1985) suggested that diapause may be a consequence of signals given to the developing females and eggs by the host plant during its growing season. This signal was suggested to be light (photoperiod) acting on the potato plant which affects developing females and which subsequently influences the hatching mechanism of her juvenile progeny (Hominick, 1986). The amount, intensity anchor quality of light acting on the host plants are suggested to affect the hatching of plant parasitic nematodes (Franco and Evans, 1979, Bird et al., 1980 and Hominick, 1986). Assuming this hypothesis to be correct, the hatching of PCN in vitro, in PRD, may suggest that we are missing a vital contribution from the potato host plant, which is contributed by the continuous activity of photoperiodism on the growing potato plants. This may further suggest that there are gene(s) in the potato plant which are influenced by the activity of light. Since potato plants are known to be responsive to photoperiod which influences the production of exudates (Franco and Evans, 1979 and Evans, 1982). These exudate (PRD), in turn may send putative signals to switch on some gene(s) in the juveniles of the PCN, which controls both the process(es) of induction anchor termination of diapause. PRD production is a dynamic process in the physiology of the potato plant, and the use of PRD at a specified stage of the development of the potato plant may result in under utilization of the full potentials

107 of the PRD. In most hatching tests, most workers are only interested in high emergence, and as a consequence, the details of PRD activity are neglected. This therefore suggests that when cysts are removed from the field the necessary signal(s) for the induction of diapause has already been received and begun to be acted upon in the juveniles of the PCN, due to the long seasonal association between the potato plant and the nematode. The reason why diapause is not terminatedin vitro maybe because the necessary signal(s) required over the season, by the continuous interaction of both host plant and nematode to switch off the gene(s) controlling diapause in the juveniles of the PCN is missing. These sorts of gene-mediated responses are not uncommon in nature. Each plant-nematode association has a unique gene combination such that any physiologi­ cal input by the nematode results in a positive response by the host plant and vice versa. Evidence that gene(s) from host plants and the nematodes are involved in host responses can be postulated from the existence of resistant cultivars and biotypes which can "break the resistance" of the cultivars (Jones and Pawelska, 1963, Jones and Parrott, 1965, Jones, 1974, Jones etal., 1981, Fassuliotis, 1987, Lewis, 1987 and Triantaphyllou, 1987). In animal nematodes, Sommerville and Rogers (1987) also thought gene-medi­ ated responses in development to be the norms, and reported that development is delayed and resumed on a signal from the host. The intensity of the response is thought to be related to the intensity of the signals, and it starts and stops roughly in step with the signal. They further suggested that the signal activates a "gene set" which is largely responsible for physiological changes, morphological responses are minimal, temporary and reversible. Like other workers, they also failed to identify exactly the nature of the signal(s) from any particular host, and how it governs changes in the developmental process(es) in the nematodes. However, they pointed out that the effect of the signal from the host is rapid, irreversible and specific. The process of hatching and the phenomenon of diapause are two distinct but closely related processes. All second stage juveniles of G.rostochiensis and G.pallida found in soil after harvest of a crop may be regarded as being in "obligate" quiescence, and with the arrival of favourable conditions, such quiescence is overcome and hatching processes (as detailed in hatching mechanism) start. In diapause, there is no evidence of set(s) of condition(s) which initiates or terminates diapause at least in G.pallida. The only evidence that can be clearly deduced from hatching tests is the presence of diapause itself. In this state, all hatching processes are inoperative. Each of these two processes are significant and complimentary in the

108 survival of the plant parasitic nematode. Obligate quiescence followed by active hatching in the presence of favourable conditions is essential for the reproductive success of the nematodes. On the other hand, obligate quiescence following diapause is essential for the nematode to overcome unfavourable internal and external conditions which may last for hours or years. This generally improves the nematode’s adaptive qualities and enhances its chances of existence which may be inherited by its offspring. The structural simplicity of plant parasitic nematodes and the diversity of their environment and hosts imposes great pressure for their existence, but since natural selection has favoured those species with genes which delay their hatch, the ability to enter diapause has become an important factor in evolution of potato cyst nematodes. In this work and in Hominick et al. (1985), it has been possible to demonstrate the presence and termination of diapause inG.rostochiensis, which appears to be dependent on the age of the cysts. This observation fits well with the description and definition of Evans and Perry (1976) as obligate diapause. However, in G.pallida, the same conditions as presented to G.rostochiensis failed to overcome diapause in some cysts. A similar observation was made by de Guiran (1979b) in Meloidogyne incognita, where he failed to achieve complete hatching under optimum hatching conditions. This suggests that the classification of types of dormancy in nematodes as advanced by Evans and Perry (1976) and Evans (1987), are not comprehensive enough to cover all nematodes (especially plant parasitic nematodes). It might be more helpful at this stage to limit the classification of dormancy to quiescence and diapause only, until more studies are accomplished on other nematodes. Quiescence would therefore be any arrest in development caused by unfavourable environmental conditions which can be resumed by return of favourable environmental conditions (readily reversible). Diapause would be any arrest in development which could not be readily overcome by return of favourable environmental conditions. The practical implication of this present work is the confirmation thatG.pallida has a more persistent diapause thanG.rostochiensis, which means it hatches slowly and over a longer period. The reason for dominance of G.rostochiensis could be faster hatching in a seasonal environment. The evidence to support this is seen in the field where mixed populations of PCN exist, the reduction of G.rostochiensis populations by the use of resistant varieties (e.g. Maris Piper) has led to an increased abundance of G.pallida. In nature, this mode of hatching in G.pallida may be the product of evolution in a less seasonal environment (e.g. around the equatorial area of Andes). This is known from Evans et al. (1975) and Evans and Stone (1977) to be

109 the major habitat for G.pallida types of populations. These qualities will suggest added advantages for G.pallida with the implication of replacing the much studied G.rostochiensis, thereby causing a more serious problem in the field in the future. The almost complete emergence of juveniles of G.rostochiensis under wet storage, may suggest the possibility of incorporating "wet fallowing" (Goodell and Ferris, 1989) where possible in control strategies. However, differences in hatching patterns observed over a hatching period of one year in G.rostochiensis and G.pallida suggest practical difficulties in conventional cultural cultivations and control measures where both species exist. Though the advantages of using resistant varieties are enormous, strong selection pressure may shorten the useful field life of resistant genes in resistant varieties in today’s sophisticated modem agriculture. Usage of chemicals is less attractive, due to the current politics and realities of environmental concerns. Therefore, the most feasible effective method of control remains through an integrated approach, and recognizing the potentials of diapause in nematodes will make such models more robust. To advance our understanding of diapause in plant parasitic nematodes, hatching mechanisms and plant-nematode interactions will require further studies. At the moment there is inadequate information and circumstantial evidence supporting various theories. The challenge is to produce hard evidence to back up the proposed theories. The way forward will require intensive studies at cellular, molecular and genetic levels. Fortunately the techniques and the personnel for such works are abundant, what is required is the will to finance it.

110 REFERENCES.

1. Antoniou, M. (1983). Diapause in the nematode Meloidogyne naasi (Franklin, 1965). Ph.D. thesis, University of London, pp. 202. 2. Atkinson, H. J. and Ballantyne, A. J. (1977a). Changes in the oxygen consumption of cysts of Globodera rostochiensis associated with the hatching of juveniles. Ann. appl. Biol. 87: 159-166. 3. Atkinson, H. J. and Ballantyne, A. J. (1977b). Changes in the adenine nucleotide content of cysts of Globodera rostochiensis associated with the hatching of juveniles. Ann. appl. Biol. 87: 167-174. 4. Atkinson, H. J. and Ballantyne, A. J. (1979). Evidence for the involvement of calcium in the hatching of Globodera rostochiensis. Ann. appl. Biol. 93: 191-198. 5. Atkinson, H. J. and Taylor, J. D. (1980). Evidence for a calcium-binding site on the eggshell of Globodera rostochiensis with a role in hatching. Ann. appl. Biol. 96: 307-315. 6. Atkinson, H. J. and Taylor, J. D. (1983). A calcium binding sialoglycoprotein associated with an apparent egg-shell membrane of Globodera rostochiensis. Ann. appl. Biol. 102: 345-354. 7. Atkinson, H. J.; Taylor, J. D. and Ballantyne, A. J. (1980). The uptake of calcium prior to the hatching of the second stage juvenile of Globodera rostochiensis. Ann. appl. Biol. 94: 103-109. 8. Atkinson, H. J.; Taylor, J. D. and Fowler, M. (1987). Changes in the second stage juveniles of Globodera rostochiensis prior to hatching in response to potato root diffiisate. Ann. appl. Biol. 110: 105-114. 9. Ayoub, S. M. (1980). Plant nematology, an agricultural training aid. NemaAid Publication, Cal. USA, pp. 195. 10. Bakker, J. and Bouwman-Smits, L. (1988a). Genetic variation in polypeptide maps of two Globodera rostochiensis pathotypes. Phytopathology 78: 894-900. 11. Bakker, J. and Bouwman-Smits, L. (1988b). Contrasting rates of protein and morphological evolution in cyst nematode species. Phytopathology 78: 900-904.

Ill 12. Bakker, J.; Schots, A.; Bouwman-Smits, L. and Gommers, F. J. (1988). Species-specific and thermostable proteins from second stage larvae ofGlobod- era rostochiensis and G.pallida. Phytopathology 78: 300-305. 13. Banyer, R. J. and Fisher, J. M. (1971a). Effect of temperature on hatching of eggs of Heterodera avenae. Nematologica 17: 519-534. 14. Banyer, R. J. and Fisher, J. M. (1971b). Seasonal variation in hatching of eggs of Heterodera avenae. Nematologica 17: 225-236. 15. Banyer, R. J. and Fisher, J. M. (1980). Mechanisms controlling hatching of Heterodera avenae. Nematologica 26: 390-395. 16. Beane, J. and Perry, R. N. (1983). Hatching of the cyst nematode Heterodera goettingiana in response to root diffusate from bean {Vida faba). Nematologica 29: 360-362. 17. Behrens, E. (1975). Globodera Skarbilovic, 1959, eine selbstandige gattung in der unterfamilie Heteroderinae Skarbilovic, 1947 (Nematoda: Heteroderidae). Aktuellen Probl. Phytopath. 29.5.1975, Rostock, 12-26. 18. Bhatt, B. D. and Rohde, R. A. (1970). The influence of environmental factors on the respiration of plant parasitic nematodes. J. Nematology 2: 277-285. 19. Bird, A. F. (1968). Changes associated with parasitism in nematodes. ID. Ultrastructure of the egg-shell, larval cuticle, and contents of the subventral esophageal glands in Meloidogyne javanica, with some observation on hatch­ ing. J. Parasitology 54: 475-489. 20. Bird, A. F. (1974). Suppression of embryogenesis and hatching in Meloidogyne javanica by thermal stress. J. Nematology 6: 95-99. 21. Bird, A. F. (1976). The development and organization of skeletal structures in nematodes, pp. 107-137. In: The organization of nematodes, (Croll, N. A. ed.). Academic Press, London, pp. 439. 22. Bird, A. F. and McClure, M. A. (1976). The Tylenchid (Nematoda) egg-shell structure, composition and permeability. Parasitology 72: 19-28. 23. Bird, A. F.; Sauer, M. R.; Chapman, R. M. and Loveys, B. R. (1980). The influence of light quality on the growth and fecundity of Meloidogyne infesting tomato. Nematologia Mediterranea 8: 59-66.

