STUDIES ON THE ECOLOGY OF TRICHODQRUS VIRULIFERUS
HOOPER, 1963 (NEMATODA: TRICHODORIDAE)
by
Keith Oohn Derrett, B.Sc.
March 1983
A thesis submitted for the degree of Doctor of Philosophy of the University of London and for the Diploma of Membership of the Imperial College
East Mailing Research Station Maidstone Kent 1
ABSTRACT
The attraction of Trichodorus viruliferus towards apple roots and carbon dioxide (CO^) was studied on agar plates. A single nematode, apparently attracted towards a CO^ source, demonstrated two types of movement which produced either "sinusoidal" or "broad tracks" on the agar surface. The use of agar plates was limited because J. viruliferus was prone to physical damage during handling and/or inoculation.
A simple model, closer to the actual soil system, was developed where the attraction of nematodes towards roots was studied in a
"soil-box". Using this system it was possible to show that:
• Nematodes were positively attracted towards apple
plant roots and if nematodes were inoculated into the
soil at increasing distances from a plant, the
percentage of nematodes activated into movement
declined. This was interpreted as J_. viruliferus
existing in one of two activity states, either an
"attracted" state (stimulated by plant attractant),
or a Sluggish, non-attracted" state. The existence
of this sluggish state was presumed to be an energy-
conserving mechanism for use in the absence of host
plants.
• In fallow soil nematodes did not move far from the
inoculation point. This was assumed to result from a
lack of stimulation by plant-root exudates.
• When nematodes were inoculated between two plants
some nematodes moved towards each plant.
e Partially sterilized soil, in the soil-box, interfered
with the attraction phenomenon.
• Further analyses of data were possible and these 2
demonstrated that in T_. viruliferus males and females
moved towards a plant at similar rates; adults moved
faster than larvae and that nematodes with the greater
quantity of food reserves were more active - see
appendix 6.3.
The energy-conserving mechanism was investigated in greater depth: i* viruliferus was found capable of surviving at least 25 months without plants. Nematode food reserves were slowly catabolized during storage but the rate was increased in soil previously heated to 70°C. Starved nematodes could replenish food reserves by feeding on host plants, but not if stored in plant-free, field soil. Survival time was apparently affected by soil micro organisms. Five hypotheses were proposed including those of other workers to explain this latter phenomenon. These were critically examined in light of experimental results. It was considered most likely that chemicals, unfavourable to nematode survival, were produced by thermoduric microorganisms or possibly that certain soil microorganisms were producing chemicals which induced nematode quiescence.
The possibility of _T. viruliferus utilizing microorganisms as a secondary food source was considered unlikely. ACKNOWLEDGEMENTS
I gratefully acknowledge the studentship given to me by the
Agricultural Research Council which enabled this study to be made.
My sincere thanks are due to Drs 3.3.M. Flegg and D.G. McNamara who gave much assistance, both practical and academic, during this study and offered great encouragement in times of difficulty.
I am grateful to many members of the staff at East Mailing Research Station for assistance, in particular to Mr G.P. Barlow for statistical advice; to Miss 3. Parsons and Mrs P. Edwards for help in the production of figures and photographs; to Mrs D. Cookson and Mr D. Richardson for typing and printing this thesis and to the Director, Dr A.F. Posnette and his successor Dr 1.3. Graham-Bryce for allowing me to use the facilities of the research station.
I would also like to thank Dr D.3. Wright, my University Super- visor, and Dr A.A.F. Evans for their continued interest, consideration and assistance.
Finally, I would like to record my greatest thanks to the late
Dr R.S. Pitcher for initiating my interest in nematology and for being instrumental in arranging for me to carry out this study. His help will be remembered always. 106 4
CONTENTS Page
Abstract 1 Acknowledgements 3
1. Introduction 10
2. Attraction of' T. viruliferus to apple plants
A. Experiments on agar 19
2 • A • 1 • Introduction 19
2.A.2. Materials and methods 21
2. A. 3. Attraction to apple roots 24
2.A. Attraction to carbon dioxide 27
B. Experiments in soil-boxes 30
2.B.I. Intrdduction 30
2.B.2. Materials and methods 31
i Nematodes 31
ii Plants 31
iii Soil 32
iv Soil-boxes 32 2.B.3. "Pitcher Model" of T. viru- liferus attraction 37 2.B .4. Effect of different inoculation distances 41 2.B.5. Movement of nematodes in absence of plant 47
2.B.6. Effect of inoculating between two plants in a soil-box 49
2.B.7. Effect of activated charcoal in soil-box 51
2.B.8. Effect of partial sterilization of soil in a soil-box 54 107 Page
3. Survival of X* viruliferus in plant-free soil 60
3.1. Introduction 60
3.2. Materials and methods 64
i Nematodes 64
ii Soil 64 iii Pot experiments 64
iv Optical density estimation 65
3.3. Survival of JT. viruliferus stored 67 in a polythene sack of soil
3.4. Comparison of the survivability of pale and dark nematodes 70
3.5. Effect of soil heating on nematode survivability 72
3.6. Investigation of the temperature required to destroy survival factor 76
3.7. Investigation of the ability of X* viruliferus to regain stored food reserves after feeding 79 3.8. Investigation of the ability of i- viruliferus to regain food reserves in field soil 80
3.9. Distribution of J. viruliferus with depth 81
3.10. Discussion of results 84
4. General Discussion 95
5. References 112
6. Appendices 126
6.1. Extraction efficiency and handling deaths of J. viruliferus 126
6.2. Taxonomic authors of organisms cited in text 129 107 Page
6.3. Other ecological data 132
i Sex ratio with distance 133
ii Ratio of larvae to adults with distance 135
iii Nematode motility in relation- ship to stored food reserves 138
6./L Raw data Inside back cover 109 7
LIST OF PLATES
Page
Plate 1. Aggregation of _T. viruliferus at an apple root-tip feeding site 11
Plate 2. Response of _T. viruliferus to handling 22 Plate 3. The method of applying carbon dioxide to the surface of agar plates 26 Plate Soil^-box system with apple plant iji situ 33 Plate 5. Colour-score indexing system for X* viruliferus 66 Plate 6. Female T\ viruliferus showing three developing oocytes within the reproductive tract. 68 110 8
LIST OF FIGURES
Page
Figure 1. Tracks produced by a single nematode on the surface of agar 28
Figure 2. Movement of viruliferus in response to an apple plant 38
Figure 3. Effect of inoculation distance on the movement of J. viruliferus towards an apple plant (non-replicated experiment) 42
Figure 4. Effect of inoculation distance on the movement of T_« viruliferus towards an apple plant (replicated experiment) 44
Figure 5. Movement of J. viruliferus in the absence of a plant 48
Figure 6. Position of heated soil bands in soil-boxes 56
Figure 7. Effect of bands of heated soil on the movement of T. viruliferus towards an apple plant 58 Figure 8. The survivability indices for batches of J. viruliferus stored in either heated or unheated soil for different time intervals 73
Figure 9. Effect on percentage nematode survival of heating soil to different temperatures 77
Figure 10. The variation in percentage of larvae with distance from the inoculation point 136
Figure 11. Mean colour scores of X* viruliferus plotted against distance from inoculation point 139 Ill 9
LIST OF TABLES
Page
Table 1. Movement of nematodes towards or away from a plant in a soil-box experiment designed to test the Pitcher Theory 39 Table 2. Percentage of nematodes "activated" when inoculated at different distances from a plant 45 Table 3. Results from the effect of inoculating nematodes between two plants 49 Table 4. Distribution of nematodes towards a plant when inoculated into activated charcoal/ soil medium 52 Table 5. Differences in the movement of nematodes towards a plant in heated and unheated soil 54 Table 6. Differences in the movement of nematodes when bands of heated soil were set into soil-boxes of unheated soil 57 Table 7. Length of nematode survival in plant-free soil 61 Table 8. The numbers and population structure of J. viruliferus stored in a polythene sack of soil for 25 months 67 Table 9. The survivability of pale and dark ]_. viru- liferus stored in plant-free conditions for six weeks 70 Table 10. The effect of nematode survival in heated or unheated soil after three periods of storage 74 Table 11. Ability of J. viruliferus to survive in soil from different depths compared to natural distribution in relation to depth 82 Table 12. Experimental findings on extraction efficiency and effect of mortality factors in pot experiments 127 Table 13. The mean cumulative percentages of dark and paler nematodes for different time periods and the level of significance between the emergence of the two classes 141 1 INTRODUCTION
Trichodorus viruliferus* is an obligate plant-parasitic nematode
(Hooper, 1976) which causes direct damage to many agricultural crops, e.g. Docking Disorder of sugarbeet (Whitehead & Hooper, 1970) and causing 'stubby-root' symptoms (Christie & Perry, 1951) on cereals
(Hooper, 1963), peas (Gibbs & Harrison, 1964) and apple (Pitcher &
Flegg, 1965). As a vector this nematode can transmit pea early browning virus (Hooper, 1963) and tobacco rattle virus (van Hoof,
1968).
In a root-observation laboratory (Rogers & Head, 1963) at East
Mailing Research Station, Kent, _T. viruliferus was observed to concen-
trate its attacks on one type of apple root (Pitcher & Flegg, 1965).
These roots, which were termed 'white extending roots', ranged In
diameter from 0.5-2.5 mm and grew quickly when young (Pitcher, 1967).
Such roots were few in number and most of the visible apple roots were
fine feeder-roots which were found to be unimportant for nematode
feeding. Pitcher observed the development of nematode attack and found
that nematodes congregated 1-3 mm behind the apical meristem, in the
zone of elongation, to form aggregations of over 100 individuals within a
short time (4-20 days). Superficial yellowing occurred on the root
surface which became brown after a further 3-10 days, at a time when
epidermal cracks were observed extending into the outer cortex. A
marked decline in the growth of the root usually occurred at this stage,
accompanied by a fall in nematode numbers and a tendency for the attack
to be transferred to the apical meristem, hitherto virtually unscathed.
Nematodes dispersed into the soil once root growth had ceased.
*A11 taxonomic authorities are listed in Appendix 2. Plate 1. Aggregation of ]_. viruliferus at an apple root-tip feeding site. (R.S. Pitcher) Pitcher (1967) suggested that the aggregations of _T. viruliferus could have resulted from reproduction at the feeding site, although he expressed some doubts because this would have required a faster breeding rate than that normal for most plant-parasitic species. Subsequently,
Pitcher & McNamara (1970) investigated the breeding biology of ]_. viruliferus and found that, although the root feeding sites were the most important for reproduction, the rate of reproduction was not sufficient to account for the observed aggregation, i.e. reproduction followed aggregations, rather than causing them.
Pitcher (1967) suggested that an alternative explanation for aggregation was that nematodes moved towards root-tip feeding sites in response to an attractant stimulus produced by that root. He proposed a theory, subsequently referred to as the 'Pitcher Theory", to account for the numbers of _T. viruliferus observed in an aggregation, in which all of the nematodes surrounding a root-tip, to a radius of 8 cm, were attracted towards the centre. Time-lapse cinematographic films were made of the development of attack of apple roots by T. viruliferus (Pitcher &
McNamara, 1971) and these supported the theory of attraction.
. Seven species of Trichodorus and Paratrichodorus have been found to form aggregations at feeding sites (Christie & Perry, 1951; Anon., 1967;
Whitehead, 1967; and Whitehead & Hooper, 1970). Any understanding of the aggregation phenomenon could therefore be significant to all these species.
At the time of Pitcher's studies of J_. viruliferus on apple, the attractiveness of plants to some plant-parasitic nematodes had long been known, e.g. Stewart (1921), Steiner (1925) and Linford (1939), yet the maximum distance to which attraction had been shown to occur was only
4.5 cm in sand for Heterodera schachtii (Wallace, 1958). However, Luc
(1961) had shown that chemicals in millet soil could affect movement of Hemicycliophora paradoxa up to 40 cm away. Only recently have nematodes been shown to respond to plants at greater distances in soil: 25 cm by
Trichodorus proximus and T. christiei (Harrison, 1975 and Harrison &
Smart, 1975) and 75 cm for Heloidogyne spp (Prot, 1978b).
KUhn (1959) believed that orientation towards roots was not an active process in which nematodes sensed and followed chemical concen- tration gradients in soil towards a plant. He suggested that location of host plants was a random process, and that on contact with a root surface, nematodes remained on that root. The 'random movement hypo- thesis' was supported by Sandstedt et al. (1961), Sandstedt & Schuster
(1962) and Schuster & Sandstedt (1962). It was suggested by Rohde
(1960) that carbon dioxide concentrations greater than those of the atmosphere inhibited nematode movement, and that as nematodes reached the immediate vicinity of the root they would become inactive. This mechanism (attraction occurring through locomotory inhibition) supported
KUhn's beliefs. However, Bird (196Z) reanalysed KUhn's results and concluded that they provided evidence against the random movement hypothesis.
Some of the most concrete evidence to show that attraction occurs is that of Blake (1962), who presented diagrams of tracks made by
Ditylenchus dipsaci on agar moving towards oat seedlings. Since
Blake's work, the positive attraction of nematodes to the vicinity of plant roots has rarely been disputed. However, host plants were not found to be attractive to schachtii (Bergman & van Duuren, 1959b) whereas the bacteria collected from the rhizosphere of these plants were
(Bergman & van Duuren, 1959a) and it was suggested that chemicals secreted by bacteria in the rhizosphere surrounding the roots were the real attractive agents. This is not necessarily true for all nematode/ plant attractions because some nematodes are attracted to plants growing in sterile systems where no rhizosphere bacteria are present (Azmi &
CJairajpuri, 1977; Prot, 1975 and Wyss, 1977). Fungal infections of roots can also influence their attractiveness, e.g. Fusarium oxysporum- infected alfalfa roots are more attractive to Pratylenchus penetrans than uninfected roots (Edmunds & Mai, 1966 and 1967), whereas barley roots infected with Gaeumannomyces graminis are less attractive to Heterodera avenae (Cook, 1975).
Nematodes are not unique in their ability to respond to stimuli produced by plant roots. Motile zoospores have been shown capable of responding to a root attractant, e.g. Phytophthora cinnamoni (Zentmeyer,
1960 and 1961) and Pythium aphanidermatum (Royle & Hickman, 1964a,
1964b and Kraft & Endo, 1966) and so have some species of rhizophagous insects, e.g. wireworms (Thorpe et al_., 1947 and Doane et al., 1975) and collembola (Klingler, 1958).
Nematodes are attracted and/or repelled by a variety of agents, not just host plants (Gofman-Kadoshnikov et al., 1955). Indeed, nema- todes can be attracted to non-hosts (Dickinson, 1959 and Lownsbery &
Viglierchio, 1961) as well as to a variety of chemicals, but in concen- trations unlikely to be encountered naturally in soil (Clapham, 1931;
Taniguchi, 1933; Ibrahim & Hollis, 19b7 and Prot, 1978a). However, the attractiveness of rice varieties to Aphelenchoides besseyi has been shown to be positively correlated with host susceptibility (Goto &
Fukatsu, 1956; Lee, 1973 and Lee & Evans, 1973). Alby & Russel
(1975) also found a weak correlation between host attractiveness and host susceptibility for Helicotylenchus di gonicus. No such corre- lation was found for Meliodoqyne hapla (Viglierchio, 1961), Hemicyclio- phora similis (Khera & Zuckerman, 1963) or D. dipsaci (Griffin, 1969 and Griffin & Waite, 1971). In most instances the attractant produced by a plant appears to be not very specific and attraction could result from the combined effects of several chemicals.
Some evidence has accumulated to suggest that the attractant stimulus is chemical in nature. This has been demonstrated in experi- ments where plants were grown for a period in sand or soil and then removed. Nematodes added to the substrate, after the removal of the plant, were able to orientate themselves to the point where the plant previously was growing (Wallace, 1958). Nematodes will also positively orientate towards root diffusate (Bird, 1959) collected in water
(Weischer, 1959 and Lavallee & Rohde, 1962). It is not clearly under- stood whether nematodes respond to a concentration gradient of a stimu- latory chemical or to an oxidising/reducing chemical gradient (Bird,
1959). While nematodes have also been demonstrated to respond to physical stimuli, e.g. thermotaxis (Bird & Wallace, 1965) and galvano- taxis Pones, 1960) these are considered by many to be of secondary importance in root attraction by nematodes.
Some attractants have been shown to pass through a dialysis mem- brane (Lownsbery & Viglierchio, 1958; Lownsbery & Viglierchio, 1960 and Viglierchio, 1961) i.e. attraction towards the plants still occurred if a membrane was positioned between plant and nematodes. Such sub- stances have been shown to be relatively non-polar and to have a low molecular weight (Viglierchio, 1961 and Lownsbery & Viglierchio, 1961).
The greatest concentration of attractant was found to be produced in the elongating zone of tomato root (Wieser, 1955) i.e. the most meta- bolically active region of a growing root, and at the origins of the lateral roots in tomato (Widdowson et al., 1958) and sugar beet (Kiimpfe,
1960).
These features of the attractant, along with the knowledge that it can be non-specific, led Klingler (1965) to emphasize the importance of carbon dioxide ((XL) as an attractant agent for plant-parasitic nematodes. A number of species of nematodes have been shown to respond positively
to C02, e.g. D. dipsaci P. penetrans (Klingler,
1972), M. javanica and H. schachtii (Bird, 1960). Other inverte- brates have also been found to orientate successfully to a CO^ source, e.g. Collembola - Sinus coeca (Klingler, 1975), Diptera - Psila rosae
(Stadler, 1971) and various arthropods (Moursi, 1962). Gadd & Loos
(1941) observed that injured parts of a root were the most attractive to nematodes and Croll (1970) suggests that this might be explained by the injured parts of plants producing an increased amount of CO2.
Besides C^, other plant-produced chemicals have been shown attractive for some nematodes, e.g. amino acids (Bird, 1959 and Oteifa
& Elgindi, 1961). Several varieties of rice were shown to differ in their attractiveness to A. besseyi and, because of the experimental conditions, Lee & Evans (1973) were able to show that this was due to subtle differences in root exudates rather than by differences in CO^ production. Other results such as these have led some workers to hypothesize that C0^ may be important as an attractant over long dis- tances, but with other chemicals becoming more important as nematodes approach a root (Blake, 1962 and Klingler, 1965).
The attractiveness of plant roots growing under different environ- mental conditions have been compared one with another (Gillard & van den
Brande, 1956; Moussa, 1971 and Moussa et al., 1972) and healthy plants compared with diseased ones (Rebois & Feldmesser, 1966). All these results support the theory of Wieser (1955), that the attractiveness of roots is dependent upon their rate of growth, i.e. the physiological activity of roots. Increasing concentrations of root exudates have been found to increase not only the attractiveness of that root but the rate of nematode movement as well (Weischer, 1959). In fact, in some
species of nematode no movement is detected in the absence of a host 10 17
plant (Endo, 1959; Luc et al., 1969 and Alby & Russel, 1975).
Differences in rates of movement have also been shown to exist
between juveniles and adult nematodes of the same species. Azmi &
Oairajpuri (1976) believed this to result from differences in "percept-
ual ability or agility" in Hoplolaimus indicus and Helicotylenchus
indicus. This situation is apparently more complex in Pratylenchus
scribneri. In the absence of a host plant Black & Van Gundy (Van
Gundy, 1965) found that £. scribneri larvae moved less than adults but
when stimulated by a plant root exudate jjveniles moved faster than
adults.
Other aspects of nematode attraction to plants that have been
investigated include the effect of nematicides on attraction. Tomato
plants which were treated with aldicarb were more attractive to Globo-
dera rostochiensis than untreated plants (de Grisse & Moussa, 1971).
Other nematicides were not found to have the same effect (Moussa & de
Grisse, 1972) and in contrast, some nematicides can reduce attraction,
e.g. 2,Dichloropheny 1 methanesulphonate (McBeth et al., 1964), ForVVv^rv*®*"*-
oxamyl appears to prevent orientation of Meloidogyne incognita juveniles
to cucumber foots (Wright et al., 1980).
An interesting observation was made by Loewenberg et al. (1960) on
the attraction of M. incognita towards mustard seedlings on agar. On
plain agar medium, M. incognita did not appear to be able to locate
mustard roots, but in a medium containing sucrose or cellobiose they
could. The authors suggested that sugars may have induced the plants
to excrete an attractant or counteracted a compound that prevented
nematodes from locating the host, or that the juveniles utilized sugars
to overcome this latter compound. It is also possible that there
could have been an interaction with the rhizosphere bacteria of the mustard seedlings effecting the attraction response. 18
It was against this background, and in the knowledge of its relatively spectacular ability to form large aggregations at feeding sites in a short time, that the attraction of _T. viruliferus to apple plants was studied.
As a second part to this thesis a number of experiments were carried out to investigate the ability of J. viruliferus to survive"in plant-free soil. These were conducted to compare data obtained from a related species of nematode, Xiphinema diversicaudatum (McNamara &
Derrett, 1976, and McNamara, 1980). Neither of these two species have specific resistant life stages yet X.* diversicaudatum had been shown capable of surviving in plant-free conditions for at least 47 months
(Pitcher & McNamara, 1977). This ability to survive, however, was greatly reduced if the soil had been heated to 70° for one hour prior to the addition of nematodes. McNamara (1980) suggested that diversi- caudatum was most probably interacting with a component of the soil microflora and proposed an hypothesis that some microbes, the "survival factor", represented an alternative food source and/or an agent which could induce nematode quiescence. The nature of this interaction was tested further with viruliferus in this investigation.
Note
The experimental data within this thesis are presented in two distinct formats which reflect differences in experimental approach.
The first part of the investigation (Chapter 2) involved discrete experi- ments in chronological sequence. The experiments did not overlap and one experiment could be planned in the light of a previous one. Thus it seems most relevant to discuss each experiment in turn. In the second part (Chapter 3) a number of long-term investigations were carried out on
T* viruliferus concurrently and here all experimental data are considered in one discussion. 2.A. EXPERIMENTS ON AGAR 2.A.1 INTRODUCTION
Widdowson, Doncaster & Fenwick (1958) first used agar plates as a substrate on which to study the movements of a nematode (Heterodera rostochiensis) towards a host plant. Other workers have since used agar to study the phenomenon of attraction in a wide range of nematode genera. The attraction of Trichodorus spp, however, had not been investigated on this medium although Wyss (1971b) used agar in his studies of the feeding behaviour of _T. similj s.
In attraction studies the advantages of using agar are three- fold: • Results can be obtained quickly, usually within hours. • Nematodes can be observed directly throughout the course of
the experiment and their tracks can be easily observed.
• The experimental system is simpler than soil because of
interactions with soil microorganisms and complex physico-
chemical systems are absent.
The disadvantage of using agar is:
• The system may sometimes be too simple and results may not
be indicative of the situation occurring in soil.
There is much to be learnt from agar plate studies, but any conclusions extrapolated to cover attraction in soil need carefully designed follow-up experiments in soil to validate them.
Using agar as a medium Klingler (1959) was able to demonstrate
that Ditylenchus dipsaci moved towards a carbon dioxide source which
produced gas at a similar rate to a growing root (c. 2 ml/hour).
From this observation he concluded that carbon dioxide might be one of
the most important factors in attraction of nematodes to plants. Other plant-parasitic nematodes were also shown to be attracted towards a carbon dioxide source, including Meloidogyne javanica, Heterodera schachtii, Pratylenchus minyus (Bird, 1960), M. hapla, M. javanica
Oohnson & Viglierchio, 1961), P. penetrans (Edmunds & Mai, 1967) and
Aphelenchoides fragariae (Klingler, 1970). 21 47
2.A.2. MATERIALS & METHODS
Trichodorus spp are notorious for their sensitivity to various
chemicals, especially metal ions (Van Gundy & Thomason, 1962) and to
handling (Winfield & Cooke, 1975). Thus first consideration was given
to finding a suitable agar, and to inoculation techniques.
The agar selected for these experiments was that available with
the lowest copper levels, "Agar No. 1" (Oxoid Ltd, London). Although
this contains 1 ppm copper, the metal is bound in complex organic mole-
cules and considered harmless.
Various agar concentrations were tested to determine the gel
density most suitable for nematode movement. Very wet agars (0.5-0.8%)
allowed nematodes to move within the agar rather than on the surface,
whereas concentrations in excess of 2% apparently provided too little
surface moisture for movement. Optimum agar concentration lay between
1.3 and 1.5%, and movement seemed greater if the plate was exposed to
the air for one hour before use to allow some surplus surface moisture
to evaporate.
One of the major problems in using agar plates was to inoculate
nematodes successfully on to the agar surface. At first, nematodes
were picked out of water suspension with a pointed quill and placed
into a drop of water on the agar surface. This caused high nematode
mortality. The nematodes first became short and thickened, and the
cuticle appeared loose; subsequently they became inactive (see plate
2). Similar symptoms were observed at other times if nematodes were
damaged mechanically or encountered unfavourable circumstances, e.g.
immersion in anaesthetizing liquid (Ellenby & Smith, 196^).
Other methods were tested in an attempt to overcome this hand-
ling sensitivity. Neither sucking up nematodes from water suspension 22
(b)
Plate 2. Response of J. viruliferus to handling (a) "short and thickened" form (b) normal appearance 47 23
in a fine capillary, which does not harm T_. similis (Wyss, pers. comm.)
nor using Ford's continuous-suction apparatus (Ford, 1957) was success-
ful. The most satisfactory method was to use a normal Pasteur pipette
with a rubber teat. Nevertheless a high degree of inactivation
remained despite the care used in the inoculation, with most nematodes
remaining at the inoculation site once the water drop had dispersed.