112 24. Bishop, D. (1953). Hatching the contents of cysts of Heterodera rostochiensis with alternating temperature conditions. Nature, London 172: 1108. 25. Bishop, D. (1955). The emergence of larvae of Heterodera rostochiensis under conditions of constant alternating temperature. Ann. appl. Biol. 43: 525-532. 26. Bridge, J.; Page, S. L. J. and Jordan, S. M. (1982). An improved method for staining nematodes in roots, pp. 171. In: Rothamsted Experimental Station, report for 1981, part 1. Lawes agricultural trust, Harpenden, Herts. UK, pp. 322. 27. Calam, C. T.; Raistrick, H. and Todd, A. R. (1949). The potato eelworm hatching factor. 1. The preparation of concentrates of the hatching factor and a method of bioassay. Biochem. J. 45: 513-519. 28. Clarke, A. J. (1968). The chemical composition of the cyst wall of the potato cyst nematode, Heterodera rostochiensis. Biochem. J. 108: 221-224. 29. Clarke, A. J.; Cox, P. M. and Shepherd, A. M. (1967). The chemical composition of the egg-shells of the potato cyst nematode Heterodera rostochiensis Woll. Biochem. J. 104: 1056-1060. 30. Clarke, A. J. and Hennessy, J. (1976). The distribution of carbohydrates in cysts of Heterodera rostochiensis. Nematologica 22: 190-195. 31. Clarke, A. J. and Hennessy, J. (1978). Osmotic stress and the hatching of Globodera rostochiensis. Nematologica 24: 384-392. 32. Clarke, A. J. and Hennessy, J. (1981). Calcium inhibitors and the hatching of Globodera rostochiensis. Nematologica 27: 190-198. 33. Clarke, A. J. and Hennessy, J. (1983). The role of calcium in the hatching of Globodera rostochiensis. Revue. Nematol. 6: 247-255. 34. Clarke, A. J. and Hennessy, J. (1987). Hatching agents as stimulants of movement of Globodera rostochiensis juveniles. Revue. Nematol. 10: 471-476. 35. Clarke, A. J. and Perry, R. N. (1977). Hatching of cyst nematodes. Nematologica 23: 350-368. 36. Clarke, A. J. and Perry, R. N. (1980). Egg-shell permeability and hatching of Ascaris suum. Parasitology 80: 447-456.

113 37. Clarke, A. J. and Perry, R. N. (1985a). The role of eggshell calcium in the hatching of Heterodera schachtii. Nematologica 31: 151-158. 38. Clarke, A. J. and Perry, R. N. (1985b). Eggshell calcium and the hatching of Globodera rostochiensis. Int. J. parasitology 15: 511-516. 39. Cooper, A. F. and Van Gundy, S. D. (1971). Senescence, quiescence and cryptobiosis, pp. 297-318. In: Plant parasitic nematodes, Vol. ID. Cytogenetics, host parasite interactions and physiology, (Zuckerman, B. M.; Mai, W. F. and Rohde, R. A..eds.). Academic Press, New York, pp. 347. 40. Cooper, A. F.; Van Gundy, S. D. and Stolzy, L. H. (1970). Nematode reproduction in environments of fluctuating aeration. J. Nematology 2: 182-188. 41. Cunningham, P. C. (1960). An investigation of winter dormancy in Heterodera rostochiensis. Scientific proceedings of the royal Dublin society, series B 1, 1-4. 42. Demeure, Y. and Freckman, D. W. (1981). Recent advances in the study of anhydrobiotic nematodes, pp. 205-226. In: Plant parasitic nematodes, Vol. Ill (Zuckerman, B. M. and Rohde, R. A. eds.). Academic Press, New York and London, pp. 508. 43. Demeure, Y.; Freckman, D. W. and Van Gundy, S. D. (1979). In vitro response of four species of nematodes to desiccation and discussion of this and related phenomena. Revue. Nematol. 2: 203-210. 44. Doncaster, C. C. and Shepherd, A. M. (1967). The behaviour of second-stage Heterodera rostochiensis larvae leading to their emergence from the egg. Nematologica 13: 476-478. 45. Dropkin, V. H. (1976). Nematode parasites of plants, their ecology and the process of infection, pp. 222-246. In: Encyclopedia of plant physiology (Pisson, A. and Zimmermann, M. H. eds.). New series Vol. 4. Springer-Verlag, Berlin. 46. Dropkin, V. H.; Martin, G. C. and Johnson, R. W. (1958). Effect of osmotic concentration on hatching of some plant parasitic nematodes. Nematologica 3: 115-126. 47. Dunn, E. (1954). Factors influencing the emergence of Heterodera rostochiensis larvae. Nature, London 173: 780.

114 48. Dunn, E. (1962). Preconditioning of the cyst contents of potato root eelworm, Heterodera rostochiensis Woll., by temperature and its effect on the subsequent emergence of the larvae in water and root diffusate. Nematologica 7: 177-185. 49. Eissa, M. F. A.; Beinhart, G. and Al-Uyayed, S. (1979). Cooperation with the international Meloidogyne project in the Kingdom of Saudi Arabia, pp. 105-112. In: Proceedings of the second research planning conference on root knot nematodes, Meloidogyne spp., Nov. 26-30, 1979. Athens, Greece. North Carolina State University Press. 50. Ellenby, C. (1946a). Nature of the cyst wall of the potato-root eelworm Heterodera rostochiensis Woll., and its permeability to water. Nature, London 157: 302-303. 51. Ellenby, C. (1946b). Ecology of the eelworm cyst. Nature, London 157: 451-452. 52. Ellenby, C. (1946c). The influence of potato variety on the cyst of the potato-root eelworm Heterodera rostochiensis Woll. Ann. appl. Biol. 33: 433-446. 53. Ellenby, C. (1955). The seasonal response of the potato-root eelworm Heterod­ era rostochiensis Woll.: Emergence of larvae throughout the year from cysts exposed to different temperature cycles. Ann. appl. Biol. 43: 1-11. 54. Ellenby, C. (1974). Water uptake and hatching in the potato cyst nematode, Heterodera rostochiensis, and the root knot nematode, Meloidogyne incognita. J. Exp. Biology 61: 773-779. 55. Ellenby, C. and Perry, R. N. (1976). The influence of the hatching factor on the water uptake of the second stage larvae of the potato cyst nematode Heterodera rostochiensis. J. Exp. Biology 64: 141-147. 56. Ellenby, C. and Smith, L. (1967a). Emergence of larvae from new cysts of the potato-root eelwormHeterodera rostochiensis. Nematologica 13: 273-278. 57. Ellenby, C. and Smith, L. (1967b). Soil leachings and the hatching of larvae of the potato-root eelworm, Heterodera rostochiensis Woll. Ann. appl. Biol. 59: 283-288. 58. Ellis, P. R. and Hesling, J. J. (1975). Larval emergence from cysts of twelve isolates of Heterodera rostochiensis and H.pallida reared on resistant and susceptible tomatoes. Plant Pathology 24: 77-83.

115 59. El-Shatoury, H. (1978). Genetic control of dormancy in the potato cyst nematode. Experientia 34: 448-449. 60. Evans, A. A. F. (1974). The effect of climate on plant and soil nematode, pp. 53-77. In: Effects of meteorological factors upon parasites, (Taylor, A. E. R. and Muller, R. eds.). Blackwells, Oxford. 61. Evans, A. A. F. (1987). Diapause in nematodes as a survival strategy, pp. 180-187. In: Vistas on nematology: A commemoration of the twenty-fifth anniversary of the Society of Nematologists, (Veech, J. A. and Dickson, D. W. eds.). Society of Nematologists Inc. Maryland, USApp. 509. 62. Evans, A. A. F. and Perry, R. N. (1976). Survival strategies in nematodes, pp. 383-424. In: The organization of nematodes (Croll, N. A. ed.). Academic Press, New York pp. 439. 63. Evans, K. (1969). Changes in a Heterodera rostochiensis population through the growing season. Ann. appl. Biol. 64: 31-41. 64. Evans, K. (1982). Effects of host variety, photoperiod and chemical treatments on hatching of Globodera rostochiensis. J. Nematology 14: 203-207. 65. Evans, K.; Franco, J. and de Scurrah, M. M. (1975). Distribution of species of potato cyst nematodes in South America. Nematologica 21: 365-369. 66. Evans, K. and Stone, A. R. (1977). A review of the distribution and the biology of the potato cyst nematodes Globodera rostochiensis and G.pallida. PANS 23: 178-189. 67. Farrer, L. A. and Phillips, M. S. (1983). In vitro hatching of Globodera pallida in response to Solanum vernei and S. tuberosum #X S. vernei hybrids. Revue. Nematol. 6: 165-169. 68. Fassuliotis, G. (1987). Genetic basis of plant resistance to nematodes, pp. 364-371. In: Vistas on nematology: A commemoration of the twenty-fifth anniversary of the Society of Nematologists (Veech, J. A. and Dickson, D. W. eds.). Society of Nematologists, Inc. Maryland USA pp. 509. 69. Fenwick, D. W. (1949). Investigations on the emergence of larvae from cysts of the potato-root eelworm Heterodera rostochiensis. 1. Technique and variabil­ ity. J.Helminthology 23: 157-170.

116 70. Fenwick, D. W. (1956). The breakdown of potato root diffusate in soil. Nematologica 1: 290-302. 71. Fenwick, D. W. and Reid, E. (1953a). Population studies on the potato-root eelworm (Heterodera rostochiensis Woll.). J.Helminthology 27: 119-128. 72. Fenwick, D. W. and Reid, E. (1953b). Seasonal fluctuations in the degree of hatching from cysts of the potato-root eelworm. Nature, London 171: 47. 73. Foot, M. A. (1978). Temperature response of three potato cyst nematode populations from New Zealand. Nematologica 24: 412-417. 74. Forrest, J. M. S. and Phillips, M. S. (1984). The effect of Solanum tuberosum X S. vernei hybrids on hatching of the potato cyst nematode Globodera pallida. Ann. appl. Biol. 104: 521-526. 75. Fox, F. C. and Atkinson, H. J. (1984). Isoelectric focussing of general protein and specific enzymes from pathotypes ofGlobodera rostochiensis and G.palli- da. Parasitology 88: 131-139. 76. Franco, J. (1979). Effect of temperature on hatching and multiplication of potato cyst nematode. Nematologica 25: 237-244. 77. Franco, J. and Evans, K. (1979). Effects of daylength on the multiplication of potato cyst nematode {Globodera spp.) populations. Nematologica 25: 184-190. 78. Franklin, M. T. (1937). The effect on the cyst contents of Heterodera schachtii on the cultivation of maize on potato sick land. J. Helminthology 15: 61-68. 79. Franklin, M. T. (1951). The cyst-forming species of Heterodera. Commonwealth Agric. Bureaux, Famham Royal pp. 147. 80. Fushtey, S. G. and Johnson, P. W. (1966). The biology of the oat cyst nematode, Heterodera avenae in Canada. 1. The effect of temperature on the hatching of cysts and emergence of larvae. Nematologica 12: 313-320. 81. Gemmell, A. R. (1940). Studies in the biology and control of Heterodera schachtii (Schmidt). 1. A comparison of the H.schachtii cyst population of two potato growing districts. Bull. W. Scot, agric. coll. No. 139, pp. 1-15. 82. Gemmell, A. R. (1943). The resistance of potato varieties to Heterodera schachtii Schmidt, the potato-root eelworm. Ann. appl. Biol. 30:61-10.

117 83. Goodell, P. B. and Ferris, H. (1989). Influence of environmental factors on the hatch and survival of Meloidogyne incognita. J. Nematology 21: 328-334. 84. Gooris, J. and D’Herde, C. J. (1972). Le cycle de developpement de Meloidogyne naasi Franklin sur cereales de printeemps et d’hiver et sur betteraves. Revue de 1’Agriculture 4: 651-657. 85. Green, C. D.; Greet, D. N. and Jones, F. G. W. (1970). The influence of multiple mating on the reproduction and genetics of Heterodera rostochiensis and H.schachtii. Nematologica 16: 309-326. 86. de Guiran, G. (1979a). Survie des nematodes dans les sols secs et satures d’eau: Oeufs et larves de Meloidogyne incognita. Revue. Nematol. 2: 65-77. 87. de Guiran, G. (1979b). A necessary diapause in root-knot nematodes. Observa­ tions on its distribution and inheritance in Meloidogyne incognita. Revue. Nematol. 2: 223-231. 88. de Guiran, G. (1980). Facteurs induisant chezMeloidogyne incognita un blocage du developpement des oeufs considere comme une diapause. Revue. Nematol. 3: 61-69. 89. de Guiran, G. and Demeure, Y. (1978). Influence du potentiel hydrique des sols sur les masses d’oeufs de Meloidogyne incognita (Nematoda: Meloidogynidae). Revue. Nematol. 1: 119-134. 90. de Guiran, G. and Germani, G. (1980). Fluctuations des populations de Meloidogyne incognita dan un sol cultive en climat tropical humide. Revue. Nematol. 3: 51-60. 91. de Guiran, G. and Villemin, M. (1980a). Specificite de la diapause embryonnaire des oeufs de Meloidogyne (Nematoda). Revue. Nematol. 3: 115-121. 92. de Guiran, G. and Villemin, M. (1980b). Etude de la diapause embryonnaire des oeufs de Meloidogyne incognita en culture monoxenique: Technique d’inocula- tion, influence de l’age de la mere et de la nutrition en potassium. Revue. Nematol. 3: 161-166. 93. Hesling, J. J. (1959). The emergence of larvae of Heterodera rostochiensis Woll. from single cyst. Nematologica 4: 126-131.

118 94. Hominick, W. M. (1979). Selection for hatching at low temperatures in Globodera rostochiensis by continues cultivation of early potatoes. Nemato- logica 25: 322-332. 95. Hominick, W. M. (1986). Photoperiod and diapause in the potato cyst nematode, Globodera rostochiensis. Nematologica 32: 408-418. 96. Hominick, W. M.; Forrest, J. M. and Evans, A. A. F. (1985). Diapause in Globodera rostochiensis and variability in hatching trials. Nematologica 31: 159-170. 97. Huijsman, C. A. (1961). The influence of resistant potato varieties on the soil population ofHeterodera rostochiensis Woll. Nematologica 6: 177-180. 98. Hussey, G. and Stacey, N. J. (1981). In vitro propagation of potato{Solanum tuberosum L.). Annals of Botany 48: 787-796. 99. Ishibashi, N.; Kondo, E.; Muraoka, M. and Yokoo, T. (1973). Ecological significance of dormancy in plant parasitic nematodes: 1. Ecological difference between eggs in gelatinous matrix and cyst of Heterodera glycines Ichinohe. (Tylenchida: Heteroderidae). Appl. Entom. Zool. 8: 53-63. 100. Janssen, R.; Bakker, J. and Gommers, F. J. (1987). Circumventing the diapause of potato cyst nematodes. Neth. J. PI. Pathology 93: 107-113. 101. Johnson, P. W. and Potter, J. W. (1980). Winter survival of root-knot nematodes (Meloidogyne incognita and M.hapla) under selected host crops in Southern Ontario. Can. J. Plant Sci. 60: 203-207. 102. Jones, F. G. W. (1974). Host parasite relationships of potato cyst nematodes: A speculation arising from the gene-for-gene hypothesis. Nematologica 20: 437-443. 103. Jones, F. G. W. (1975a). The soil as an environment for plant parasitic nematodes. Ann. appl. Biol. 79: 113-139. 104. Jones, F. G. W. (1975b). Accumulated temperature and rainfall as measures of nematode development and activity. Nematologica 21: 62-70. 105. Jones, F. G. W. (1978). The soil-plant environment, pp. 46-62. In: Plant nematology (Southey, J. F. ed.). HMSO, London pp. 440.

119 106. Jones, F. G. W. and Parrott, D. M. (1965). The genetic relationships of pathotypes of Heterodera rostochiensis Woll. which reproduce on hybrid potatoes with genes for resistance. Ann. appl. Biol. 56: 27-63. 107. Jones, F. G. W. and Parrott, D. M. (1969). Population fluctuations of Heterodera rostochiensis Woll. when susceptible potato varieties are grown continuously. Ann. appl. Biol. 63: 175-181. 108. Jones, F. G. W.; Parrott, D. M. and Perry, J. N. (1981). The gene-for-gene relationship and its significance for potato cyst nematodes and their solana- ceous hosts, pp. 23-36. In: Plant parasitic nematodes, Vol. ID. (Zuckerman, B. M. and Rohde, R. A. eds.). Academic Press Inc. New York pp. 508. 109. Jones, F. G. W. and Pawelska, K. (1963). The behaviour of populations of potato-root eelworm (Heterodera rostochiensis Woll.) towards some resistant tuberous and other solanum species. Ann. appl. Biol. 51: 277-294. 110. Juhl, M. (1968). Temperaturvariationers indflydelse pa havrenematodens {Heterodera avenae) Klackning. Tidsskr. PlAvl. 72: 42-63 (English summary, pp. 61). 111. Kaul, R. (1962). Untersuchungen Uber einen aus Zysten des Karfoffelnema- toden {Hetetodera rostochiensis Woll.) isolierten phenolischen Komplex. Nematologica 8: 288-292. 112. Keilin, D. (1959). The problem of anabiosis or latent life: History and current concept. Proc. Roy. Soc. B, 150: 149-191. 113. Kerry, B. R. and Jenkinson, S. C. (1976). Observations on emergence, survival and root invasion of second stage larvae of the cereal cyst nematode, Heterodera avenae. Nematologica 22: 467-474. 114. Khan, W. M. and Siddiqui, Z. A. (1986). Some comments on root-knot nematodes infecting vegetables in Libya. Int. Nematol. Network Newsl. 3: 18-20. 115. Krusberg, L. R. and Sardanelli, S. (1989). Survival of Heterodera zeae in soil in the field and in the laboratory. J. Nematology 21: 347-355. 116. Lane* A. and Holliday, J. M. (1974). Potato cyst-eelworm pathotype competi­ tion. In: Report on field experiments, Agricultural Development and Advisory Service, East Midland Region: 115-116.

120 117. Laudien, H. (1973). Resting stages in development and their induction or termination by the effect of temperature, pp. 390-399. In: Temperature and life (Precht, H.; Christopherson, J.; Hensel, H. and Larcher, W. eds.). Springer verlag, Berlin and New York. 118. Laughlin, C. W.; Lugthart, J. A. and Vargas, J. M. (1974). Observations of the emergence of Heterodera iri from the egg. J. Nematology 6: 100-101. 119. Lees, A. D. (1955). The physiology of diapause in arthropods. Cambridge University Press, pp. 151. 120. Lewis, F. J. Von M. and Mai, W. F. (1957). Survival of encysted eggs and larvae of the golden nematode to alternating temperatures. Phytopathology 47: 527. 121. Lewis, F. J. Von M. and Mai, W. F. (1960). Survival of encysted and free larvae of the golden nematode in relation to temperature and relative humidity. Proc. Helminth. Soc. Wash. 27: 80-85. 122. Lewis, S. A. (1987). Nematode-plant compatibility, pp. 246-252. In: Vistas on nematology. A commemoration of the twenty-fifth anniversary of the Society of Nematologists (Veech, J. A. and Dickson, D. W. eds.). Society of Nemato- logists Inc. Maryland, USA. pp. 509. 123. Lownsberry, B. F. (1951). Larval emigration from cysts of the golden nematode of potatoes, Heterodera rostochiensis Woll. Phytopathology 41: 889-896. 124. Magnusson, M. L. (1986). Development of Globodera rostochiensis under simulated Nordic conditions. Nematologica 32: 438-446. 125. Mansingh, A. (1971). Physiological classification of dormancies in insects. The Canadian Entomologist 103: 983-1009. 126. Martin, G. C. (1967). Longevity of Meloidogyne javanica under conditions of bare fallow in Rhodesia. Rhodesia Agric. Journal 64: 112-114. 127. McKenna, L. A. and Winslow, R. D. (1972). Differences in hatch and development rates of potato cyst nematode pathotypes. Ann. appl. Biol. 71: 274-278. 128. Mugniery, D. (1978). Vitesse de developpement en fonction de la temperature, de Globodera rostochiensis et G.pallida (Nematoda: Heteroderidae). Revue. Nematol. 1: 3-12.

121 129. Ogunfowora, A. O. (1973). Studies on cereal root-knot nematode, Meloidogyne naasi. Ph.D. Thesis, University of London, pp. 241. 130. Ogunfowora, A. O. and Evans, A. A. F. (1977). Factors affecting the hatch of eggs of Meloidogyne naasi, an example of diapause in a second-stage larva. Nematologica 23: 137-146. 131. Okada, T. (1972a). Hatching inhibitory factor in the cyst contents of the soybean cyst nematode Heterodera glycines. Appl. Ent. Zool. 7: 99-102. 132. Okada, T. (1972b). Hatching stimulant in the egg of the soybean cyst nematode Heterodera glycines. Appl. Ent. Zool. 7: 234-237. 133. Okada, T. (1974). Effects of hatching stimulants obtained from the cyst contents of Heterodera spp. (Tylenchida: Heteroderidae) on the hatching of other species. Appl. Ent. Zool. 9: 49-51. 134. Okada, T. (1977). Studies on the hatching factors of the soybean cyst nematode Heterodera glycines Ichinohe. Jap. J. Nematol. 6(special issue): pp. 50. 135. Onions, T. G. (1955). The distribution of hatching within the cyst of the potato-root eelworm, Heterodera rostochiensis. Quart. J. Micr. Sci. 96: 495-513. 136. Oostenbrink, M. (1967). Studies on the emergence of encysted Heterodera larvae. Mededelingen Rijksfaculteit Landbouw-Wetenschappen Gent. 32: 503-539. 137. den Ouden, H. (1960). Periodicity in spontaneous hatching of Heterodera rostochiensis in the soil. Nematologica suppl. 11: 101-105. 138. Oydvin, J. and Hammeraas, B (1973). Lethal low temperatures for the cyst content of the potato-root eelworm. Nematologica 19: 568-569. 139. Parrott, D. M. (1972). Mating of Heterodera rostochiensis pathotype. Ann. appl. Biol. 71: 271-273. 140. Parrott, D. M. and Berry, M. M. (1976). Hatching of encysted eggs. Rep. Rothamsted Exp. Stn. for 1975, 198. 141. Parrott, D. M.; Berry, M. M. and Farrell, K. M. (1976). Competition between Globodera rostochiensis Rol and G.pallida Pa3. Rep. Rothamsted Exp. Stn. for 1975, 197.

122 142. Perry, R. N. (1977). Water content of the second-stage larva of Heterodera schachtii during the hatching process. Nematologica 23: 431-437. 143. Perry, R. N. (1986). Physiology of hatching, pp. 119-131. In: Cyst nematodes (Lamberti, F. and Taylor, C. E. eds.). Plenum Press, New York and London, pp. 467. 144. Perry, R. N. (1987). Host-induced hatching of phytoparasitic nematode eggs, pp. 159-164. In: Vistas on nematology. A commemoration of the twenty-fifth anniversary of the Society of Nematologists (Veech, J. A. and Dickson, D. W. eds.). Society of Nematologists Inc. Maryland, USA. pp. 509. 145. Perry, R. N. (1989). Dormancy and hatching of nematode eggs. Parasitology Today 5: 377-383. 146. Perry, R. N. and Beane, J. (1983). The hatching of Heterodera goettingiana in response to brief exposure to pea root diffiisate. Nematologica 29: 34-38. 147. Perry, R. N. and Clarke, A. J. (1981). Hatching mechanisms of nematodes. Parasitology 83: 435-449. 148. Perry, R. N.; Clarke, A. J. and Beane, J. (1980). Hatching of Heterodera goettingiana in .vitro Nematologica 26: 493-495. 149. Perry, R. N.; Clarke, A. J. and Hennessy, J. (1980). The influence of osmotic pressure on the hatching of Heterodera schachtii. Revue. Nematol. 3: 3-9. 150. Perry, R. N. and Feil, J. J. (1986). Observations on a novel hatching bioassay for Globodera rostochiensis using fluorescence microscopy. Revue. Nematol. 9: 280-282. 151. Perry, R. N.; Hodges, J. A. and Beane, J. (1981). Hatching of Globodera rostochiensis in response to potato root diffusate persisting in soil. Nematologi­ ca 27: 349-352. 152. Perry, R. N.; Wharton, D. A. and Clarke, A. J. (1982). The structure of the egg-shell of Globodera rostochiensis (Nematoda: Tylenchida). Int. J. Parasi­ tology 12: 481-485. 153. Perry, R. N.; Zunke, U. and Wyss, U. (1989). Observations on the response of the dorsal and subventral oesophageal glands of Globodera rostochiensis to hatching stimulation. Revue. Nematol. 12: 91-96.