In only a few instances did nematodes move any distance, but when
movement occurred, it could involve long distances, e.g. 25 cm in 72
hrs.
Many techniques have been reported for observing and recording
the tracks made by nematodes moving on the agar surface. By project-
ing light through the plate the tracks can be copied on to tracing
paper (Lee, 1973) or photographic film (Ward, 1973). Sandstedt,
Sullivan & Schuster (1961) found that if the agar was allowed to dry
out for one week then the tracks could be more easily photographed.
Other techniques were investigated (Green, 1966b; Kunz & Klingler,
1976 and Prot, 1976) but were considered to be unnecessarily compli-
cated for the present purposes. A modified form of Rode & Staar's
(1961) technique was ultimately selected in which a vertical beam of
light was projected on to the agar surface, via a split prism, to give
an image of dark lines (nematode tracks) on a pale background (agar
surface), when observed through a binocular microscope. 2.A.3. ATTRACTION TO APPLE ROOTS
The In vitro attraction of T. viruliferus towards apple plants was investigated using three different techniques:
(1) A well (diameter 0.5 cm) was cut into the agar and filled
with either
(1) macerated apple roots, or
(ii) root exudate (extracted according to Bird, 1959).
(2) A small plant was set into the agar on one side of the plate (Azmi & Oairajpuri, 1977).
Ten nematodes were added to plates of 1.5% agar, inoculated
1 or 2 cm from the source of possible attractant, and examined over a period up to 48 hours. Five replicates were used for each treatment.
No nematodes moved towards the apple seedlings or apple-root preparations in any of the replicates. In some replicates 1 or 2 nematodes moved in what appeared to be a random manner away from the inoculation point, often moving towards the edge of the plate.
From this evidence no positive conclusions could be drawn on the movement of J. viruliferus towards root material. One suggestion to explain these results is that the root macerations may have contained chemicals noxious to nematodes, such as phenolic compounds. The role of these compounds in plant roots is not fully understood but it is thought possible that they may be responsible for disinfecting wounds in roots or in plant defence (Flegg, 1965) and hence are likely to have some nematicidal action. The root "exudate" Bird^ 1959) is not known to contain noxious chemicals, indeed, Bird found that Meloidogyne spp were attracted towards it when tested in agar. It was therefore assumed that the failure to obtain a positive attraction response in this experiment resulted from either basic experimental problems, such 25 47
as inoculation damage or unsuitability of the agar preparation, or else
an absence of an attraction stimulus by the plant root preparations.
Since carbon dioxide has been supported by a number of workers as being
the primary attraction source it was decided to investigate the response
of IT. viruliferus to this gas within the same experimental restraints in
an attempt to elucidate the results above. 26
Plate 3. The method of applying carbon dioxide to the surface of agar plates 2.A.ATTRACTION TO CARBON DIOXIDE
In order to study the effects of carbon dioxide on J. virulif- erus an electro-mechanically operated gas syringe was used to administer carbon dioxide on to the surface of an agar plate at a steady rate of
2 ml/hour. (See plate 3.) A series of agar plates was tested, using different distances between the gas source and the inoculation point to determine the optimal inoculation distance for the planned experiments.
At an inoculation distance of 1.7 cm an individual nematode produced the track in Fig. 1. In all other tests, at various inoculation distances, no nematode movement towards the source was observed.
The one nematode track leading towards the carbon dioxide source showed two distinct types of movement, normal "sinusoidal tracks", as observed in random wanderings, and "broad tracks", resulting from frequent head waving during the course of forward motion. Klingler (pers. comm.) reported that D. dipsaci also shows two distinct track forms, similar to J. viruliferus. He interpreted the broad tracks as those made during orientated movement, the frequent head waving aiding detection of a gradient of chemical attractant, and the sinusoidal movement as the tracks produced when either the nematode was moving in a random manner, or moving towards an attractant source, after having positively oriented its body to it. Another plant- parasitic species of nematode, Globodera rostochiensis, was reported to produce two distinct types of track on agar (Green, 1966a).
Greater significance was given to this result after carrying out experiments on nematode attraction in soil boxes (see section
2.B.4) because J. viruliferus appears to exist in two activity states and it may be that the two types of track are related to this.
Samoiloff et al. (1974) believe that Panagrellus redivivus adopt a
similar change in locomotory activity when males approach females. INOC POINT
(a) C02 SOURCE
(b) broad track
normal track
Figure 1. Tracks produced by a single nematode on the surface of agar (a) Sketch of track between the inoculation point and the carbon dioxide source, and (b) detailed differences in the two track forms. 47 29
This has been termed as "Samoiloff's two-state model of behaviour" by
Croll (1976).
Since only a single nematode appeared to respond positively to a
CO2 source this experimental series could be said to suggest that
i* viruliferus does not usually respond to CO^, in its attraction to plant
roots. However, this interpretation could only be made if all possible
artefacts of the experimental system were eliminated. Such factors as
agar type, light intensity or temperature could be important; the
extraction time for nematodes used in the experiment (T. Alphey, pers.
comm.) or the existence of volatile and noxious chemicals emanating from
the plastic petri dishes (Miller, 1978). Some of these variable were
investigated but the situation could not be resolved in the time
available.
As a result of the two experimental series in sections 2.A.3
and 2.A.it was considered that agar was not generally a suitable
medium on which to study the attraction response of _T. viruliferus. 2.B. EXPERIMENTS IN SOIL-BOXES
2.B.I. INTRODUCTION
Most of the difficulties in maintaining T_. viruliferus on the surface of agar were presumed to have arisen from mechanical damage to the nematode. The genus Trichodorus is particularly sensitive to physical damage (Bor & Kuiper, 1966) and also the genus' susceptibility to desiccation (Rttssner, 1971; Simons, 1973 and Wyss, 1970) make it a difficult subject to work with on an agar surface exposed to the air.
T. similis has been successfully studied on agar by Wyss (1971a and b) but here the nematodes were able to live within the agar rather than on its surface. Because of the unresolved difficulties in handling
X* viruliferus, and thus of obtaining reproduceable results on agar, further experimental work was carried out in soil. Considerable
variation in results was expected, because of the influence of other chemicals in the soil, but soil was a medium in which experiments with
T. viruliferus were known to be possible (Pitcher & McNamara, 1970). 10 31
2.B.2. MATERIALS & METHODS (i) Nematodes
X* viruliferus were obtained from a light loam soil on the
East Mailing Research Station, Kent (plot WE108), immediately under
the boles of a number of plum rootstock varieties. Nematodes were
collected here for the life-cycle studies of Pitcher & McNamara (1970).
X* viruliferus occurred in high numbers, 200 per 100 ml soil
(measured by the displacement of water), for much of the year with a
maximum in spring of up to 520 per 100 ml soil. J. viruliferus com-
prised c. 95% of the plant-parasitic nematodes present (some Tylenchor-
hynchus spp and Paratylenchus spp were present, too): no other species
of the genera Trichodorus or Paratrichodorus were found. To minimize
deaths due to physical damage (Winfield & Cooke, 1975), T. viruliferus
were not hand-picked but inoculated in a water suspension, along with
all the other nematodes extracted.
The nematodes were extracted using a Seinhorst Elutriator
(Seinhorst, 1962) running with a flow-rate of 17 ml per minute. A
maximum of 200 ml of soil could be elutriated in any one extraction,
any more soil than this caused blockages in the apparatus and hence
reduced the elutriation flow rate. The material collected from the
upper section of the elutriator was washed on a bank of three 53p
sieves and then suspended on plastic screens (Pitcher & Flegg, 1968) in
glass-distilled water contained within a glass petri-dish (Petri-dishes
provided better oxygenation for the nematodes compared to using a
Baerman Funnel (Baerman, 1917).) Nematodes were collected 20 hours
later after having crawled through the screen. No less than 200 nema-
todes were added to any soil-box.
(ii) Plants
The plants used in all experiments were apple seedlings, var. 47 32
Worcester Pearmain, germinated and grown under glasshouse conditions
(a temperature of 17-1.0°C with a 16-hour day-length maintained with
artificial lighting when necessary). Plants were used at the eight
true-leaf stage (3-4 months old).
(iii) Soil
Soil was collected from a fallow plot alongside the plum root-
stock nematode-source at East Mailing Research Station. Prior to use
the soil was air-dried at laboratory temperature for two days. The
dried soil was sieved through a 2 mm sieve and remoistened
This treatment of the soil was 100% effective in removing any viruliferus, checked by screening soil samples, and was a homogeneous medium, free from stones, suitable for use in small-scalt experiments.
(iv) Soil-boxes
A system was developed to study the attraction of J. viruliferus
to apple plants in soil. This was termed a "soil-box" and based on a
system used by Harrison (1975) to study the movement of _T. christiei and i T. proximus towards tomato plants. (See plate 4.)
The box, containing loosely packed soil, was a plastic gravel
tray (with no drainage holes) measuring 25 x 20 x 5 cm. Towards one
end of the box an apple seedling was planted, either in a bag made of
nylon bolting cloth (53p aperture) or, in later experiments, behind a
screen of the same cloth, supported on a wire frame. The bolting
cloth acted as a "root-screen", to prevent roots penetrating it, but
allowed any chemicals produced by the plant to diffuse out, equally in
all directions.
The soil-box was sealed in a plastic bag, slit to allow the
foliar parts of the plant to project. This reduced the need for Plate Soil-box system with apple plant In situ NB Some soil has been removed to expose root-screen. 10 34
* watering the system. However, it was found necessary to add water to
the boxes from time to time, using a hypodermic needle and syringe, to
maintain the rhizosphere at a steady moisture level because of water
loss due to transpiration. The soil-box was kept in a constant temper-
ature growth chamber for two weeks at 17°C and with a 16-hour light and
8-hour dark cycle to allow the plant to establish itself in the soil.
Once the plant had become established nematodes were inoculated
into the soil at specific distances from it by cutting a hole into the
soil, c. 3 cm deep, with a 5 mm cork borer. A water suspension of
nematodes (containing _T. viruliferus) was poured into the hole and then
the hole filled with soil.
The soil-box was returned to the growth chamber and left for a
further 14 days unless specified otherwise. After this time the soil
within the box was assayed by cutting 1 cm sections across the box using
a shaped piece of metal, from the back of the box towards the plant,
the soil being carefully removed with a spatula. The soil sections
(c. 100 ml) were soaked in beakers of water for one hour before being
screened, using a modification of Flegg's (1967) technique. This
consisted of stirring the water to suspend soil particles and nematodes
and, after a settling period of 10 seconds (to allow heavier soil
particles to fall out of suspension), the supernatant was poured through
a bank of three 53jj sieves. More water was added to the beaker and the
process of stirring, settling and decanting repeated. After gently but
thoroughly cleaning the sample by washing on the bank of sieves, it was
placed on to a plastic screen (Pitcher & Flegg, 1968), suspended in a
Petri-dish filled with glass-distilled water. Twenty hours later the
nematodes were collected in water suspension and counted under a
binocular microscope (x 50).
*A minimal amount of water was added to a soil-box at one time in order to reduce the chances of upsetting any gradient of plant-produced nematode attractant which existed. The system was developed to approximate as closely as possible to the situation in soil, where J. viruliferus was originally observed
(Pitcher & Flegg, 1965 and Pitcher, 1967). Certain differences did occur, the most important of these being:
• Moisture content was not constant throughout the experi-
ment in proximity to the plant. Because of the watering
technique at times the moisture content was occasionally
less than the rest of the soil or occasionally greater.
Watering was carried out once every 24 hours to reduce the
amount of variation and to minimize the effect of nematodes
being attracted to an area of dry or wet soil. There was
no apparent correlation with increased/decreased nematode
attraction in soil-boxes which contained moister soil
throughout, compared to others. It was, therefore, con-
cluded that moisture content variation had little effect
on nematode attraction in \rWesw\ev\Vs.
• Nematodes were not being attracted towards a single root
but towards a plant root system. The roots which were
shown to be attractive to _T. viruliferus (white extending
roots) (Pitcher, 1967) and the seedling root system were
both centres of extreme metabolic activity, both producing
similar metabolic by-products. Because of the difficulty
in using living, white extending roots, a seedling root
system was considered to be an effective and convenient
substitute (Pitcher & McNamara, 1970).
• The soil-box was very shallow so nematodes were at the
most 5 cm below the soil surface whereas T. viruliferus in
soil is usually found at 10-40 cm (Richter, 1969). This 36
might allow a greater diffusion of plant-produced, volatile compounds to escape from a soil-box than in soil at a greater depth. Also, it was not possible for compounds to diffuse downwards into the soil (greater than 5 cm down).
Differences in chemical concentration gradients therefore probably exist in soil-boxes compared with in soil.