123 154. Peters, B. G. (1952). Toxicity tests with vinegar eelworm. 1. Counting and culturing. J. Helminthology 26: 97-110. 155. Reversat, G. (1980). Effect of in vitro storage time on the physiology of second-stage juvenile of Heterodera oryzea. Revue. Nematol. 3: 233-241. 156. Rivoal, R. (1978). Biologie d’ Heterodera avenae Woll. en France. 1. Differ­ ences dans les cycles d’eclosion et de developpement des deux races Frl et Fr4. Revue. Nematol. 1: 171-179. 157. Rivoal, R. (1979). Biologie d 'Heterodera avenae Woll. en France. 2. Etude des differences dans les conditions thermiques d’eclosion des races, Frl et Fr4. Revue. Nematol. 2: 233-248. 158. Rivoal, R. (1983). Biologie d'Heterodera avenae Woll. en France. 3. Evolution des diapauses des races Frl et Fr4 au cours de plusiers annees consecutives; influence de la temperature. Revue. Nematol. 6: 157-164. 159. Rivoal, R. (1986). Biology of Heterodera avenae Woll. in France. IV. Comparative study of the hatching cycles of two ecotypes after their transfer to different climatic conditions. Nematologica 9: 405-410. 160. Robinson, M. P.; Atkinson, H. J. and Perry, R. N. (1985). The effect of delayed emergence on infectivity of juveniles of the potato cyst nematode Globodera rostochiensis. Nematologica 31: 171-178. 161. Robinson, M. P.; Atkinson, H. J. and Perry, R. N. (1987a). The influence of soil moisture and storage time on the mobility, infectivity and lipid utilization of second-stage juveniles of the potato cyst nematodes Globodera rostochiensis and G.pallida. Revue. Nematol. 10: 343-348. 162. Robinson, M. P.; Atkinson, H. J. and Perry, R. N. (1987b). The influence of temperature on the hatching, activity and lipid utilization of second-stage juveniles of the potato cyst nematodes Globodera rostochiensis and G.pallida. Revue. Nematol. 10: 349-354. 163. Rode, H. (1971). Der Einflus versschiedener Temperaturen und Wechselreize auf das Schlupfen von larven des Kartoffelnematoden. Pedobiologia 11: 143-158.

124 164. Sasser, J. N. and Freckman, D. W. (1987). A world perspective on nematology: The role of the society, pp. 7-14. In: Vistas on nematology. A commemoration of the twenty-fifth anniversary of the Society of Nematologists (Veech, J. A. and Dickson, D. W. eds.). Society of Nematologists Inc. Maryland, USA. pp. 509. 165. Satchell, J. E. (1967). Lumbricidae, pp. 259-322. In: Soil biology (Burges, A. and Raw, F. eds.). Academic Press, London and New York, pp. 532. 166. Shepherd, A. M. (1962). The emergence of larvae from cysts in the genus Heterodera. A review of factors affecting hatching. C. A. B. England, pp. 90. 167. Shepherd, A. M. (1963). The emergence of larvae of Heterodera goettingiana Liebs. in vitro and a comparison between field populations ofH.goettingiana and H.rostochiensis Woll. Nematologica 9: 143-151. 168. Shepherd, A. M. and Clarke, A. J. (1971). Molting and hatching stimuli, pp. 267-287. In: Plant parasitic nematodes, Volume II (Zuckerman, B. M.; Mai, W. F. and Rohde, R. A. eds.). Academic Press, London and New York, pp. 347. 169. Shepherd, A. M. and Cox, P. M. (1967). Observations on periodicity of hatching of eggs of the potato cyst nematode, Heterodera rostochiensis Woll. Ann. appl. Biol. 60: 143-150. 170. Sommerville, R. I. and Rogers, W. P. (1987). The nature and action of host signals. Adv. in Parasitology 26: 239-293. 171. Stone, A. R. (1973). Heterodera pallida n. sp. (Nematoda: Heteroderidae), a second species of potato cyst nematode. Nematologica 18: 591-606. 172. Stone, A. R. (1979). Co-evolution of nematodes and plants. Symb. Bot. Uppsala XXII: 4: 46-61. 173. Stelter, H. and Sager, I. (1987). Der larvenschlupf von Globodera rostochiensis Woll., 1923, pathotype 1, in Bodenablaufwasser. Archiv. Fur Phytopathologie und Pflanzenschutz 23: 69-76. 174. Storey, R. M. J. (1983). The initial neutral lipid reserves of juveniles of Globodera spp. Nematologica 29: 144-150. 175. Storey, R. M. J. (1984). The relationship between neutral lipid reserves and infectivity for hatched and dormant juveniles of Globodera spp. Ann. appl. Biol. 104: 511-520.

125 176. Tefft, P. M. and Bone, L. W. (1984). Zinc-mediated hatching of eggs of soybean cyst nematode, Heterodera glycines. J. Chem. Ecol. 10: 361-372. 177. Tefft, P. M. and Bone, L. W. (1985a). Leucine aminopeptidase in eggs of the soybean cyst nematode Heterodera glycines. J. Nematology 17: 270-274. 178. Tefft, P. M. and Bone, L. W. (1985b). Plant-induced hatching of the soybean cyst nematode Heterodera glycines. J. Nematology 17: 275-279. 179. Thomason, I. J. and Caswell, E. P. (1987). Principles of nematode control, pp. 87-130. In: Principles and practice of nematode control in crops (Brown, R. H. and Kerry, B. R. eds.). Academic Press, Sydney and London, pp. 447. 180. Thomason, I. J. and Fife, D. (1962). The effect of temperature on development and survival of Heterodera schachtii. Nematologica 7: 139-145. 181. Triantaphyllou, A. C. (1987). Genetics of nematode parasitism on plants, pp. 354-363. In: Vistas on nematology. A commemoration of the twenty-fifth anniversary of the Society of Nematologists (Veech, J. A. and Dickson, D. W. eds.). Society of Nematologists Inc. Maryland, USA. pp. 509. 182. Triffit, M. J. (1930). On the bionomics of Heterodera schachtii on potatoes, with special reference to the influence of Mustard on the escape of the larvae from the cysts. J. Helminthology 8: 19-48. 183. Trudgill, D. L. (1967). The effect of environment on sex determination in Heterodera rostochiensis. Nematologica 13: 263-272. 184. Trudgill, D. L. and Carpenter, J. M. (1971). Disk electrophoresis of proteins of Heterodera spp. and pathotypesoiH.rostochiensis. Ann. appl. Biol. 69: 35-41. 185. Turner, S. J. (1985). Potato cyst nematodes (eelworms) in Northern Ireland. Biology (1). Agric. in N. Ireland Vol. 60 Part 7: 230-232. 186. Tyler, J. (1933). Development of root-knot nematode as affected by tempera­ ture. Hilgardia 1: 391-415. 187. Van Gundy, S. D. (1965). Factors in survival of nematodes. Ann. Rev. phytopathology 3: 43-68. 188. Van Gundy, S. D.; Bird, A. F. and Wallace, H. R. (1967). Aging and starvation in larvae of Meloidogyne javanica and Tylenchulus semipenetrans. Phytopatho­ logy 57: 559-571.

126 189. Wallace, H. R. (1955). Factors influencing the emergence of larvae from cysts of the beet eelworm, Heterodera schachtii Schmidt. J. Helminthology 29: 3-16. 190. Wallace, H. R. (1959). Further observations on some factors influencing the emergence of larvae from cysts of the beet eelworm Heterodera schachtii Schmidt. Nematologica 4: 245-252. 191. Wallace, H. R. (1961). The orientation of Ditylenchus dipsaci to physical stimuli. Nematologica 6: 222-236. 192. Wallace, H. R. (1966). The influence of moisture stress on the development, hatch and survival of eggs of Meloidogyne javanica. Nematologica 12: 57-69. 193. Wallace, H. R. (1968a). The influence of soil moisture on survival and hatch of Meloidogyne javanica. Nematologica 14: 231-242. 194. Wallace, H. R. (1968b). Undulatory locomotion of the plant-parasitic nematode, Meloidogyne incognita. Parasitology 58: 377-391. 195. Watson, T. R. and Lownsberry, B. F. (1970). Factors influencing the hatching of Meloidogyne naasi, and a comparison with M.hapla. Phytopathology 60: 457-460. 196. Wharton, D. A. (1980). Nematode egg-shells. Parasitology 81: 447-463. 197. Wharton, D. A. (1986). A functional biology of nematodes. Croom Helm, London and Sydney, pp. 192. 198. Whitehead, A. G.; Tite, D. J.; Fraser, J. E. and Nichols, A. J. F. (1984). Differential control of potato cyst-nematodes, Globodera rostochiensis and G.pallida by oxamyl and the yields of resistance and susceptible potatoes in treated and untreated soils. Ann. appl. Biol. 105: 231-244. 199. Williams, T. D. (1978). Cyst nematodes: Biology of Heterodera and Globod­ era, pp. 156-171. In: Plant nematology (Southey, J. F. ed.), third edition. HMSO, London, pp. 440. 200. Williams, T. D. and Beane, J. (1979). Temperature and root exudates on the cereal cyst nematode Heterodera avenae. Nematologica 25: 397-405. 201. Winslow, R. D. (1956). Seasonal variations in the hatching responses of the potato-root eelworm, Heterodera rostochiensis Woll., and related species. J. Helminthology 30: 157-164.

127 202. Wright, D. J. and Newall, D. R. (1976). Nitrogen excretion, osmotic and ionic regulation in nematodes, pp. 163-210. In: The organization of nematodes (Croll, N. A. ed.). Academic Press, New York pp. 439.

128 Appendix A. An example of Chi-squared test (using two-by-two table) on the hatching patterns of "new" and "old" cysts of G.rostochiensis. A November hatch. B August hatch. C January hatch. D July hatch.

A PEARSON’S CHI SQUARE 0 .5 4 YULE’S Q - 0 . 2 9 P (PEARSON'S) 0 .4 6 1 9 SE (Q) 1 .4 4 2 E - 0 1 YATES’ CORRECTED CHI SQ 0.10 SE (HO: Q = 0 ) 1 .7 1 3 E - 0 1 P (YATES) 0 .7 4 7 5 YULE’S Y - 0 . 1 5 LOG ODDS RATIO - 6 . 0 6 1E ~0I SE (Y) 4 .1 2 7 E - 0 2 SE (LOR) O .9 1 3 E -0 1 SE (HO: Y = 0 ) 4 .2 8 2 E - 0 2 SE (HO: LOR = 0) r>,H 52E-01 max 0 . 4 3 CROSS PRODUCT RATIO 5.455E-01 PHI - 0 . 1 1 CONTINGENCY COEFF 0 .1 1 PHI MAX - 0 . 4 8 B PEARSON’S CHI SQUARE 0 .6 3 YULE’ S Q - 0 . 2 7 P (PEARSON’S) 0.4270 SE (Q) 1.022E-01 YATES’ CORRECTED CHI SQ 0 .2 1 SE (HO: Q = 0 ) 1 .1 4 2 E - 0 1 P (YATES) 0.6482 YULE’S Y - 0 . 1 4 LOG ODDS RATIO - 5 .4 4 1 E - 0 1 SE (Y) 2 .8 5 1 E - 0 2 SE (LOR) 4 .7 3 4 E - 0 1 SE (HO: Y = 0) 2.856E-02 SE (HO: LOR = 0) 4.570E-01 C MAX 0.55 CROSS PRODUCT RATIO 5 .8 0 4 E - 0 1 PHI - 0 . 13 CONTINGENCY COEFF 0.1.2 PHI MAX - 0 . 6 6

c PEARSON’S CHI SQUARE 1 7 . 4 7 ^ 'ilJLE’ S Q - 0 . 9 5 P (PEARSON’S) 0 .0 0 0 0 SE (Q) 3 .5 0 6 E - 0 3 YATES’ CORRECTED CHI SQ 1 4 .4 9 SE (HO: Q = 0) 1.393E-01 P (YATES) 0 .0 0 0 1 YULE’S Y —0.71 LOG ODDS RATIO - 3 . 5 8 4 SE (Y) 1 .9 0 0 E - 0 2 SE (LOR) 1 .2 6 7 SE (HO: Y = 0 ) 3 .4 8 2 E - 0 2 SE (HO: LOR = 0 ) 5 .5 7 0 E - 0 1 C MAX 0 . 5 7 CROSS PRODUCT RATIO 2 .7 7 8 E - 0 2 PH I - 0 . 5 9 CONTINGENCY COEFF 0 .5 1 PH I MAX - 0 . 7 0

D PEARSON’S CHI SQUARE 3 . 9 4 * YULE’S Q - 0 . 7 7 P (PEARSON’S) 0.0470 SE (Q) 5 .5 0 1 E - 0 2 YATES’ CORRECTED CHI SQ 2.37 SE (HO: Q = 0 ) 1 . 9 8 1 E -0 1 P (YATES) 0 .1 2 3 4 YULE’S Y - 0 . 4 7 LOG ODDS RATIO - 2 . 0 4 0 SE (Y) 5 . 0 3 4 E -0 2 SE (LOR) 1 .3 2 7 SE (HO: Y = 0 ) 4 . 9 5 4 E -0 2 SE (HO: LOR = 0) 7.926E-01 C MAX 0 . 4 2 CROSS PRODUCT RATIO 1 .3 0 0 E - 0 1 PHI - 0 . 3 2 CONTINGENCY COEFF 0 . 3 0 PHI MAX - 0 . 4 6

129 Appendix B. An example of Chi-squared test (using two-by-two table) on the hatching patterns of "new" and "old" cysts of G.pallida. A October hatch. B September hatch. C November hatch. D December hatch.

A PEARSON’S CHI SQUARE 1 .5 5 Y U LE'S Q 0 .4 5 ■' (PEARSON’S) 0 .2 1 3 8 SE (Q) 1 .0 1 6 E -0 1 r'ATES ’ CORRECTED CHI SQ 0 .7 4 SE (HO: q = 0) 1 .4 6 9 E -0 1 p (YATES) 0 .3 8 9 9 YULE'S 0 .2 4 LOG ODDS RATIO 0 .9 8 1 SE (Y) 3 .5 8 3 E -0 2 SE (LOR) 6 .4 5 8 E - 0 1 SE (HO: Y = 0) 3 .6 7 3 E - 0 2 SE (HO: LOR = 0 ) 5 .8 7 7 E - 0 1 C MAX 0 .5 0 CROSS PRODUCT RATIO 2 .6 6 7 PHI 0 .2 0 CONTINGENCY COEFF 0 .2 0 PH I MAX 0 .5 8 B PEARSON’S CHI SQUARE 0 .4 1 YULE’ S Q - 0 . 2 5 P (PEARSON’S) 0 .5 1 9 7 SE (Q ) 1 .4 4 7 E - 0 1 YATES’ CORRECTED CHI SQ 0 .0 6 SE (HO: Q = 0) 1 .6 3 3 E -0 1 P (YATES) 0 .8 1 0 6 YULE’S Y - 0 . 1 3 LOG ODDS RATIO - 5 .1 9 5 E - 0 1 SE (Y) 3 . 9 9 6 E -0 2 SE (LOR) 6 .6 1 3 E - 0 1 SE (HO: Y = 0 ) 4 . 0 8 2 E -0 2 SE (HO: LOR = 0 ) 6 .5 3 0 E - 0 1 C MAX 0 .3 7 CROSS PRODUCT RATIO 5 .9 4 8 E - 0 1 PHI - i ). 08 CONTINGENCY COEFF 0 .0 8 PHI MAX - 0 . 4 0 C PEARSON’S CHI SQUARE 28.91^ YULE'S Q -0.97 P (PEARSON’S) 0.0000 SE (Q) 7 .2 1 7 E - 0 4 YATES’ CORRECTED CHI SQ 25.65 SE (HO: Q = 0) 9 .7 1 5 E - 0 2 P (YATES) 0.0000 YULE’S Y -0.79 LOG ODDS RATIO -4.248 SE (Y) 8 .5 1 0 E - 0 3 SE (LOR) 9.357E-01 SE (HO: Y = 0) 2 . 4 2 9 E -0 2 SE (HO: LOR = 0) 3.886E-01 C MAX 0.69 CROSS PRODUCT RATIO 1.429E-02 PHI* -0.78 CONTINGENCY COEFF 0 .6 1 PH I MAX - 0 . 9 6 D PEARSON’S CHI SQUARE 2 7 . 1 9 ^ YULE’S Q - 0 . 9 6 P (PEARSON’S) 0 .0 0 0 0 SE (Q ) 1 .0 4 0 E - 0 3 YATES’ CORRECTED CHI SQ 2 4 .2 5 SE (HO: Q = o) 8 . 3 9 2 E -0 2 P (YATES) 0 .0 0 0 0 YULE’S Y - 0 . 7 6 LOG ODDS RATIO - 4 . 0 0 7 SE (Y) 9 .2 6 8 E - 0 3 SE (LOR) 8 .4 5 5 E - 0 1 SE (HO: Y = 0) 2 . 0 9 8 E -0 2 SE (HO: LOR = 0) 3 .3 5 7 E - 0 1 C MAX 0 .6 8 CROSS PRODUCT RATIO 1 .8 1 8 E - 0 2 PHI - 0 . 7 5 CONTINGENCY COEFF 0 . 6 0 PH I MAX - 0 . 9 2

130 Appendix C. An example of one way analysis of variance (ANOVA) on number of eggs in "new" and "old" cysts of G.rostochiensis and G.pallida. G.rostochiensis. A October hatch B November hatch SAMPLE GROUP A VARIABLE MEAN SIZ E VARIANCE

RN1 1.A95E+0A A 2 . 81.9F.+06 R01 2.773E+0A A 2.131E+07 TOTAL 2 . 13AE+OA 8

SOURCE DF SS MS F P

BETWEEN 1 3 . 269E+08 3.269E+08 27.09 0 .0 0 2 0 ★ WITHIN 6 7.239E+07 1 .207E+07 TOTAL 7 3.993E+08

SAMPLE GROUP B VARIABLE MEAN SIZE VARIANCE

RN2 2 . 018E+0A A 9.9A2E+06 R02 2 . A62E+0A A 5.A39E+07 TOTAL 2 . 2A0E+0A 8

SOURCE DF SS MS F p

BETWEEN 1 3 . 9AAE+07 3.9AAE+07 1.23 0 .3 1 0 5 WITHIN 6 1.930E+08 3 . 216E+07 TOTAL 7 2.32AE+08 G.pallida. A October hatch B November hatch SAM:PLE GROUP A VARIABLE MEAN SIZE VARIANCE

PN1 2 . 100E+OA A 3.180E+06 POl 1 . 899F.+0A A 1.155E+07 TOT AI 1 .999E+0A 8

SOURCE DF SS MS F P

BETWEEN 1 8 . 088E+06 8.088E+06 1.10 0 .3 3 5 0 WITHIN 6 A.A18E+07 7 . 36AE+06 TOTAL 5 . 227F+07 SAMPLE GROUP B VARIABLE MEAN SIZE VARIANCE

PN2 2 . 950E+0A - 2. 329E+0'7 P02 1.888E+0A A 1.011E+07 TOTAL 2 . A19E+0A 8

SOURCE DF SS MS F P

BETWEEN 1 2.257E+08 2.257E+08 13.51 0 .0 1 0 A WITHIN (> 1.002E+08 1 .670E+07 TOTAL 3 . 259E+08 131 Appendix D. Mean temperature and rainfall at Silwood Park Ascot, 1986 to 1989. Latitude 51°28N. Mean sea level 220 feet. (Source Department of Biology, Imperial College, Silwood Park, Ascot).

Year Monthly Mean Temperature °C. J a n .. F e b . M ar. Apr. May J u n . J u l . Aug. S e p . O c t . N ov. , D e c .

198 6 3 .8 1.1 4 . 9 6.1 11.2 1 5 .6 1 6 .6 1 4 .2 11.6 1 1 .7 8.1 6 .3

1987 0.6 4 . 1 4 . 2 1 0 .5 1 0 .9 13.7 16.8 16.5 14.1 9.8 6 . 3 5 . 9 1988 5.8 4.8 6.7 7.9 1 2 .4 1 4 .0 1 4 .6 1 5 .6 1 3 .5 1 0 .5 4 . 8 7 . 4

1 9 8 9 5 .8 5.8 7.6 6.5 14.2 1 5 .4 1 9 .3 1 7 .2 1 5 .2 12.1 5 .8 5 . 8

Y e a r Monthly Mean Rainfall in mm. J a n . Feb. Mar. Apr. May Jun. Jul. Aug. Sep. Oct. Nov. Dec. 1 9 8 6 1 2 5 .5 15.9 54.8 76.5 54.0 17.3 41.6 79.8 23.8 85.7 88.4 63.8 1 9 8 7 2 3 .0 34.5 57.9 48.5 51.0 79.9 47.3 53.3 57.0 174.6 49.8 11.0 19 8 8 1 0 6 .0 41.5 61.5 24.6 35.7 38.0 95.1 40.2 57.7 67.0 18.1 12.9 1 9 8 9 2 9 .1 54.5 73.0 69.1 20.7 24.6 49.9 45.3 22.6 56.5 28.2 123.4

132 Appendix E. An example of two way analysis of variance (ANOVA)G.rostochiensis on cysts stored dry at 5°C.

ANALYSIS OF VARIANCE TABLE FOR HI A First hatch (HI and H2)

SOURCE DF SS MS F P

R (A) 3 3 .8 5 8 7 E - 0 2 1.2862E-02 0.44 0 .7 3 0 7 T (B ) 5 4 .1 5 8 9 E - 0 1 8.3177E-02 2.82 0 .0 5 4 7 A*B 15 4.4293E-01 2.9528E-02

TOTAL 23 8 .9 7 4 0 E - 0 1 GRAND AVERAGE 1 1 7 .6 3 6

ANALYSIS OF VARIANCE TABLE FOP H2A Second hatch (H3 and H4)

SOURCE DF SS MS FP

R (A ) 3 9 .2 1 1 2 E - 0 4 3 .0 7 0 4 E - 0 4 0.21 0 .8 9 0 9 T (B ) 5 9 .8 7 3 2 E - 0 2 1 .9 7 4 6 E - 0 2 1 3 .2 3 0 . 0 0 0 0 ^ A*B 15 2.2396E-02 1 .4 9 3 1 E - 0 3

TOTAL 23 1.2205F-01 GRAND AVERAGE 1 1 .6 1 8 8 E -0 1

ANALYSIS OF VARIANCE TABLE FOP H3A Third hatch (H5 and H 6)

SOURCE DF SS MS F p

R (A) 3 1.2504E-04 4.1681E-05 1.00 0 .4 1 9 9 T (B ) 5 2.0840E-04 4.1681E-05 1.00 0 .4 5 0 9 A*B 15 6.2521E-04 4.1681E-05

TOTAL 23 9 .5 8 6 5 E - 0 4 GRAND AVERAGE 1 4 . 1 6 8 1 E -0 5

ANALYSIS OF VARIANCE TABLE FOP H4A Unhatched eggs

SOURCE DF SS MS F p

R (A) 3 3.8492E-02 1.2831E-02 0.41 0 .7 4 6 2 T (B ) 5 3 .8 0 4 9 E - 0 1 7 .6 0 9 8 E - 0 2 2 .4 5 0 .0 8 2 0 A*B 15 4.6623E-01 3 .1 0 8 2 E - 0 2

TOTAL 23 8 . 8 5 2 1 E -0 ] GRAND AVERAGE 1 1 1 .7 8 6 133 Appendix F. An example of two way analysis of variance (ANOVA)G on .pallida cysts stored dry at 5°C.