The moisture content of soil used in soil-box experiments was less than "field capacity" (maximum amount of water that can be held by freely draining soil). This might reduce the mobility of T_. viruliferus, as demonstrated by van Hoof (1976). Using an indirect method he measured the apparent mobility of trichodorids in a sandy soil maintained, at most, with the moisture content encountered during August in the Netherlands
(16.7% or less). Mobility seemed to be positively related with moisture content over the range tested. In soil-box experiments, however, although soil was maintained at less than the maximum, the implications of van Hoof's experimen- tation remains unclear. 37 47
2.B.3. "PITCHER THEORY" OF T. VIRULIFERUS ATTRACTION
Pitcher (1967) suggested that aggregations of J. viruliferus
on apple roots arose because of a chemical attraction (produced by those
roots) acting on nematodes in the surrounding soil. In order to explain
the numbers recorded at one of the aggregations a theory was proposed in
which all of the nematodes from a hemisphere of soil, radius 8 cm
(Pitcher worked on root tips growing against a glass plate in an under-
ground root observation laboratory), surrounding a suitable root tip were
attracted towards the centre. The fastest time for an aggregation to
reach maximum numbers was 10 days, from the first nematode to arrive at that root. This was the time tested in this experiment (cf 14 days in all other soil-box experiments). To test this theory it was necessary to design a soil-box
experiment with the two variables of time and inoculation distance
approximating to Pitcher's suggestions. Since 8 cm was considered to be
the furthest distance that nematodes moved it was decided to use an
inoculation distance of 7 cm, a little short of the maximum. Eight
soil-boxes were used in two series of four replicates each.
The results of the experiment are presented graphically in
figure 2. The results were analysed using the parameter of "nematode-
cm" (a nematode scores one for every centimetre it moves from the
inoculation point) and considering whether the movementwfts towards the
plant or away from it, the following measurements wire obtained (see
table 1). 30.
20.
I Q O
o.
i INOC - -2 -3-4 -5 -6 P 6 5 4 3 2 POINT DISTANCE (cm) travelled from inoculation point
Figure 2. Movement of T. viruliferus in response to an apple plant (P = plant).
Number of replicates = 8 Nematodes recovered = 1954 Experimental period = 10 days, only 39 47
No. of Movement Movement Mean % Standard Replicates towards away from movement Error plant plant towards (Nem. cm) (Nem. cm) plant
Expt. 1 4 1313 239 85.4 i 1.0
Expt. 2 4 2174 103 95.3 - 1.7
Total 8 3487 342 90.35 ± 1.7
Table 1. Movement of nematodes towards or away from a plant in a soil-box experiment designed to test the Pitcher Theory.
The form of the data presentation in Table 1 above, i.e. the
mean of the percentage-movement towards the plant, with its standard
error, was chosen in consultation with the Statistics Department of
East Mailing Research Station for two reasons. Firstly, this form of
analysis was simple and could demonstrate the generalised progression
of nematodes towards and away from a plant. Other analyses would have
been unworkable because of the complexities of nematode movement in
soil-box experiments, e.g. differences between replicates of the same
experiment; differences between individual nematodes; the inactivity
of those nematodes remaining about the inoculation point, etc.
Secondly, this form of analysis could be used in much of the study of
nematode attraction to plants and hence direct comparison is possible
with other tables in this thesis.
Using this technique it was not considered necessary to trans-
form the data before analysis.
The results of this experiment clearly show that T. vi ruliferus 10 40
is attracted towards apple rootsw Under purely random movement,
approximately 50% would have been expected to move each side of the
inoculation point. It also shows that in soil some T. viruliferus
can move at least 7 em towards a plant within a period of 10 days.
However, not all of the nematodes moved towards the plant: indeed, a
significant number remained at the point of inoculation and some even
moved away from the plant. Thus, the theory proposed by Pitcher
(1967) needed to be modified to account for these anomalies.
It is important to note that few nematodes pass through the
53>j bolting cloth screen (a total of six nematodes in only two of the
eight replicates), and so little or no feeding occurred on the plant
roots. Feeding could be an important factor, releasing root sap into
the soil, part of which is suggested as a nematode attractant (Gadd &
Loos, 1941). If feeding had occurred, as in the original observations,
attraction might have been greater since increased quantities of
attractant can cause nematodes to move faster (Azmi & Oairajpuri,
1976).
Besides reducing the effect of feeding to a minimum, the bolting
cloth, by acting as a nematode barrier (but not a chemical one) produces
an accumulation of nematodes in the soil section immediately behind it. « It also appears as if nematodes turn around and move back into
iVvAnattempt to find a route to the plant. Such a response must
be necessary in natural circumstances to overcome the effects of
obstructions in the soil such as cracks and stones. The percentage
movement towards the plant can therefore be considered as an under-
estimate . 2.BA. EFFECT OF DIFFERENT INOCULATION DISTANCES
In experiment 2.B.3, it was shown that not all of the nematodes inoculated at 7 cm from a plant root move towards that root. To explain the numbers of nematodes recorded aggregating at a root-tip,(?\V«k** nematodes must be travelling from distances in excess of 7 cm, i.e. from a sphere exceeding 7 or even 8 cm in diameter.
This experiment was designed to investigate the response of nematodes to a plant when inoculated at different distances spanning
Pitcher's maximum, viz: 5 cm, 8 cm (maximum proposed in Pitcher's theory) and 10 cm. The longer experimental period of 14 days was used to allow more marked differences between the treatments to appear.
The first experiment of this series was non-replicated for each distance. The second was replicated five-fold.
The results for the first experiment are presented graphically in figure 3. They suggest that the activity of the nematodes at one of the three inoculation distances falls into one of two categories. A nematode is either attracted towards the plant or else it remains in close proximity to the inoculation point, possibly wandering in a random manner. ArveAW*- activity state may exist, that of no movement, quiescence. Such differences in activity would cause the bimodal shape of the distribution histograms. It can be argued that the peak around the inoculation point contains those nematodes moving little and the second peak being a wave of nematodes moving towards the plant in an orientated manner. These two categories can be better defined as:
"attracted nematodes" - nematodes moving towards a plant root and orientated towards it by detection of chemical attractant produced by the root. 42
20 % n = 106
IO
TJ f-tCD >CD O a C(-D1 20
"«D % O -CPO E CD 10 C ct- O CTICD +C3O C CGD U aCD 30 •
20 %
79 10 -I r
INOC -I -O -Q P 4 3 2 I POINT * J DISTANCE (cm) travelled from inoculation point
Figure 3. Effect of inoculation distance on the movement of X* viruliferus towards an apple plant (non-replicated experiment).
Nematodes recovered = n Experimental period = 14 days 43 30
" -aWW nematodes" - nematodes not stimulated by plant root
exudates, either because there is no plant present or because the con-
centration of exudate is not high enough to exceed the threshold level
to which that individual nematode responds. These nematodes appear
to be slow moving and may be in a state of quiescence.
Synonym - "sluggish nematodes^
There are clear differences in the comparative sizes of these
peaks between the treatments, i.e. the 5 cm inoculation distance gives a
large peak for the activated and a small peak for the non-attracted
nematodes, the 10 cm inoculation distance the opposite. At the 8 cm
distance the two peaks are of size.
It is not possible to analyse the results using a nematode.cm
measure for each treatment because of the accumulation of nematodes
behind the root-screen in the 5 cm inoculation distance, and possibly in
the 8 cm box, too. Analysis was possible in the second (replicated)
experiment to measure the differences in the percentage of nematodes
moving towards the plant for the different inoculation distances. At
the beginning of the experiment presumably as many sluggish nematodes
are randomly moving towards the plant as away from it. A method of
measuring the number of nematodes remaining in the sluggish phase,
therefore, lies in assuming that the number of sluggish nematodes on the
non-plant side of the inoculation point is symmetrical with that on the
plant side. This is obviously a simplified analysis because it does
not allow for any nematodes becoming activated as they move towards the
plant through random wanderings. Graphs for the pooled results of the 44
30. "Attracted nematodes"
20 n = 600 %
IO
-O 03 P98765 43 2 u POINT >CD Q O (D t-t 20J CO CD X) % O -P CO n = 569 E CD C IO J Ct- 0 CD 01 CO -P CcD O CUD P 7 6 5 4 3 2 INOC -I -2 -3 -4 a POINT
20 J %
n = 589 IO J
lllgl
P 4 3 2 I woe -2 -3 -4 POINT DISTANCE Ccm) travelled from inoculation point
Figure 4. Effect of inoculation distance on the movement of T. viruliferus towards an apple plant (replicated experiment). Number of replicates = 3 Nematodes recovered = n Experimental period = 14 days replicated experiments are presented in figure 4, showing the segre- gation of activated and sluggish nematodes, as used in the analysis.
See table 2. NB The results for the second experiment do not show
the bimodal distribution.
Inoculation Percentage Standard Distance Activated Error
5 cm 65.2 - 2.5
8 cm 53.9 - 3.3
10 cm 37.8 ± 1.3
Table 2. Percentage of nematodes "activated" when inoculated at different distances from a plant.
If the distribution of percentage-nematodes activated against
distance is sigmoidal, as might be expected for this type of biological
response, then the maximum distance for activation by a plant root,
under the conditions prevailing during this experiment, could be about
12 cm. This was not tested.
Within the sluggish group there may well be some individuals
which were not attracted because of physical damage sustained prior to
inoculation of the soil-box. This, however, will be constant for all
inoculation distances and is likely to be small (few unhealthy nema-
todes would be capable of crawling through the 53(j sieves used in the
soil-box nematode extraction procedure). The higher the value,
though, of percentage damaged nematodes, the greater the actual differ-
ences in the percentage of nematodes attracted. 10 46
Such results could be explained if T. viruliferus within soil
demonstrated an energy-conserving mechanism by existing in two states of
activity, i.e. an activated state or a non-attracted, sluggish state.
The nematode could enter the activated state as a result of chemical
stimulation from a potential food source whereas when there is no plant
present, or else the nearest plant is "too far away", the nematodes exist
in the energy-saving, sluggish phase. A two-phased activity state has
been reported in the stimulation of male nematodes migrating towards
females of the same species, for example, Pelodera teres (Oones, 1966) and
Heterodera and Globodera spp (Green, Greet & Evans, 1968). Other plant-
parasitic nematodes have been shown to move most in the presence of host
plants and it is suggested that these nematodes also exist in one of two
activity states e.g. Pratylenchus spp (Endo, 1959), Hemicycliophora
paradoxa (Luc, Lespinat & Souchaud, 1969) and Helicotylenchus digonicus
(Alby & Russel, 1975). Unlike Pratylenchus spp and H. digonicus, there
is some movement in T. viruliferus when in the non-activated phase but
because of the long periods of time for which T. viruliferus can remain
inactive (more than 30 months) without depleting all of its food reserves
(see section 3.3) it is presumed that any such movement is slow. It may
be that movement decreases with time after a successful feed. 47 47
2.B.5. MOVEMENT OF NEMATODES IN ABSENCE OF PLANT
If viruliferus only move long distances (up to 10 cm in 14
days) when activated by root exudates then, in the absence of a host
plant, it might be expected that small movements only will be made from
the inoculation point, ie all of the nematodes will remain in the sluggish
phase.
Soil-boxes were inoculated with nematodes in the normal manner at
a point equivalent to 8 cm from a "plant". The point chosen was not in
the centre of the boxes, so that it was possible to investigate whether
any "edge-effects" existed. The experiment was replicated five times
and left 14 days before assay.
Figure 5 shows that 93 - 2% of nematodes moved no further than
3 cm, the maximum distance being 5 cm, and that nematode distribution
around the inoculation point was uniform (316 nematode.cm one side of the
inoculation point and 320 nematode.cm the other).
The results indicated that in the absence of a host plant,
nematode movement was, indeed, very considerably less than that in the
presence of a plant. All the nematodes were considered to be in the
sluggish, non-activated phase when no plant was present.
NB It is considered that nematodes can change from one activity state to
another in a relatively short period of time, hours rather than days. On
this assumption all nematodes will be in the same activity state after the
extraction procedure since this takes about 24 hours to complete. This,
however, could be the "activated" state if the extraction conditions are
physically adverse or "non-attracted" state because there are no positive
signals emanating from an attractant source. 48
•D 30 (-CD1 >CD O a uCD 1 20 •p CO E CD o % CD CT CO -cP IO CD ua CD Q.
I INOC POINT DISTANCE (cm) travelled from inoculation point
Figure 5. Movement of J. viruliferus in the absence of a plant.
Number of replicates = 5 Nematodes recovered = 494 Experimental period = 14 days 49 47
2.B.6. EFFECT OF INOCULATING BETWEEN TWO PLANTS IN A SOIL-BOX
Whilst in soil nematodes must often be in a position where they
are between two roots. Presumably, in such a choice situation, nematodes
move towards one or other of the roots. To simulate this situation two
plants were grown in the same soil-box and nematodes inoculated midway
between.