A First hatch (HI and H2) B Second hatch (H3 and H4) C Third hatch (H5 and H6) D Unhatched eggs

A ANALYSIS OF VARIANCE TABLE FOR III A

SOURCE DF SS MS F P

R (A) 3 4 .3 4 1 1 E - 0 3 1.4470E-03 0.36 0 .7 8 3 8 T (B ) 5 I .1 4 2 1 2 .2 8 4 3 E - 0 1 5 6 .5 8 0 . 0 0 0 0 * A*B 15 6 .0 5 5 4 E - 0 2 A . 0 3 6 9 E - 0 3

TOTAL 23 1 .2 0 7 0 GRAND AVERAGE 1 1 3 .2 7 5

ANALYSIS OF VARIANCE TABLE FOR ]I2A

SOURCE DF SS MS F P

R (A) 3 4.6277E-03 1.5426E-03 0.92 0.4544 T (B) 5 4 .5 3 9 3 E - 0 1 9.0786E-02 54.21 0 . 0 0 0 0 * A*B 15 2.5120E-02 1.6746E-03

TOTAL 23 4 .8 3 6 8 E - 0 1 GRAND AVERAGE 1 6 .2 5 4 7 E -0 1

C ANALYSIS OF VARIANCE TABLE FOR H3A

SOURCEDF SS M3 I' P

R (A) 3 5 .1 5 3 8 E - 0 4 1 .7 1 7 9 E - 0 4 1 .5 8 0 .2 3 5 1 T (B ) 5 1 .0 2 5 9 E - 0 2 2 .0 5 1 9 E - 0 3 1 8 .9 0 0 . 0 0 0 0 * A*B 15 1.6282E-03 1 .0 8 5 5 E - 0 A

TOTAL 23 1 .2 4 0 3 E - 0 2 GRAND AVERAGE 1 4 .6 1 6 1 E - 0 3

DANALYSIS OF VARIANCE TABLE FOR H4A

SOURCEDFSS MS F P

R (A) 3 5 .0 5 7 3 E - 0 3 1.6858E-03 0.42 0 .7 4 2 0 T (B ) 0 1 .2 9 4 5 2.5891E-01 64.36 0 . 0 0 0 0 * A*B 15 6 .0 3 4 4 E - 0 2 4 .0 2 2 9 E - 0 3

TOTAL n o 1 .3 5 9 9 GRAND AVERAGF l 1 4 .6 1 1 134 Appendix G. An example of two way analysis of variance (ANOVA) G.rostochiensison cysts stored wet at 5°C.

A First hatch (HI and H2) B Second hatch (H3 and H4) C Third hatch (H5 and H6) D Unhatched eggs

A ANALYSIS OF VARIANCE TABLE FOR H1A

SOURCEDF SS MS F P

R (A) 3 2.1351E-02 7 . 1 1 6 9 E -0 3 1.00 0 .4 2 1 7 T (B) 5 1.4761 2.9522E-01 41.30 0 . 0 0 0 0 ^ A*B 15 1.0723E-01 7 .1 4 8 8 E - 0 3

TOTAL 23 1 .6 0 4 7 GRAND AVERAGE 1 2 7 .0 5 6

B ANALYSIS OF VARIANCE TABLE FOR H2A

SOURCEDF SS MS F p

R (A) 3 1 . 5 7 9 1 E -0 2 5.2637E-03 0.63 0.6093 T (B) 5 9 .3 0 8 3 E - 0 1 1 .8 6 1 7 E -0 1 2 2 . 1 4 0 . 0 0 0 0 ^ A*B 15 1.2614E-01 8 .4 0 9 3 E - 0 3

TOTAL 23 1 .0 7 2 8 GRAND AVERAGE 1 3 .4 5 5 6

c ANALYSIS OF VARIANCE TABLE FOR IL3A

SOURCE DP SS MS F p

R (A) 3 1.3832E-03 4.6106E-04 0.50 0.6878 T (B) 1 . 1 6 1 8 E -0 1 2.3235E-02 25.21 O.OOOO^ A*B 15 1.3828E-02 9 .2 1 8 4 E - 0 4

TOTAL 23 1 .3 1 3 9 E -0 1 GRAND AVERAGE 1 3 .3 4 4 7 E - 0 2

D ANALYSIS OF VARIANCE TABLE FOR !H4A

SOURCE DF SS MS F P

R (A) 3 1.4493E-02 4.8310E-03 0 . 5 8 0 .6 3 6 3 T (B) 5 3.1985E-01 6.3970E-02 7 . 7 0 0 . 0 0 0 9 ^ A*B 15 1 .2 4 6 5 E - 0 1 8 .3 1 0 3 E - 0 3

TOTAL 23 A.5900E-01 GRAND AVERAGE 1 1 .8 9 2 4 135 Appendix H. An example of two way analysis of variance (ANOVA) Gon .pallida cysts stored wet at 5°C.

A First hatch (HI and H2) B Second hatch (H3 and H4) C Third hatch (H5 and H6) D Unhatched eggs

A a n a l y s i s o f v a r ia n c e t a b l e f o r H1A

SOURCE DF SS MS F P

R (A) 3 A . 2 0 5 9 E -0 3 1 . A 0 2 0 E -0 3 0 . 3 9 0 . 7 6 3 2 T (B) 5 3 . 9 5 A3 7 . 9 0 8 6 E -0 1 2 1 8 .9 3 0 . 0 0 0 0 ^ A*B 15 5 .A 1 8 7 E - 0 2 3 .6 1 2 A E -0 3

TOTAL 23 A .0 1 2 7 GRAND AVERAGE 1 6 .6 3 2 9

B ANALYSIS OF VARIANCE TABLE FOR H2A

SOURCE DFSS MS F P

R (A) 3 A.7876E-02 1.5959E-02 1 .8 1 0 .1 8 9 2 T (B ) 5 1 .0 0 3 1 2 . 0 0 6 1 E -0 1 2 2 .7 1 0 . 0 0 0 0 ^ A*B 15 1.3252E-01 8.83A8E-03

TOTAL 23 1 .1 8 3 5 GRAND AVERAGE 1 A .8 1 2 9

ANALYSIS OF VARIANCE TABLE FOR !H3A

SOURCEDF SS MS F P

R (A) 3 A.1530E-03 1.38A3E-03 0.52 0 . 6 7 6 3 T (B) 5 3 .0 9 6 2 6 .1 9 2 3 E - 0 1 2 3 1 .6 3 0 . 0 0 0 0 ^ A*B 15 A .0 1 0 0 E - 0 2 2 .6 7 3 3 E - 0 3

TOTAL 23 3.1A0A GRAND AVERAGE 1 1 .0 3 6 5

D ANALYSIS OF VARIANCE TABLE FOR HAA

SOURCE DF SS MS F P

R (A) Q 8 .5 7 6 5 E - 0 2 2 .8 5 8 8 E - 0 2 2.88 0 .0 7 0 7 T (B ) 5 1 .2 2 5 6 2.A511E-01 2A.71 0 . 0 0 0 0 ^ A*B 15 1 .A 8 8 1 E -0 1 9 .9 2 0 8 E - 0 3

TOTAL 23 1.A 6 0 1 GRAND AVERAGE 1 8 .5 3 2 7

136. Appendix J. An example of two way analysis of variance (ANOVA) G.rostochiensison cysts stored humid at 5°C.

A First hatch (HI and H2) B Second hatch (H3 and H4) C Third hatch (H5 and H6) D Unhatched eggs A ANALYSIS OF VARIANCE TABLE FOR H1A

SOURCEDF SS NS F P

R (A) 3 1 . A 5 5 5 E -0 2 A .8 5 1 8 E - 0 3 0 . 3 6 0 .7 8 1 5 T (B ) 6 . AAA5E-01 1 .2 8 8 9 E - 0 1 9 .6 1 0 . 0 0 0 3 ^ A*B 15 2.0117E-01 1 .3 A 1 1 E -0 2

TOTAL 23 8.6017E-01 GRAND AVERAGE 3. 2 A .5 3 2

B ANALYSIS OF VARIANCE TABLE FOR H2A

SOURCE DF SS NSFP

R (A) 3 A .9 0 A 2 E -0 3 1 .6 3 A 7 E -0 3 0 .5 3 0 . 6 6 8A T (B) 5 8.1681E-01 1 .6 3 3 6 E - 0 1 5 2 .9 8 0 . 0 0 0 0 ^ A*B 15 A .6 2 A 9 E -0 2 3 .0 8 3 3 E - 0 3

TOTAL 23 8.6796E-01 1 GRAND AVERAGE j. 2 .1 1 6 1

ANALYSIS OF VARIANCE TABLE FOR H3A

SOURCEDFSSNSFP

R (A) 3 8 .7 2 8 9 E -0 A 2 . 9 0 9 6 E -0 A 1.00 0 .A 1 9 9 T (B) 5 9.8360E-03 1.9672E-03 6 .7 6 0 . 0 0 1 7 ^ A*B 15 A.36AAE-03 2 . 9 0 9 6 E -0 A

TOTAL 23 1 .5 0 7 3 E - 0 2 GRAND AVERAGE 3 1 .9672E-03

D ANALYSIS OF VARIANCE TABLE FOR HAA

SOURCEDF SSNS u P

R (A) 3 1 .A939E-02 A.9798E-03 0 . A1 G .7A 85 T (B ) Z) 3 .5 6 1 3 E - 0 1 7 .1 2 2 7 E - 0 2 5 . 8 6 0 . 0 0 3 A ★ A*B 15 1 .8 2 2 5 E - 0 1 1 .2 1 5 0 E - 0 2

TOTAL 23 5.5333E-01 GRAND AVERAGE i A. 2 5 8 9 137 Appendix K. An example of two way analysis of variance (ANOVA)G.pallida on cysts stored humid at 5°C.

A First hatch (HI and H2) B Second hatch (H3 and H4) C Third hatch (H5 and H6) D Unhatched eggs A ANALYSIS OF VARIANCE TABLE FOR IilA

SOURCEDFSS MS I' P

R (A ) 3 4.7443E-03 1.5814E-03 0.22 0 .8 8 1 4 T (B ) 5 3 .2 0 3 8 6.5676E-01 91.12 0 . 0 0 0 0 ^ A*B 15 1.0812E-01 7 .2 0 7 7 E - 0 3

TOTAL 23 3 .3 9 6 7 GRAND AVERAGE 1 6 .6 1 9 2

BANALYSIS OF VARIANCE TABLE FOR H2A

SOURCEDFSS MS F P

R (A) 3 2.0090E-02 6.6965E-03 1.38 0.2865 T (B ) 5 7.3537E-01 1.4707E-01 30.36 0 . 0 0 0 0 ★ A*B 15 7.2668E-02 4.8445E-03

TOTAL 23 8.2812E-01 GRAND AVERAGE 1 3 .9 8 4 7

C ANALYSIS OF VARIANCE TABLE FOR H3A

SOURCE DF SS MS F P

R (A) 3 6.4741E-05 2.1580E-05 1.00 0 .4 1 9 9 T (B ) 5 1 .7 4 4 0 E - 0 1 3 .4 8 7 9 E - 0 2 1 6 1 6 .2 6 0 . 0 0 0 0 ^ A*B 15 3 .2 3 7 1 E - 0 4 2 . 1 5 8 0 E -0 5

TOTAL 23 1.7479E-01 GRAND AVERAGE 1 3 . 4 8 7 9 E -0 2

D ANALYSIS OF VARIANCE TABLE FOR HAA

SOURCE DF SS MS F P

R (A) 2.1281E-02 7.0936E-03 0.62 0.6133 T (B ) 5 1.2862 2.5725E-01 22.46 0 . 0 0 0 0 ^ A*B 15 1.7183E-01 1.1455E-02

TOTAL 23 1 .4 7 9 3 GRAND AVERAGE 1 1 4 .7 5 3 138 Appendix L.

Hear. total natcheci ana unhatchsri eggs or G .rostochiensis stored dry at various temperatures for various periods.

5 t o . te m p . ■ to. period. '.lean tot. niton lean t ct . unhat.

1 0 0 6 7 •6475 • , ^ - __ _ * \J O / u

6 6 9 3 9 5413 . _ 0 1 c> - - -> *. \ j 110 4 3 53 3 3

.1 z, 4 2 5 3 3 0 3 8 •p 2.U £ 1 1 3 8 4 472 5 4 1 1 5 7 9 4 7 6 3

6 1 0 3 4 8 2225 13217 5663

0 4 4 4 4 -eta: a 5 5 0 - *0 Z 110 6 9 5150 4 9 9 9 4 3525

6 1 0 2 2 4 3125 U 1 4 2 7 3 2375 J. u 11411 1333 _ ^ ^ _ t o o o ,• 6e

C. V 1 1 2 6 5 1750 ci 5109 5938 5 7 4 1 4 4 7 3 8 9 6 6 4 a- Z ^ „ .-.0 i. u i t : 0 6

2 6 4 8 5 7 5 5 0 “? r 5371 : U'J’J - 2937 3138

6 5 5 3 9 5263 3 380 3 10525

10 2405 17863 t Z. 590 1 5 7 6 3

139 Appendix LI.

Ilean totai hatched and unhatched eggs of G.pallida stored dry at various temperatures for various oeriods.

S t o . te m p . 5to. period. Mean tot. hatch Mean tot. unhat.

5 2 1 5 3 2 5 9 3 2 5 4 13631 9 2 2 5 6 1 5 5 4 0 8 7 0 0 8 2 0 6 2 6 8 5 0 0 10 1 3 1 1 7 1 8 9 0 0 - 'A 1841 1 7 7 0 0 10 £ 179 9 4 66 3 8 4 14201 1 1 9 7 5 6 1 2 4 2 3 852 5 8 1 9 0 8 2 6 5 7 5 10 1 5 2 9 5 1 0 7 7 5 12 255 7 1 6 6 1 3 5 n 1 3 4 4 9 10563 4 1 1 2 5 4 95 2 5 6 1 1 5 5 3 6 8 1 3 8 1 3 9 9 6 5325 10 1 5 4 7 7 113 7 5 i n L ^ 3 5 3 6 1 3 5 5 0 2 G 1 1 6 1 2 1 5 0 3 8 4 816 2 1 5 4 0 0 6 4 2 3 3 1 4 5 5 0 8 6 6 9 6 1 3 0 3 8 i r\ XU 6 2 2 5 3 6 1 3 -i 2 776 2 2 3 1 3 d 2> 1 1 4 9 0 763 8 4 1 1 9 0 5 9 5 5 8 5 8 8 9 5 1 0 6 5 0 8 1 2 6 9 1 1 0 5 0 0 1 0 7636 1 2 2 7 5 32 24 5 1 4 2 7 5

140 Appendix N.

Mean total hatched and unhatched eggs of G .rostochiensis stored xet at various temperatures for various periods.

S t o . te m p . Sto. period Mean tot. hatch Mean tot. unhat.

5 2 8 6 9 1 149 4 4 5 2 4 2 1525 6 7 9 9 9 338 3 9 9 0 8 200 xU 3 0 7 4 638

12 1 0 2 3 7 6 4 4 J. 6 6 6 3 7 -625 4 5 4 5 8 1983 6 1 1 5 5 8 425 3 8 5 1 8 89 4 10 6 5 1 6 313 L x 1 1 8 4 9 11 9 4 " r - 1 0 7 3 8 66 3 4 1 0 9 9 6 250 5 1 0 7 7 4 40 0 3 1 1 5 4 5 113 10 8 6 0 3 1 88 12 1 5 3 8 0 2940

a ■■J 2 9 2 6 0 -L X 1. O 4 1 0 1 5 2 7 ^ ^ 1 1 0 3 3 9 2 38 5 1 0 9 7 2 225 10 3 7 1 6 206 -5 1 4 1 5 1 245 ; c; - 1 1 3 6 5 500 - ” 46 3 - 6 3 3 9 1 5 8 400 8 9 3 5 0 738 7 r\ iU 9 2 2 6 63 X 1 1 7 0 8 250

141 Appendix P. Mean total hatched and unhatched eggs of G.pallida stored vet s.t various temperatures for various periods.

S t o . te m p . Sto. period ilean tot. hatch ilean t o t . u n h a t .

5 2 7999 5 3 5 0 4 280 5 6 6 3 8 6 5 6 1 7 5 9 0 0 3 7 7 1 4 1 3 4 4 10 1 1 6 7 2 2025

- i ~ C t: r - .1 ^ * *' f - p 2 982 5 7083 4 2 1 3 4 9425 6 5 5 3 0 5 0 5 0 3 1 0 4 6 3 2488 10 1 0 8 7 3 1338 12 1 1 4 3 9 1863 - r. r. X '-J ~ 9 4 7 0 4 1 7 6 4 1 1 2 2 5 6 6 0 1 1 4 8 6 3 6 8 6 8 4 1363

1. 1J 40 / d 852 5 -2 5 1 0 0 2 COO .0 2 4 1 1 3 8 4 0 0

4 6 0 4 8 / wj i o 3 9 0 4 1 1312

2 9461 ^ J O 10 1463 1 3 9 4

-v r\ - 2 5531 ?• 5 2 7 3 0 1 5033 4 6 1 4 2 3513 - 5 5 0 2 10 C 5 3560 4 7 5 10 8905 788 12 10368 550

142 Appendix Q. liean total hatched and unhatched eggs of 4. rostochiensis stored humuid at various temperatures for various periods.

3 to . te m p . Sto. period llean r o t . h atch . He an ~ o t.. u n n a t

=; 116 7 5 938

4 5 6 9 4 1 0 1 3 •3 •3S54 3375 q 1 4 6 7 9 - - 3

4 n a n ,4 JL. U 3113 12 7 2 1 8 3106 10 2 9 0 5 7 1975 4 375 5 1338 6 1 0 0 6 5 2038

3 1 3 5 6 1 ” 2 0 10 1.1 31 0 ; ;1 7 1}

l 4- 6 7 7 5 3 7 5 ?, * 2 9 2 9 4 1 3 6 3 4 7 3 7 1 1363

6 6 7 7 1 2131 3 6 0 0 4 61 9

1 0 1 2 6 1 4 90G

-2 5 4 7 7 1081 *•' ^ ^ rr o 4, u L 6 4 7 9 £ w. O c

4 7 8 9 5 5 2 5 gj 6 4 0 3 ‘ O fs o

5 3 6 9 0 475 6 7 3 4 2094

L £ 3 3 6 2 1038 ^ n 2 1 0 6 7 1 4 3 5 0

4 3 3 7 6 3138

•3 4 3 9 1 - u .**'> & ^ r 3 7 1 3 1

1 0 1 0 1 9 9 1994 -*> 3 6 7 2 2544

143 Appendix R.

Mean total hatched and unhatched eggs of G.pallida stored humid at various temperatures for various periods.

S t o . te m p . Sto. period. Kean tot. hatch Heap tot. unhat.

5 2 5009 10558 4 6 3 4 6 9 9 3 8 6 1 0 5 9 2 4 1 0 0 8 1 2 5 9 9 4294 10 10727 5738 12 2311 1 0 9 2 5 10 n 460 1 1 1 8 3 8 4 6 5 3 3 132 2 5 6 1 0 6 2 9 9 6 0 0 8 137 5 3 391 3 10 1 4 7 9 8 9 0 5 0 12 3 1 4 0 1 3 8 5 0 15 n 2491 7 4 0 0 4 4 5 8 8 872 5 6 9 3 2 7 26 1 3 3 1 1 5 5 7 2 2 4 4 10 1 3 0 5 2 100 6 12 2775 7313 20 £ 8251 1 1 7 5 0 4 1915 881 3 1 6 6 0 1 3 1 4 7 q 3 24 1 1 1 1 3 10 1078 173 3 8 12 49 1 4 9 3 8 25 4 9 1 0 361 2 4 7 3 3 7 2800 6 1 1 5 3 4 1333 3 9 7 6 3 1375 10 12439 1 0 5 0 12 5749 4963

144 Appendix S. An example of two way analysis of variance (ANOVA)G.rostochiensis on infectivity assay.

ANALYSIS OF VARIANCE TABLE FOR ROSE

SOURCE DF SS MS FP

R (A ) 3 2 . 0 1 4 0 E -0 2 6 .7 1 3 4 E - 0 3 1 .2 2 0 .3 2 4 6 P (B) 8 1.0588 1.3235E-01 24.01 0.0000^ A*B 24 1 .3 2 2 7 E - 0 1 5 .5 1 1 4 E - 0 3

TOTAL 35 1 .2 1 1 2 GRAND AVERAGE 1 1 8 .4 1 3

LEAST SIGNIFICANT DIFFERENCE PAIRWISE COMPARISONS OF R0S2 BY P

HOMOGENEOUS P MEAN GROUPS

9 0 .9 7 7 I 8 8 .9 4 1 E - 0 1 II 6 8.894E-01 I I 7 8 .0 0 4 E - 0 1 I 4 6 .6 3 8 E - 0 1 . . • . I 2 6 .2 2 2 E - 0 1 , . • , I 6 .1 7 1 E - 0 1 . , I I 1 5 .0 9 8 E - 0 1 . I I 3 4 .6 3 1 E - 0 1 I

THERE ARE 5 GROUPS IN WHICH THE MEANS ARE NOT SIGNIF. DIFF. FROM ONE ANOTHER.

CRITICAL T VALUE 2.064, REJECTION LEVEL 0.050 CRITICAL VALUE FOR COMPARISON 1.0834E-01 STANDARD ERROR FOR COMPARISON 5.2495E-02

ERROR TERM USED: R*P, 24 DF

145 Appendix T. An example of two way analysis of variance (ANOVA)G.pallida on infectivity assay

ANALYSIS OF VARIANCE TABLE FOR PAL2

SOURCE DFSS v c FP

R (A) 3 1 .5 6 3 2 E - 0 2 5 .2 1 0 7 E - 0 3 3 .8 9 0 .0 2 3 5 P (B ) 7 1 .4 6 9 7 E - 0 1 2 .0 9 9 6 E - 0 2 1 5 .6 7 0 . 0 0 0 0 ^ A*B 21 2 .8 1 3 4 E - 0 2 1 .3 3 9 7 E - 0 3

TOTAL 31 1 .9 0 7 4 E -0 1 GRAND AVERAGE 1 1 4 .6 4 7

LEAST SIGNIFICANT DIFFERENCE PAIRWISE COMPARISONS OF PAL2 BY P

HOMOGENEOUS P MEAN GROUPS

8 7.854E-01 I 7 7.853E-01 I 4 6 .7 4 4 E - 0 1 I 5 6 .6 9 2 E - 0 1 I 6 6.614E-01 I I 1 6 .2 4 7 E - 0 1 III 3 6 .1 4 1 E - 0 1 II 2 5 .9 7 8 E - 0 1 I

THERE ARE 4 GROUPS IN WHICH THE MEANS ARE NOT SIGNIF. DIFF. FROM ONE ANOTHER.

CRITICAL T VALUE 2.080, REJECTION LEVEL 0.050 CRITICAL VALUE FOR COMP.ARISON 5.3823E-02 STANDARD ERROR FOR COMPARISON 2.5881E-02

ERROR TERM USED: R*P, 21 DF

146 Appendix U. An example of Chi-squared test on G.rostochiensis. a) 3TW+RA on Sir: c y s t s

O ) STW+RA on RCH c y s o s 2 ) PRD+RA on SIH c y s t s d) PRD+RA on RCH c v s t s a) _ *_ <*• PEARSON'S CHI SQUARE 1 3 .7 2 * \" I ' r T? • c;; t ) i PEARSON' S ) • 0.0002 SE (Q YATES 1 CORRECTED CHI Si./ H .OR S: (HO: 0 - ;; T 'YATES) 0 .0 0 0 ' YURT 1 r Y LOU ODDS RATIO \-r {\\ SF (LOR; V SE (HO: - i : ' SE ( HO: LOR = 0 ) V C NAY CROSS PRODUCT RATIO 0 .000 PHI . 22 CONTINGENCY COEFE 0.22 PITT V'AY . 22 b) -E'UoON'S CHI SQUARE 2 0 * ’A i.F ’ S o : U'EARSO.N • S ; 0 .0 0 2 A SE i.C; —. —r *— YATES1 CORRECTED CHI SO SE (Ho: Q = 0/■ ■ . i ){ 1 i v P (YATES) 0.0053 YULE’ S Y LOO ODDS RATIO -2 .4 9 5 SE' (Y) . 221E-02 SE (TOR) 1 .072 SE (HO: Y = 0) ].503E-02 SE (HO: LOR = 0) 2.A0AE-01 0 YAX CROSS PRODUCT RATIO 0 .252E-02 7>UT r-[ f : V * Y COVn.YGEYn COEFE \J . 1 f > r i cvA . 2:

C) PEARSON’ S CHI SQUARE 1 6 .8 4 * YULE'S 0 -0 .7 3 P (PEARSON’ S) 0.0000 SE (Q) 1 .398E-02 YATES’ CORRECTED CHI so 15.22 SE (HO: 0 = 0) A.109E-02 P (YATES) 0.0001 YULE’S Y - 0 . A A LOO ODDS RATIO -1.867 SE (Y) 1 .066E-02 ST (LOP) 2 . 598E-01 SE (HO: V = 0) 1 .027E-02 SE (HO: LOR = 0) 1.6AAE-01 C MAX 0 .3 3 CROSS PROD)"'"' RATIO 1 . 5A6E-0: PH) -1,. 23 COM i poEM ’t COEFF n PHI MAX \ ■. 3 A

d)