In this experiment two distances were used between the plant and
the nematodes, 4 cm and 7 cm (8 cm and 14 cm between the two plants
respectively). Due to technical difficulties in the growth chambers,
in which the soil-boxes were maintained, only two replicates for each
distance were possible.
Nematode.cm Dry weight of roots (gm)
To Plant A To Plant B Plant A Plant B
( 1 83 8* 0.1446 0.1471 4- cm ( ( 2 87 116 0.1460 0.1494
( 1 91 76 0.1169 0.1100 7 cm ( ( 1 72 203 0.1130 0.1807
Table 3. Results from the effect of inoculating nematodes between two plants.
*Soil had dried out around plant.
In a choice situation, nematodes moved towards either plant although in
one replicate there was some indication that the larger the plant root
system, the greater the attractiveness of that plant. From these
results it was not possible to say so with statistical backing.
If this were true 107
it might help to explain why Pitcher (1967) found larger-sized aggregations on larger roots. An increase in the diameter of a root increases both the area of epidermal cells available for nematodes to feed upon (surface area) and the numbers of cells producing attractant in the zone of elongation
(volume). However, when nematodes are in a choice situation where one plant root system is larger than the other not all of the nematodes move towards the largest root system but simply a larger proportion of those nematodes which have been activated.
Time did not permit observation of the effect of two plants, between which were large differences in size or differences in photo- synthetic rates, the latter obtained by covering one plant with a translucent material (Moussa, 1971). It would be interesting to know if, when there are such marked differences, all of the nematodes move towards the most metabolically active root system. 51
2.B.7. EFFECT OF ACTIVATED CHARCOAL IN SOIL-BOX
Peacock (1961) experimented to ascertain the relative importance
of complex organic molecules compared with carbon dioxide as the nematode
attractant substance in plant root exudates. He used a basic anion
exchange resin (Deacidite FF) mixed with sand to remove a high percent-
age of the carbon dioxide produced by an attractant plant, yet leaving complex organic molecules free; and a medium of activated charcoal and
sand to remove complex organic molecules but leaving carbon dioxide free.
As a measure of the attractiveness of the test plant to Meloidogyne
incognita larvae within the different media he recorded the numbers of
galls appearing on a test plant after 13 and 40 days: no absolute
measure of nematode numbers was made. The addition of activated charcoal
considerably reduced the numbers of galls on the test plant and so
Peacock concluded that complex organic molecules were the most important
substances in root exudates causing nematode attraction.
From work with T. viruliferus on agar (see section 2.A.4) and
from work by Klingler (1959) on Ditylenchus dipsaci and Bird (1960) on
Meloidogyne spp, there appears to be strong evidence that, at least in
part, carbon dioxide is important in the attraction mechanism.
To test thempo*W^of carbon dioxide in the attraction of
T. viruliferus to apple 4% w/v activated charcoal was mixed uniformly
with the soil of a soil-box. Five replicates were set up in which the
nematodes were inoculated 7 cm away from the plant and the soil-boxes
left for 14 days before screening.
On examination it appeared that most of the nematodes in each
soil-box had died. Only 2% of the original inoculum survived and all of
these remained close to the inoculation point. It may be worth noting
that no nematode moved to the non-plant side of the inoculation point, 52
whereas a few nematodes did move towards the plant. See table
Distance moved (cm) No. of nematodes
Total from 5 replicates -1 0
(inoculation total = INOCULATION POINT 15
c 1500 nematodes) +1 11
+2 1
+3 1
+4 to +6 0
PLANT 0
Table Distribution of nematodes towards a plant when inoculated into activated charcoal/soil medium.
Despite the small numbers involved it appeared as if some attrac- tion to the plant might have occurred. It may be, therefore, that activated charcoal did not prevent the attraction of nematodes to plants by absorbing complex organic molecules of the attractant substance.
However, the treatment did result in a large nematode mortality since all other important factors remained constant and yet a kill of 98% was obtained. It would be interesting to know if such an effect occured with other genera. Trichodorus spp. are more sensitive than most to changes in their physical environment (Clones et al., 1969; Wyss, 1970;
Rttssner, 1971; Van Gundy & Thomason, 1972 and Simons, 1973) and to lower concentrations of toxic materials (Hafkenschied, 1971 and Pitcher
& McNamara, 1972) than any other genera tested. These tests above included Meloidogyne spp., thus in Peacock's (1961) experiments it is uncertain whether activated charcoal reduced the attraction of M. incogni towards a host plant or whether the treatment affected the nematodes' habitat so that the sense of orientation was affected.
It is suggested that a controlled experiment in which lower levels of activated charcoal were used « 4%) may help elucidate the effect of this soil additive, whether it is chemically toxic to _T. viru- liferus (not so to many other forms of life) or whether it changes physical and/or chemical conditions within the soil. Both remain a possibility. 47 54
2.B.8 EFFECT OF PARTIAL STERILIZATION OF SOIL IN A SOIL-BOX
It has been reported that nematode attraction is dependent upon,
or increased by, the soil microflora (Bergman & van Duuren, 1959a).
This experiment was designed to investigate the effect of partially
sterilizing the soil in a soil-box and to allow observation of whether
microorganisms do modify the attraction response.
The soil used was heated at 70°Cfor one hour, a standard treat-
ment for soil where it is necessary to give a maximal kill of micro-
organisms yet cause as little amendment as possible to the soil's
chemistry (Pitcher, 1957). At 70°Csome thermoduric microorganisms can
survive and hence the treatment does not always achieve a complete
sterilization of the soil. The treated soil was left for about a
month in a paper bag before use to allow unstable and possibly toxic
chemicals produced by the heating to volatilize. Four replicates of both
the treated soil and the unheated control were used.
After 14- days the nematodes were extracted and the percentage
attracted towards the plant was calculated. This is expressed in table
5 below as the mean of the percentage of nematode movement towards the
plant and is presented with its standard error and range of values.
% movements Mean Standard towards the plant Percentage error
Unheated soil 83.1, 85.5, 88.1, 97.8 88.6 + 3.2
Heated soil 42.2, 43.1, 71.0, 87.3 60.9 +11.1
Table 5. Differences in the movement of nematodes towards a plant in heated and unheated soil.
(No. of replicates = 4; experimental period = 14 days; number of nematodes recovered - heated = 626; unheated = 798)
NB ^50% * attraction; 50% = no effect; <^50% = repulsion 55
The results showed that in three of the four replicates of
heated soil there was little attraction of nematodes towards their
respective plants: in two of these replicates there was evidence of
repulsion. One replicate showed an attraction response almost as great
as that of the unheated control. If the loss of the attraction response
in heated soil was due to the removal of essential microorganisms then
the following might help explain the variability in the results of the
heated treatments:
• Chance recolonization of "essential" microorganisms might
have occurred from the air, the plant or from the nematode inoculum.
• Thermoduric microorganisms might have survived and in some
soil-boxes become dominant in the absence of natural competitors.
Certain thermoduric species of microorganism are capable of producing
toxins or nematode repellents (Bergman & van Duuren, 1959b and Andrew &
Nicholas, 1976) and these could have created a less favourable environ-
ment for nematodes. The role of soil microorganisms in connection with the attraction response was investigated further in the following set of experimental
treatments. A singie band of heated soil, three centimetres wide, was added
to normal soil-boxes of unheated soil, in one of three positions. See
Fig. 6 below. The three regions tested and the reasons for their
testing were as follows:
• Around the plant -
- microorganisms might be modifying plant root exudates to
produce greater quantities of the primary attractant through their
normal metabolism (Bergman & van Duuren, 1959a) or ^ producing a
secondary attractant (Edmunds & Mai, 1967). 10 56
• Between the plant and the nematode -
- microorganisms might be responsible for augmenting and
amplifying the detectable range of attractant concentration gradient
which might assist detection by nematodes (Prot & Van Gundy, 1981).
• Around the nematode inoculation point -
- there might be some direct influence of microorganisms on
the nematodes' physiology, as suggested by Van Gundy et al. (1967) who
reported that the nematode ageing rate was increased in the absence of a
normal soil microflora.
/ 1 / K /
/ z
N • " nematode inoculation point
P - plant i i i ~ i root screen m heated soil Fig. 6. Position of heated soil bands in soil-boxes. (Band width 3 cm in all treatments.)
The results of nematode extractions from these soil-boxes, after
14 days, are given in Table 6 (see Fig. 7 for histograms of nematode
distribution).
Technical difficulties in the growth chambers eliminated two
replicates of the five from consideration, resulting in a total of
three replicates per treatment. Position of % movements Mean Standard % heated soil towards the plant error
Plant 70.3, 93.3, 97.6 87.1 + 9.1
Between 12.1, 28.6, 34.1 25.0 + 6.6
Nematodes 58.6, 73.9, 95.6 76.0 +10.7
Table 6. Differences in the movement of nematodes when bands of heated soil were set into soil-boxes of unheated soil.
NB >50% = attraction; 50% = no effect; <50% - repulsion
(for experimental details see Fig. 7)
Heated soil around the nematode inoculation point and around the plant seemed to have no, or only a small, effect of decreasing normal attraction. However, there seemed to be a marked repellent effect when heated soil was put as a barrier between the plant and the nematodes.
Such a clear result could have arisen from a toxic or repellent effect of the heated soil, possibly caused by an unbalanced microflora1
system of thermoduric species. Even in the presence of noxious chemi-
cals it appears that^/nematodes can still enter the activated phase in
response to plant root exudates and move towards a course of the latter,
i.e. treatments 1 and 3 in Table 6. The noxious effect of the heated
soil must be great enough, though, at short distances (treatment 2) to
repel the nematodes when plant root exudate levels are low, i.e. at a
distance from the plant. Where the repellent effects are in direct
competition with the attractant effects of a plant, originating at the
same place, then the repellency is not strong enough to override the
attraction response (treatment 1). Indeed, when plants are grown in
heated soil it may be that the rhizosphere microflora can quickly
restore the "normal" balance. 58
>i?< Heated soil 20,
IO
P 6 5 4 3 2 I ,NOC -I -2 -3 -4 POINT
(D f-l >a) 20 o n = 646
' % TCJD O -P IO . CO E CD C <+- O CJCD1 CO -p c aCD u aCD 20 J
% n = 807
IO
DISTANCE CcnO travelled from inoculation point
Figure 7. Effect of bands of heated soil on the movement of T. viruliferus towards an apple plant (P = plant).
Number of replicates = 3 Nematodes recovered = n Experimental period = 14 days 59 47
On close inspection of Fig. 7 it is possible to observe different
patterns of movement existing between treatments 1 and 3: the nematodes
in treatment 1 seem to have moved further, on average, towards the plant
than those in treatment 3. This could possibly imply that there was
some negative interaction occurring with chemicals in the inoculum area
of treatment 3 and normal nematode movement. This was not investigated
further.
The variation between replicates shows clearly the instability of
the soil-box system compared to field soil where, presumably, a balanced
and more completely buffered environment exists.
NB Direct physical effects of heating soil could possibly be affecting
nematode movement. The patterns would, however, be more difficult to
explain and could invalidate many other scientists' experimental results
if the suggestion that heating had more than marginal effects on the
soil's chemistry was proven. 60
3. SURVIVAL OF T. VIRULIFERUS IN PLANT-FREE SOIL
3.1 INTRODUCTION
1* viruliferus appears to exist in two activity states (see
section 2.BA)f an activated phase when a plant is present and a sluggish, inactivated phase when no plant is present, or at least not close enough to the nematode. This was interpreted as an energy-saving mechanism which prevented JT. viruliferus depleting its food reserves in unnecess- ary movement when no plant was present. In the absence of a host plant, ]_. viruliferus was shown to move little (see section 2.B.5), or not at all, remaining at the inoculation point for
(Ly. cXjC^^s. • If a nematode is not very active then its rate of stored-food catabolism will be slow, enabling it to survive in soil for longer periods of time than if it were continuously active.
Some plant-parasitic nematodes have specific mechanisms to overcome adverse conditions in soil, usually to a physical adversity rather than to an absence of food. Thescinclude the "eelworm wool" of
Ditylenchus dipsaci and the tanned cysts of Globodera spp, the latter being able to remain viable for up to 8 years in soil (Franklin, 1937).
A number of plant-parasitic nematodes, apparently without a specialized survival stage, have been reported to be capable of surviving for long periods of time in plant-free soil (see Table 7). In many of these instances it was reported that the amount of food reserves declined with time (Krusberg, 1959; Harrison & Hooper, 1963; Van Gundy et al., 1967;
Schmitt, 1973 and McNamara, 1980), death occurring when most of these reserves had been catabolized. 10 61
Nematode Length of Reference survival*
Criconemoides xenoplax 24 months Bird & Jenkins, 1965
Helicotylenchus dihystera 8 months McGlohan et al., 1961
Hemicycliophora arenarla 6 months Van Gundy & Rackham, 1961
Heterodera schachtii 12 months Golden & Schafer, 1960 (larvae)
Longidorus africanus 3 months Lamberti, 1968
JL. caespiticola 47 months McNamara & Pitcher, 1977
J_. elongatus 29 months Harrison & Hooper, 1963
J_. elongatus 47 months) ) goodeyi 47 months) McNamara & Pitcher, 1977 ) leptocephallus 47 months) martini Yagita, 1976 30 months Pratylenchus thornei Baxter & Blake, 1968 12 months Radopholus similis Tarjan, 1960 6 months Xiphinema americanum Elmiligy, 1971 4 months X. americanum Griffin & Barker, 1966 5 months Xv bakeri Sutherland & Ross, 1971 6 months X. bakeri Sutherland & Sluggett, 197*f 8 months X. diversicaudatum van Hoof, 1970 37 months _X. diversicaudatum McNamara & Pitcher, 1977 47 months X,. index Raski & Hewitt, 1963 24 months _X. index 54 months Raski et al., 1965
Table 7. Length of nematode survival in plant-free soil(b<*^Ovx tAcMcv^ccv,^.
indicates longest known survival period, either by direct measuring or by the length of an experiment. 62 47
In the studies of McNamara & Derrett
(1976) and McNamara (1980) it was shown that in Xiphinema spp survival
seemed to depend on the presence or absence of a normal soil microflora.
In pot experiments, McNamara (1980) was able to show a reduction in the
survival time of X. diversicaudatum when added to soil which had been
fumigated with the nematicide Dichloropropane:Dichloropropene (D.D.)
five years previously. He was able to return the "survival factor"
(factor allowing survival in soil) to heated soil by replacing the
fraction of soil passing through a 50p aperture sieve in heated soil
with the same quantity of appropriate fraction from unheated field soil.
McNamara suggested three possible ways in which X.* diversicaudatum
might utilize the survival factor to maintain food reserves over such
long periods o€ time when stored in field soil:
1. The nematodes fed directly upon bacterial particles, fungal
spores or on the hyphae produced by these spores.
2. The nematodes obtained nutrients from metabolites secreted by
the survival factor.
3. The survival factor produced exudates which inhibited nematode
movement thereby reducing metabolism.
The apparent interaction of nematode survivability and microflora
could also be explained by the following suggestions:
1. Nematodes become translucent when they age, having utilized all
of their stored food reserves. It has been shown for Caenorhabditis
elegans that the ageing rate is controlled in part by the presence of
"antioxidants", e.g. Vitamin E (Epstein & Gershon, 1972). Microflora in
the soil are known to synthesize some of these compounds and may, there-
fore, naturally slow down the ageing rate of soil-living nematodes. 112 63
2. When soil is heated to 70°Cthe complete microflora is not
destroyed. Thermoduric microorganisms (capable of withstanding high
temperatures) which remain could become dominant in the absence of
natural competitors. Some of the residual microflora might produce
materials either toxic or stimulatory to nematodes. In the first
instance, stored energy will be consumed in an attempt to escape, and in
the second, "wasted" by the stimulation into greater activity (Bhatt &
Rohde, 1970). In both cases, resources will be used much more quickly.
The present work suggests that J. viruliferus may possess a
physiological adaptation to extend its survival time in plant-free soil.
The behaviour of J. viruliferus has also been shown to be different in
heated soil to that in unheated soil (see section 2.B.8) and this was
suggested to result from an interaction with the microflora in heated
soil. It therefore seemed important to compare the performance of
I- viruliferus with McNamara's (1980) experimental results on the survival
of X*. diversicaudatum in plant-free soil: to compare the lengths of time
each were able to survive in plant-free soil; to determine whether the
survivability of T_. viruliferus was reduced when stored in heated soil
and to investigate the possible reasons permitting survival in plant-
free9 field soil for prolonged periods of time. 10 64
3.2 MATERIALS AND METHODS
3.2.i. Nematodes
I* viruliferus were extracted from soil under plum rootstocks
by means of a Seinhorst Elutriator. See section 2.B.2.i. for full
details.
3.2.ii. Soil
Soil used in this series of experiments came from the same
locality as that used to supply nematodes. To eliminate nematodes the
soil was air dried for 2 days at laboratory temperatures: the success
of this process was regularly checked by screening some of the remoistened
soil. No J. viruliferus were found. Whilst in the dried state the
soil was passed through a 2 mm aperture sieve to remove stones and large
pieces of organic debris, and to give a more even texture. The soil was
remoistened with glass-distilled water to resemble field conditions and
stored in polythene bags ready for use. The soil formed the substrate
for all treatments and was used as a control throughout.
3.2.iii.Pot experiments
All survival experiments were carried out by storing T_. viru-
liferus in small clay pots (70 ml) of soil maintained in a "moist
chamber" (Valdez, 1972), i.e. a tray of moist sand covered with a canopy
of clear plastic in which humidity remained relatively constant. The
clay pots had a base of plaster of Paris, added to give a completely
porous pot with no outlet through which nematodes could escape. The
porosity of the pot ensured a constant soil moisture content when set
into the sand of a moist chamber. After the pots were loosely packed
with 60 ml soil (loosely so as to leave plenty of soil spaces for
nematode movement), a soil-core (diameter 5 mm), extending half-way down
the pot was removed. Nematodes in water suspension (c. 1 ml) were 80 65
poured into this hole and the soil-core replaced. The moist chamber was
maintained in a constant temperature room of 17°C during the period of
the experiment. Dark, plastic sheets were placed over the top of the
pots to inhibit any plant growth in the soil.
At the termination of the experiment, "survival time", nematodes
were extracted by a modified form of Flegg's (1967) technique - the pot
of soil was soaked in 1 litre of water for one hour and then treated as
in section 2.B.2.iv.
3.2.iv. Optical density estimation
McNamara (1980) used a code system to categorize the optical
density of X_. diversicaudatum as a measure of stored food reserves. The
code was based on the apparent degree of "darkness" of a nematode when
viewed through a binocular microscope (x 50) with a transmitted light
source. The same system was used for T. viruliferus with the following
criteria.
Dark - most of gut appears black.
Inter - gut appears grey, but often with black and/or translucent
patches.
Pale - gut appears completely translucent.
See plate 5.
The categories were scored as follows: dark = 3, inter = 2 and
pale =1. By accumulating the scores for each pot at the termination of
the experiment a "survivability index" was obtained, provided that the
same number of nematodes were inoculated to all pots originally. This
index was used to compare treatments. (b)
• x
Plate 5. Colour-score indexing system for J. viruliferus (a) "dark" nematode: score = 3 (b) "intermediate" nematode: score = 2 112 67
3.3. SURVIVAL OF T. VIRULIFERUS STORED IN A POLYTHENE SACK OF SOIL
Soil containing J. viruliferus for use in experiments was
stored in black polythene sacks in a cold room at 2°C. The sacks,
although tied, were occasionally opened and this allowed a change of air
to take place. One such sack was stored for 25 months and, by screening
samples from this soil, it was possible to determine whether T_. virulif-
erus was capable of surviving for long periods of time in the absence
of plants. The total numbers of T. viruliferus present after this time
and the percentage of larvae are given in Table 8.
Initial Population after Percentage Population* 25 months* survival
Total numbers 321.0 112.8 35.1
Numbers of larvae 206.4 21.0
Numbers of adults 114.6 91.8
Percentage of larvae 64.3 23.8
Table 8. The numbers and population structure of T. viruliferus stored in a polythene sack of soil for 25 months.
*Mean from five replicates of 100 ml soil each (measured by displacement
of water).
Thirty-five per cent of the nematodes survived in plant-free soil
for 25 months. The population structure appeared to have
changed during storage, in that a decrease in the percentage of larval
forms was recorded. No first-stage larvae (L,) were found at the end 68
Plate 6. Female T. viruliferus showing three developing oocytes within the reproductive tract 69 112
of the storage time.
After 25 months no females were observed with developed oocytes;
whereas about 18$ of females in a field population would normally be in
such a condition (Pitcher & McNamara, 1970), irrespective of season,
providing they were not taken from a rhizosphere. See plate 6.
NB It is not possible to discuss the survivability of larvae or adults
separately because it seems possible that some larvae might have matured
into adults during the storage period. The percentage of these remains
unknown. 80 70
3.4. COMPARISON OF THE SURVIVABILITY OF PALE AND DARK NEMATODES
Having demonstrated that T. viruliferus can survive for long
periods of time comparable to Xiphinema and Longidorus spp in the absence
of plants, the following experiment was conducted to determine whether
J* viruliferiis iost body reserves and became paler with storage, and
hence whether dark nematodes were in a better condition to survive
plant-free periods.
Single pots of the control soil were inoculated with either 23
pale nematodes or 112 dark nematodes and left for six weeks. Nematodes
were extracted by the normal process, after this period, and gave the
results below. Also, the organic material retained on the final screens
at the end of the experimental period was searched to determine whether
any nematodes had survived the duration of the experiment yet failed to
pass through the screens. No such nematodes were found.
Pale Inter Dark Pale Inter Dark
Initial No. of nematodes 23* 0 0 0 0 112
Final No. of nematodes 2 0 0 8 28 16
Approx. % recovery 8.6% 46.8%
Table 9. The survivability of pale and dark ]_. viruliferus stored in plant-free conditions for six weeks.
* l/ery low numbers of "pale" nematodes are found in a field population (<2%), hence a small inoculum was used and in the time available only a single pot was tested. 112 71
Thus, dark nematodes seemed able to survive plant-free periods, considerably more successfully than pale. The results also indicated a
change in body "darkness" with a pronounced shift towards the inter-
mediate category or beyond. It seems sensible to suggest that this
change resulted from the catabolization of stored food materials (which
gave the "darkness") during the course of the experiment so that pale
nematodes were those with energy reserves nearly exhausted. 3.5. EFFECT OF SOIL HEATING ON NEMATODE SURVIVABILITY
McNamara (1980) showed that the ability of X.. diversicaudatum to survive in plant-free soil was considerably reduced if that soil had been previously heated at 70°C for one hour (partial sterilization).
He concluded that the heat treatment destroyed some biological entity, the "survival factor" (see section 3.1), and suggested that this agent, necessary for prolonged nematode survival, was probably a microorganism.
To compare the responses of J. viruliferus and X,. diversicaudatum to storage in heated soil the following experiment was conducted. Soil was heated at 70°C for one hour, left exposed to the air of a sterile cabinet for two hours and then kept in bags, at room temperature, for two months. These treatments were intended to allow any volatile, noxious materials, which might have been produced during the heating process, to disperse, e.g. ammoniacal compounds. The heated soil treatment was compared with a control of unheated soil and the experiment was repli- cated twofold for three storage periods of 4, 6 and 8 weeks. One
hundred T. viruliferus with an optical density index totalling 240 were
added to each pot in the standard manner.
After the experimental periods, nematodes were extracted from
the pots (see section 3.2) and their number recorded. Each nematode
was also categorized for optical density to produce a "survivability"
index i.e. the sum of all nematode colour-scores per pot (Table 10 and
Fig. 8). Figure 8. The survivability indices for batches of T_. viruliferus stored in either heated or unheated soil for different time intervals. (S.I. = survivability index) 80 74
4 weeks 6 weeks 8 weeks
Mean % Mean % Mean % recovery S.I.* recovery S.I.* recovery J S.I.*
Unheated soil 46 96 38 84.5 23.5 51
Heated soil 48 99.5 8 15.5 12.5 25.5
Table 10. The effect of nematode survival in heated or unheated soil after three periods of storage.
*S.I. = survivability index
The survivability indices were statistically analysed after
square-root transformation in an analysis of variance test (ANOVA).
Square-root transformed data was used on the advice of the Statistics
Department, East Mailing Research Station, and gave a more accurate
analysis than either non-transformed data or logarithm base 10 trans-
formation.
Statistically significant differences became apparent from six weeks
onwards, i.e. after four weeks1 storage no differences in either the dis-
appearance of body contents or mortality rate could be detected. The
differences between heated and unheated soil were significant at P<0.01(**)
and the week-times treatment interactions were significantly different at
P<0.05 (*), supporting the suggestion that T. viruliferus does not survive
as well in heated soil as in unheated soil.
Besides showing the similarity of response to survival in heated
soil by T. viruliferus and X. diversicaudatum the results also demon-
strated an important feature of this and all other survival experiments
carried out in pots. The survival time of nematodes, maintained in 112 75
pots, is considerably shorter than that recorded for nematodes in the
field, or those stored in polythene sacks of soil.
NB Pale nematodes rarely contribute to the survivability index because
the time that they remain in this state does not often exceed a few days.