P £'A R 5 U N ' S C HI S OU A RE 2.32 YULE ' S ■'./ « • } V > P (PEARSON'S) 0.1281 SE (Q) 1 .1 1 9E-02 YATES' CORRECTED CHI so 2.00 SE (HO: 0 = 0) 1 . 172E-02 P (YATES) 0.1576 YULE’ S Y 0 .0 8 LOO ODDS RATIO 3 . 303E-O1 ■S r ( Y ) 2 . 9t3t.-0E SC ! LOP "A ■“ T" —;; SE (HO: - 0 > _ . ' .'20E-C' SF (HO: LOP = m a . 686E- 02 ■c MAX 1.6- CROSS PRODUCT RATIO 1 .3 9 1 PHI 0 .0 8 CONTINGENCY COEFE 0.08 PHI MAX 0 .8 6

147 Appendix V. An e x a m p 1e o f C h i -squared test on G.pallida STW+PD on SIH c y s t s . b ) PRD+PD on SIH c y s t s . c ) PRD+PD on RCH c y s t s . d) STW+PD o n RCH c y s t s . a) PEARSON ‘ 5 CHI SQl/Aki. A . 2 0 * YULE’S Q P (PEARSON'S) 0 . 040 5 SE (Q) . y, <; ! 1—; • 2. YATES' CORRECTED CHI SO 2 . AH SE (HO: Q = n) ! . 0501- —0 1 P (YATES) 0.084.5 YULE'S Y LOG ODDS RATIO - 1 . 8 8 5 SE (Y) ■ • , ~ 5“ }-'-0 t SE (LOR) i. 120 SE (HO: Y = 0; J . 6 ^5 t.“ '• •• - SE (H u: LOR = ()) - .2 0 1 E -0 1 C MAN v i. 1 3 CROSS PRODUCT RATIO 1 .5 1 8 E -0 1 PHI - 0 . 0 9 CONTINGENCY COEFF 0 .0 9 PHI MAX - 0 . 12 b) PEARSON•S CHI SQUARE 1 .OQ YULE’S 0 P (PEARSON'S) ' 0 .2 9 6 4 SE (Q) 6 . 1 4 6 p -0 2 YATES’ CORRECTED CHI SQ 0 .6 1 SE (HO: Q = 0) 6 . 8 9 3 E -0 2 P (YATES) 0 .4 3 4 4 YULE' S Y -i,1. ! n LOG ODDS RATIO - 5 . 5 1 7 E -0 1 SE (Y) i .7 1 9 E -0 2 SE (LOR) 2 .8 5 7 E - 0 1 SE (HO: Y = 0) 1 . 7 2 3 E -0 2 SE (HO: LOP = 0) 2 .7 5 7 E -0 1 C MAX 0. 16 CROSS PRODUCT RATIO ■ >. 7 60 E - 0 1 PHI -0 .0 5 GO \ ! I NbE;U.) (.. (/E l: 0.05 PHI MAX -0 . 1:.:

C) PEARSON’ S CHI SQUARE 0 .0 2 YULE’ S Q -0 .0 2 P (PEARSON’ S) 0.9020 SE (Q) 3 . 835E-02 YATES’ CORRECTED CHI SQ 0 .0 0 SE (HO: 0 = 0) 3.041E -02 P (YATES) 1.0000 YULE’ S Y -0 .0 1 LOG ODDS RATIO -4 .8 2 7 E -0 2 SE (Y) 9 . 596E-03 SE (LOR) 1.536E-01 SE (HO: Y = 0) 9 . 604E-03 SE (HO: I OR = 0) 1 . 537E-01 C MAX 0 .2 7 CROSS PRODUCT RATIO 0.953 PHI -0 .0 1 CONTINGENCY COEFE 0.01 PHI VAX ~ 0 .28

d )

PEARSON’ S CHI SQUARE 8.99* YULE’S Q -0 .8 5 P (PEARSON'S) 0.0027 SE (Q) 2.221E -02 YATES' CORRECTED CHI SQ 7 .38 SE (HO: Q = 0) 7.932E -02 P (YATES) 0.0066 YULE'S Y -0 .5 5 LOG ODDS RATIO -2 .4 8 0 SE (Y) .3.31oe —or: St (LOR) 1.093 SE (HO: Y = 0) 1 .983E-02 SE (HO: LOi\ = 0 j 3.I73E-0I C MAX 0 .1 " CROSS PRODUCT RATIO 8 . 372E-02 PHI - 0 .1 4 CONTINGENCY COEFF 0 .1 4 PHI MAX - 0 . 1 "

148 Appendix W.

Electrophoresis.

Biochemical studies showed that there are differences between dormant and nondormant cysts in their lipid contents (Storey, 1984), but the differences are very small such that one and four-year old dormant cysts cannot be distinguished. This difficulty may be overcome by molecular studies using electrophoresis. Electropho­ retic separation of proteins and enzymes can make a large contribution to biochemi­ cal and physiological studies, which may possibly include distinguishing diapausing and nondiapausing cysts. However, current uses are limited to distinguishing species and pathotypes (Trudgill and Carpenter, 1971; Fox and Atkinson, 1984; Bakker and Bouwman-Smits, 1988a,b and Bakkeret al ., 1988). The versatility of electrophoresis are shown following iso-electric focussing (IEF) of potato cyst nematodes (PCN’s), where 40 protein bands and 23 enzymes were detected in the eggs of the PCN’s (Fox and Atkinson, 1984). But only one protein band and two enzymes exhibited interspecific variation. In femalesG.rostochien­ of sis and G.pallida, 245 polypeptides have been identified with two-dimensional gel electrophoresis (2-DGE), and the two species were distinguished by 70% of their polypeptides (Bakker and Bouwman-Smits, 1988b). In second stage juveniles, four major native protein bands were found to be specificG.rostochiensis to and five to G.pallida. While with thermostable proteins, there were only three discriminating bands between the two species (Bakkeret al ., 1988). The aim of the experiments in this section are to carry preliminary investigations into the behavioural and physiological responsesG.rostochiensis of and G.pallida. By using electrophoresis to determine differences between hatched juveniles, sterilized tap water (STW) soaked cysts, potato root diffusate (PRD) stimulated cysts and dormant cysts.

Materials and Methods.

Approximately 10,000 second stage juveniles, 50 STW-soaked cysts, 50 STW-presoaked cysts stimulated with PRD for 24 hours and 50 dormant cysts (from Chapter 3) ofG.rostochiensis and G.pallida were separately homogenised in a 1.5ml Biomedix microhomogeniser attached to an electric motor. Each sample was homogenised in 200m/ lOmM tris-HCl pH 7.4, 5% (v\v) 2-mercaptoethanol. Homogenates were centrifuged for 10 minutes at 11600g, the supernatant was then saturated with 213mg urea (Bakker and Bouwman-Smits, 1988b) and stored at -70°C

149 until required. Before the start of electrophoresis, samples were freeze dried and redissolved in1 0 0 m/ of distilled water. 20ul of each sample was then loaded onto the cathode of a commercially prepared gel (LKB Bromma Ampholine PAGplate pH 3.5-9.5) and run for 1 hour 30 minutes at 2000V , 25mA, 25W on a LKB Bromma Multiphor system. At the end of the running time, the gel was removed and fixed in a fixing solution (57.5g Trichloroacetic acid and 17.25g Sulphosalicylic acid made up to 500ml with distilled water) and then stained in a Bio-rad silver staining kit according to the manufacturers instructions. Destained gels were scanned with LKB Bromma Ultroscan X L Enhanced Laser Densitometer according to the manufacturers instructions.

R esults.

The results of iso-electric focussing of proteins from juveniles and cysts of G.rostochiensis and G.pallida are shown in Fig. 5.2 and 5.3. Differences in protein composition can be detected between the juvenilesG.rostochiensis of (B) and G.pallida (A), the former is distinguished by the bands B1 and B2, while the latter by the bands A l, A2 and A3. STW-soaked cysts G.rostochiensis of (D) differ from PRD-stimulated (F) and dormant cysts (H) by the band(s) Dl. However, there are no bands distinguishing between PRD-stimulated and dormant cysts. G.pallida In , STW-soaked cysts (C) differ from PRD-stimulated (E) and dormant cysts (G) by the bands C l, C2 and C3, but there were no distinguishing bands between PRD-stimu­ lated and dormant cysts.

Discussion.

The absence of any differences in protein composition between actively hatching PRD-stimulated cysts and dormant cysts was surprising. This may be because the electrophoretic system in this work was not good enough or the physiological changes were so small that they are not picked up during the electrophoresis. Bakker and Bouwman-Smits (1988b) reported that qualitative protein composition of the various developmental stages of cyst nematodes are constant and are not influenced by physiological stages or host genotypes. Results from electrophoresis, like most techniques have their shortcomings and should be treated with caution. Bakkeret al. (1988) reported that repeated experiments gave large variations in intensities of most of the species-specific protein bands and no

150 Fig. 5.2 A polyacrylamide gel (Ampholine PAGplate) showing isoelectric points of proteins of second stage juveniles G.pallida of (A) and G.rostochiensis (B), STW soaked cysts of G.pallida (C) and G.rostochiensis (D), PRD stimulated cysts of G.pallida (E) and G.rostochiensis (F), diapaused cysts ofG.pallida (G) and G.rostochiensis (H) and standard (S). 152 Fig. 5.3 Densitometer scan of the polyacrylamide gel (Fig. 5.2) showing distinguishing peaks of proteins of second stage juveniles G.pallida of (A) and G.rostochiensis (B), STW soaked cysts ofG.pallida (C) and G.rostochiensis (D), PRD stimulated cysts ofG.pallida (E) and G.rostochiensis (F), diapaused cysts of G.pallida (G) and G.rostochiensis (H).

Key: V Distinguishing peaks between juveniles. ▼ Distinguishing peaks between cysts. PH 9-51 n r |PH 3-5 CD o 154 ULL LU 0 consistent intraspecific variation was detected with one-dimensional electrophoresis. In this present work, five gels were run with the same protein extracts and the results were all consistent with the gel shown in Fig. 5.2. In this present work, three protein bands were found to be specificG.pallida to second stage juveniles and two toG.rostochiensis. In STW-soaked cysts of G.pallida, there were three protein bands distinguishing it from PRD-stimulated cysts and dormant cysts. While in STW-soaked cysts ofG.rostochiensis , there was only one protein band distinguishing it from PRD-stimulated and dormant cysts. In general, there were fewer protein bands in this work than in those reported by other workers mentioned elsewhere, and the separations and resolutions of the gel were also not good. These problems may possibly be due to the extraction technique used, even though during homogenisation complete breakage of materials was confirmed under the microscope. Another reason may be that the gel was over loaded as a result of the samples being applied as freeze-dried extracts. However, further dilution of samples with distilled water gave poorer separation and resolution. It would have been desirable to adjust some of the electrophoretic techniques to suit the purpose of this work, this proved difficult due to the limited period of time when the equipment was available. The differences in protein bands between STW-soaked cysts and PRD-stimu­ lated cysts are very interesting, and may support the suggestion that softening of the egg-shell during the hatching process involve enzymes which are activated by the hatching factor (Perry and Clarke, 1981). These differences, in this present work, are distinguished more inG.pallida than in G.rostochiensis. Changes associated with the hatching process upon stimulation by PRD include: increased oxygen consumption and decreased adenylate energy charge (Atkinson and Ballantyne, 1977a and b), changes in cAMP levels and increased activity in the nucleolus of the pharyngeal gland’s nucleus (Atkinson et al., 1987 and Perryet al., 1989), and increased metabolism indicated by depletion of lipid reserves (Robinsonet al., 1985). All these suggest significant physiological changes and the differences in the protein bands may be indications of these changes. Though the results of the electrophoresis in this preliminary work failed to conclusively show differences between cysts in diapause and non diapause for reasons mentioned elsewhere, the way forward will require such studies in order to understand the molecular basis of dormancy. Such studies may provide a more conclusive evidence of diapause in bothG.rostochiensis and G.pallida, as most behavioural studies such as hatching tests are compounded with unavoidable errors.

155