Host nematodes recovered from storage experiments are, therefore, inter or
dark and this tends to cause the survivability index to lie between 2 and
3. 80 76
3.6. INVESTIGATION OF TEMPERATURE REQUIRED TO DESTROY SURVIVAL FACTOR
The survival factor on which _X. diversicaudatum depends was
destroyed when soil was heated at between 60 and 70°C for one hour
(McNamara & Derrett, 1976). In section 3.5 it was shown that soil
heated to 70°C was less suitable for J. viruliferus survival than the
unheated control. The effect of preheating soil to different temper-
atures was investigated to determine whether an inverse relationship
existed between heat treatment and survival rate or whether there was a
threshold temperature at which the phenomenon occurred.
Soil was heated at 60, 70 and 80°C for periods of one hour, with
an additional treatment in which soil was allowed to dry in air for 2
days before being heated at 80°C. It was thought that few, if any,
thermophilic microorganisms are capable of surviving temperatures as
high as 70°C in a "normal" form whereas some species of bacteria can
withstand such heat treatment by forming endospores (Stanier et al.,
1972). Such bacteria are termed "thermoduric" and include members of
the genus Bacillus. By allowing the soil to dry prior to heating it
was hoped that an increased number of endospores would be formed. This
larger and perhaps better distributed inoculum could be expected to
colonize the soil, in the absence of natural competitors, more rapidly
and efficiently than in soils not dried before heating. All treated
soil was moistened and then stored for 2 months in bags before being
used in pot experiments.
The treatments were replicated five times and compared to a
control series of unheated soil. Approximately 120 nematodes were
added to each pot. The experiment was left for six weeks before the
surviving nematodes were extracted. See Figure •
Using an analysis of variance, the treatments in which soil was 77
60 ri rH >CO •H >P D CO "COD o 40
cn -CpO c OCD 20 . fH CD a
60 70 80 80 U.H. DRIED temperature °C to which soil was heated
Figure 9. Effect on percentage nematode survival of heating soil to different temperatures (U.H. = unheated soil). Number of replicates = 4 Nematodes inoculated to each pot = 120 Experimental period = 6 weeks " Bars = Standard Errors 78 >8
heated to 60° and 70° were found not to be statistically significantly
different from the unheated control. There were statistically signifi-
cant differences between the 80°C heated treatment and all others
(P < 0.001). Soil dried before heating was also statistically signifi-
cantly different from all others (P <0.001) (Fig. 9).
The nematodes' ability to survive was considerably reduced when
soil had been heated to above 70°C. The effect was apparently increased
when soil was dried before heating. However, the method of heating soil
to these temperatures was different to that used in section 3.5, where
the survival factor was shown to be affected by heating to only 70°C.
Subsequent investigations revealed the possibility that in this experi-
ment, all the soil may not have been uniformly heated at the desired
temperature for the appropriate time. This was thought to have arisen
through the use of containers, for holding soil, with a larger diameter
to those initially and without adequate compensation for the increased
time of heat conduction to the centre of the soil sample. As pasteur-
ization is a function of both time and temperature all of the soil may
not have been heated to the stated temperature for a full one hour.
This may explain why the differences between the treatments were not as
might have been expected. Nevertheless, two important points arise from
these experimental findings.
• There exists a critical temperature for destroying the
survival factor.
• Soil, dried and then heated, is even less suitable for nema-
tode survival than heated field soil. 112 79
3.7. INVESTIGATION OF THE ABILITY OF T. VIRULIFERUS TO REGAIN STORED
FOOD RESERVES AFTER FEEDING
During survival in plant-free soil T. viruliferus and X. diversi-
caudatum slowly lost stored food reserves. The following experiment was
performed to discover whether J. viruliferus could acquire new reserves
if allowed access to a host-plant root system growing in heat-treated
soil.
Several hundred J. viruliferus were inoculated to pots of heated
soil (70 C for one hour) and stored for a period of six weeks. When the
nematodes were extracted all pale nematodes were selected and reinocu-
lated to a pot of heated soil containing two small apple seedlings.
After a further four weeks the nematodes were again extracted.
Of the 76 pale nematodes inoculated to the apple seedlings 29
were recovered four weeks later; 18 of these were "dark" and 11 "inter"
in optical density. No pale nematodes were recovered.
The results of this experiment show that pale nematodes can feed
and regain optical density, presumably by accumulating lipids as new
food reserves. No nematodes were recovered as pale, suggesting that few
of these nematodes could have endured this period without a food supply,
i.e. they either fed successfully or died. 80 so
3.8. INVESTIGATION OF THE ABILITY OF T. VIRULIFERUS TO REGAIN FOOD RESERVES IN FIELD SOIL
One of the suggestions proposed by McNamara (1980) to explain the
apparent dependence of X. diversicaudatum on soil microflora for survival
was that nematodes may feed directly on particulate or hyphal organisms.
If J. viruliferus is capable of feeding in a similar manner it would be
expected that pale nematodes added to field soil in the absence of a
recognised host-plant could become darker with time, as their food
reserves were replenished from alternative food sources.
Pale J. viruliferus were obtained by inoculating a large number
of nematodes to heated soil and leaving them for six weeks. . After this
time 126 pale nematodes were obtained and whilst in water suspension were
added as aliquots to four pots of field soil. Four weeks later the pots
were screened. No T. viruliferus were found.
It can be concluded from this observation that T. viruliferus did
not obtain nutrients from an alternative source, in the absence of higher
plants. If such a food source does exist in this soil it is obviously
inferior to plant roots and appears most unlikely to be sufficient to
allow survival for periods exceeding 25 months (see section 3.3). 80 81
3.9. DISTRIBUTION OF T. VIRULIFERUS WITH DEPTH
The depth distribution of plant-parasitic nematodes in f«AW*So"v\
related to soil temperature; the availability of oxygen and moisture (Van
Gundy et al., 1967), and to soil structure (Ward, 1960; Harrison &
Winslow, 1961; Cohn, 1969 and Oones et al., 1969). In addition, Schmitt
(1973) showed that there were differences in the suitability of A and B
horizon soil (zones of "humification" and "mineralization", respectively
- Odum, 1971) for the survival of Xiphinema americanum and concluded that
this might affect the natural depth distribution of this nematode.
Survival was found to be better in B horizon soil than A horizon soil,
even though the two horizons were considered to be identical in texture.
However, differences were shown to exist in the levels of nitrate and
phosphorus found in each horizon. Since nitrate ions can affect the
numbers of Pratylenchus penetrans (Walker, 1971) Schmitt believed that
these ions could also be important in the depth distribution of X.* ameri-
canum.
Unpublished data of Pitcher & McNamara show that, in fallow soil,
the numbers of T. viruliferus are not uniform with depth: most nematodes
occur in the 15-30 cm layer.
An experiment was carried out to determine whether the depth
layer from which soil was taken influenced the survival of T. viruliferus,
in soils which were maintained under identical conditions. This elimi-
nated the two variables of oxygen and moisture availability which had
already been shown to be important in the distribution of Trichodorus spp
in sandy soils (Cooke & Draycott, 1971). Soil cores were taken from a plot adjacent to the nematode-
collecting site in an area where the land had been fallowed for several
years previously. Nine pots of each soil layer were prepared in the 82 112
usual manner (see section 3.2.i) and inoculated with 200 J. viruliferus.
Each soil layer was 15 cm deep, the deepest sample being taken from the
4-5-60 cm layer. The pots were maintained in a moist chamber for six
weeks and then the nematodes were extracted.
The percentage survival in each soil layer is presented in Table
11, together with the unpublished data of Pitcher & McNamara on _T. viru-
liferus distribution in fallow soil.
Soil depth
0-15 cm 15-30 cm 30-4-5 cm 4-5-60 cm
Percentage 19.6 ± 1.6 31.3 - 3.5 23.2 - 2.9 20.5 i 2.8 survival
Percentage 10.3 i 2.3 51.5 - 3.3 24-.7 - 4-.6 13.5 - 2.9 distribution*
Table 11. Ability of T_. viruliferus to survive in soil from different depths compared to natural distribution in relation to depth. Both sets of data are presented with their standard errors.
*Unpublished data of Pitcher & McNamara.
The data were statistically analysed and a correlation coefficient
calculated of 0.997 (P apparent between the two sets of data as they vary with depth. This experiment showed that the distribution of J. viruliferus in the field was unlikely to be determined solely by absolute levels of oxygen or moisture, as suggested by Van Gundy et jil. (1967) because in pot experiments these factors were constant for each soil layer. The distribution of nematodes in the field, at least in part, is, therefore, probably determined by one of the following: + The close correlation measured between these two variables is considered fortuitous. A storage time shorter or longer than that of the optimum used (from section 3.5) would be less likely to produce such a correlation i.e. this close relationship only exists at one point in time. 80 83 Soil structure. In general the clay content, although not quantified precisely, appeared to increase with depth. However, the 0-15 cm soil layer did not seem to have a higher clay fraction than the 15-30 cm layer and hence the statistical differences between these two samples could not be explained in terms of clay content alone. Win- field & Cook (1975) considered the clay content, which affects the pore space and moisture-holding capacity of the soil, to be the most important feature for determining the suitability of a habitat for a particular nematode species. Survival factor. The ability of nematodes to survive in pots and the success of nematodes at different depths in the field may be governed by the quantity or quality of microorganisms present, or by the presence of a specific organism (the survival factor). The distribution of microorganisms in soil could, however, be determined by oxygen and moisture levels, variability or extremes of temperature or availability of suitable nutrients. Any of these factors could, therefore, indir- ectly affect _T. viruliferus through the distribution of the survival factor. Ionic content. Schmitt (1973) found differences in ionic con- tent between A and B horizons of soil and it seems likely that this ionic content varies gradually since it is dependent upon the degree of humus breakdown. The parameter of pH, an indirect measure of absolute ionic content, might be easier to measure in soil than individual ionic contents but, unfortunately, it was not possible to compare the pH values for those different soil layers used in this study. Root distribution. Under field conditions in which host plants are growing it has been shown that a high percentage of ]_. viruliferus live in close proximity to roots (Pitcher, 1967). If the .host plant is removed it is considered likely that the position of roots will continue to influence the distribution of nematodes for some time. This factor is not suggested as having any influence on the above experiment. 3.10. DISCUSSION OF RESULTS X* viruliferus has been shown capable of surviving in soil for cxV W&JS.Y 25 months in the absence of the roots of vascular plants. Nevertheless, during W\\s ^ervob ©f not all nematodes remain alive, death resulting from the catabolization of stored lipid reserves below a critical level and through senescence and other natural mortal- ity factors. These lipid food reserves could have been replenished if the nematodes were allowed to feed on a host plant. Partial steriliz- ation of soil, prior to storage, reduced the nematodes' ability to survive, possibly by causing an increase in the rate of metabolism. The effect of soil sterilization suggested that there might have been a positive microbial interaction with T_. viruliferus in field soil and/or a negative interaction in heated soil. During storage of J. viruliferus in polythene sacks of field soil, changes were recorded in the population structure. After 25 months larvae constituted 23.8% of the total nematode population com- pared with 65.3% at the commencement of storage and of these larvae none of the youngest stage (L^) was found. One of the following sug- gestions might help to explain this observation. Firstly, that larvae with adequate food reserves were capable of moulting into more mature stages of nematode in the absence of a food source. Some dorylaimid nematodes of the genus Xiphinema have been described as being capable of this, e.g. X« americanum (Griffin & Barker, 1966) and X. bakeri (Sutherland & Ross, 1971). All larval stages of these two species of nematode can feed and hence are similar in this respect to J. viruliferus. However, in some other orders of nematode it is normal for a larval stage to moult to the next without feeding because these stages do not 80 85 have functional mouthparts, e.g. Meloidogyne spp and Pratylenchus spp (Corbett, 1978). Adult T. viruliferus, therefore, found in the poly- thene sack after storage could represent the surviving individuals of both the original adult class and those larvae which had moulted into the adult form during the course of storage. Larval forms might rep- resent those nematodes, which had not moulted into adults, perhaps through a lack of time or nutrients, or possibly individuals hatching from eggs after the commencement of storage. An alternative explanation for the changes in population struc- ture with storage is that the nematodes' ability to survive might be correlated with its body size. The results would suggest, therefore, that adult forms were best able to survive plant-free periods compared with larvae; the least able to survive being the smallest, the L^s. This could be related to larger forms having relatively larger amounts of stored food reserves and probably a lower rate of metabolism; having a smaller surface area to volume ratio, and possibly possessing additional physical adaptations to survival, e.g. a less permeable cuticle. The results of this experiment were not suitable for determin- ing which of these two hypotheses is correct but experience of other nematodes suggests that both these hypotheses are possible and that an interaction between the two is likely. Whilst nematodes were stored in polythene sacks it appeared unlikely that reproduction had occurred because adult females with mature oocytes in the reproductive tract (see plate 6) were not encoun- tered during the survival period and no first-stage larvae were observed after 25 months' storage. Pitcher (1967) discovered that reproduction in T. viruliferus most often occurred at a root feeding site, after feeding. Therefore, it would seem for reproduction not to 112 86 have occurred in the absence of a host plant. The ability of J. viruliferus to survive seems to be directly dependent upon the rate of energy metabolism. Nematodes with large food reserves (dark) slowly catabolize this energy source during storage appearance. Immediately prior to death T. viruliferus appears trans- lucent (pale) because all of its food reserves have been expended and hence the gut cells no longer contain the light refracting lipid glo- bules. If a nematode, even a pale one, is allowed contact with a host root, feeding will ensue and its gut cells will be replenished with fresh food reserves. T. viruliferus has not been demonstrated to regain food reserves whilst stored in fiel^soil. This gives support to McNamara's (1980) * ^VavxY-free so\\ 112 87 suggestion that the survival factor only "maintains" the nematodes' stored food reserves in the absence of roots of vascular plants. Pale nematodes thus feeding on this secondary food source might not be expected to store excess lipid in the gut cells of its body and become darker in appearance because this alternative food source (survival factor) is inferior to normal (plant roots). The effect of partially sterilizing soil before adding nematodes to it was observed to reduce considerably the suitability of that soil for nematode survival. In a series of experiments McNamara (1980) showed that this effect was almost certainly due to differences in the soil microflora of heated soil compared to unheated soil. The critical temperature (see section 3.5) of this treatment was dependent on the removal of a particular microorganism, or group of microorganisms. McNamara's interpretation of this information suggested that the soil treatment destroyed a beneficial organism for nematode survival. He did not discuss the possibility that the soil treatment, by killing natural competitors, selected microorganisms which were unfavourable to nematode survival; I believe this to be possible. Five possibilities which could explain the observations on the survival of J. viruliferus in plant-free soil are summarized below. Each is subsequently discussed in detail as a reasonable explanation of the survival phenomenon. • Nematodes feed on microorganisms as alternate food sources. • Nematodes obtain nutrients from the metabolites produced by microorganisms. • "Survival factor" induces quiescence in nematodes. • Long-term survival is a slowing down of the ageing rate: this can be by microbial production of antioxidants. 80 88 • Survival is enhanced by adopting a sluggish activity state - this is inhibited by the chemical effects of thermoduric microorganisms. Microorganisms as alternate food sources Griffin & Barker (1966) reported that Xiphinema americanum was observed "to attack" a species of Rhabditis in soil washings and suggested that X, americanum might be predacious as well as plant-parasitic. Observations of Griffin & Darling (1964) had previously shown that a population of Criconemoides xenoplax decreased at a rate correlated with an increase in population density of X. americanum in a plantation of Colorado blue spruce. These authors suggested that the decreased numbers of xenoplax was possibly related to X.* americanum's predaci- ous habit. Griffin & Barker further speculated that X. americanurn might utilize small root pieces, mycorrhizae, algae, fungi or any other micro- organism or plant tissue in the soil when no host was present. McNamara (1980) discovered that the survival factor for X. diversicaudatum was apparently in the soil fraction less than 50/j and was, therefore, unlikely to be other nematodes or root pieces, but could have been bacterial particles, fungal spores or the hyphae produced by the latter. There has been no direct evidence from any survival experiment to confirm that species of dorylaimid nematode do actually feed on microbial sources in the absence of higher plants: the evidence suggests that feeding does not occur on good alternative food sources because nematodes do not store any of this possible food source as lipid and reproduction does not seem to occur in X. americanum (Griffin & Barker, 1966), X. bakeri (Sutherland & Ross, 1971) or T. viruliferus (see above). If 80 89 nematodes are feeding on an alternative food source then it must be considered an inferior one, compared with plants, and only sufficient to slow down the rate of starvation. Furthermore, the specialized mouthparts of trichodorid nematodes with a solid spear and perhaps the need for a "saliva tube" (Wyss, 1977) wpiiild seem unsuitable for feeding on such small structures as those suggested. Indirect food source from microorganisms It was suggested by McNamara (1980) that nematodes might be able to obtain nutrients from metabolites secreted by microorganisms into the soil. Nutrients could be absorbed through oral ingestion or by diffus- ion through the cuticle. This latter process has been shown possible in some nematode species, e.g. Ditylenchus triformis (Myers & Krusberg, 1965), Aphelenchus avenae (Marks et al^., 1968), Hemicycliophora paradoxa (Luc et al., 1969), Longidorus elongatus and X.. diversicaudatum (Mayo & Thomas, 1971). Transcuticular uptake is in fact considered to be more common in dorylaimid nematodes than in other groups (Hollis & Jordan, 1962). This is supported by Taylor et al. (1970), who suggested that the greater susceptibility of dorylaimid nematodes to nematicides was due, in part, to the increased efficiency in the passage of chemicals through their cuticles because of the presence of "pore canals". Pore canals have been demonstrated to exist in several longidorid nematodes but not, so far, in trichodorid nematodes. Evidence in opposition to the theory of nutrient uptake from a microbiological source includes the observation that when nematodes were stored in field soil they could neither sexually reproduce nor replenish their gut cells with lipid. However, if nematodes could absorb chemi- cals such as simple sugars through their cuticle this might be sufficient 80 90 to supply subsistence energy. Nematodes might, therefore, use their stored lipid reserves at a slower rate than without this secondary energy source and hence retain lipid in the gut cells for prolonged periods of time. Evidence which could be interpreted as opposing this last theory comes from an experiment of McNamara (1977). He removed as much of the organic fraction as possible from field soil and discovered that nema- todes survived as well in this soil as in untreated field soil. Since humus is the major food source for most soil-living microorganisms, it seems unlikely that its removal would not reduce the amount of micro- organism metabolites produced and hence affect nematode survival. Induction of nematode quiescence Seinhorst (1950) found that movement of the normally highly active Ditylenchus dipsaci was apparently inhibited in fallow soil, although this inhibitory factor could be removed by prior heat treatment of the soil or application of particular chemicals * Wallace (1966) suggested that microorganisms in the soil could be the source of these inhibitory substances to nematodes. McNamara (1980) presented data which demonstrated that X, diversicaudatum, stored in field soil for 2 years, were apparently less active than nematodes from fresh soil, the rate of activity being measured by the technique of Lownsbery (1964), i.e. the time taken for nematodes to emerge through a 90p final separ- ation sieve. Following work carried out on J. viruliferus an alter- native interpretation could be applied to this data, suggesting that nematodes from 2-year-old soil were less active in the extraction pro- cedure because they contained smaller food reserves than freshly collected nematodes (see section 6.3.iii) rather than through having to overcome some form of induced quiescence. Although there was evidence to imply that microorganisms could affect nematode activity (Seinhorst, 1950), the results of experiments with X,. diversicaudatum (McNamara, 1980) and T. viruliferus suggested that, rather than complete quiescence being induced, microorganisms at most inhibited movement of nematodes, thereby reducing the rate of metabolism termed "hypobiosis" by Keilin (1959). In quiescence no od reserves would be utilized (Kostdk, 1965), the body's metabolism having been stopped completely until triggered to restart by environ- mental conditions (Evans & Perry, 1976). It is considered possible that microorganisms may \Jrect\.cV vmHn ~ viruliferus . reducing nematode movement in plant-free soil. Antioxidant production reducing the nematode ageing process Nematodes become paler as they age, before eventually dying (Van Gundy et £l., 1967). It is conceivable that microorganisms are capable of producing antioxidant, e.g. vitamin E, which can slow down the ageing process (Epstein & Gershon, 1972). However, two pieces of evidence argue against this suggestion. Firstly, larval forms of J. viruliferus become paler when stored. It would seem unlikely that these nematodes were becoming pale through natural ageing processes, i.e. approaching senility whilst in an immature phase, and is more likely that the nematodes are being starved rather than becoming aged. Secondly, the process of becoming pale has been shown to be reversible (see section 3.7) in that at least some nematodes are able to regain the optical density of their bodies by feeding on a host plant: ageing by definition, cannot be a reversible process. Survival in sluggish state - affected by thermoduric microorganisms T. viruliferus, in attraction experiments, |> able to exist in two states, the activated state and the sluggish 80 92 state (see section 2.B.4). In the absence of plants it is suggested that the nematodes exist in the sluggish phase and are/(able to survive for longe^periods of time (25 months, plus) before using up all stored lipid reserves. This would fulfil the role of a natural survival mechanism without necessitating any interactions with other soil microorganisms. The reduced ability to survive in heated soil might result from the production of chemicals by thermoduric microorganisms (see section 3.1) which are either nematicidal or, alternatively, stimu- latory i.e., cause nematodes to enter the activated state and use up their stored food reserves far more quickly. Few microorganisms, e.g. Bacillus spp (Stanier et al., 1972), are capable of surviving the soil-heating treatment. It has been shown experimentally by Iizuka et ad (1962) that some "strains" of Bacillus spp caused a very high level of nematode mortality. In the absence of natural competitors in soil Bacillus spp may be capable of abnormally successful growth and hence produce chemicals at a high enough level to have a direct effect on nematodes added to this soil. That nematodes are recovered from heated soil supports the view that these noxious chemicals do not kill nematodes outright but cause them to use up their food reserves quicker than when stored in fallow field soil. This would explain the greater mortality rate of nematodes added to heated soil compared to nematodes in field soil. The chemicals present might be either nematicidal (chronic level) or stimulatory, i.e. would cause nematodes to continu- ously move in attempts to escape from the chemically unfavourable areas. Since nematodes are contained within small pots, movement must be continuous in attempting to escape. The rate of energy expenditure will, therefore, be much higher than nematodes stored in untreated field soil. In soil-box experiments (see section 2.B.8) there was 112 93 evidence to suggest that a band of heated soil was repellent to j[. viruliferus. Drying soil prior to heating seemed to decrease the suitability of that soil for nematode survival (see section 3.6). It is suggested that the process of air-drying soil may have induced Bacillus-like species of microorganisms to form endospores. This would give rise to a larger inoculum after the heat treatment and perhaps a better distributed inoculum, too, thus putting this species in a strong position to recolonize the soil upon cooling. It is interesting to note that one method for initiating the germination of endospores is to expose for some minutes to a temperature of 60 to 80°C and then soak in water (Stanier et al., 1972). 1 This theory, however, does not easily explain the observation of McNamara (1980) that X. diversicaudatum did not survive well in soil treated with a soil fumigant 5 years previous. Endospores of Bacillus spp are, however, known to be more resistant to fumigants than other bacterial or fungal stages (Stanier et al., 1972) and it may, therefore, be that after five years in heavy, uncultivated soil, the natural com- petitors of Bacillus spp had either not been able to recolonize or had not been able to shift the ecological balance to its former position. In consideration of the five theories above, the balance of the various arguments seems to me to favour that survival of J. viruliferus in soil depends heavily on the ability of this nematode to exist in the sluggish, inactivated state. In field soil, in the absence of a plant, this state is naturally adopted but in "unfavourable" soils, such as heat-treated ones, nematodes may be induced by some means 112 94 to assume the activated state and in such a state use up all of their stored energy reserves quickly. An interaction with microorganisms certainly seems to occur but the evidence presented cannot determine whether this is positive (microorganisms induce nematode quiescence in field soil) or negative (thermoduric species activate nematodes whilst in heated soil). It is,however, possible that both interactions occur. Soil is an extremely complex environment physi- cally and chemically, and one in which nematodes are success- fully adapted to coexist with the soil microflora. Any major inter- vention in the system, such as heat-treatment or fumigation, is likely to have catastrophic implications to the balance of such organisms living sympatrically. The possibility that nematodes feed in their normal manner on x an alternative microbial food source in the absence of plant roots is considered unlikely, in the light of the evidence presented above. Cuticular absorption of simple sugars remains a possibility that cannot be resolved using the data from my experiments. The theory that micro- organisms affect nematodes' ageing rate is not supported for J. viru- liferus . Thus, in summary, it is speculated that J. viruliferus exists in one of two activity states depending on the presence of suitable plant- root stimuli. The hazards of the life-cycle of other migratory plant parasitic nematodes, especially members of the Hoplolaimidae and Longidoridae, would undoubtedly be reduced by a survival stratagem of this nature. Indeed, it may be that the survival times in plant-free soil of Table 7 reflect the more general existence of this type of behaviour in groups lacking a protected, resistant or resting state in the life cycle. This would seem to be a field worthy of future explor- ation. 112 95 k. GENERAL DISCUSSION Trichodorus viruliferus has been observed by Pitcher & Flegg (1965) to attack apple tree roots and often to kill those roots upon which widespread feeding had occurred (Pitcher, 1967). The mode of feeding was generally of an ectoparasitic nature, although the species did penetrate cracks which formed in the roots to exploit the inner cortical cells. The common feature to the preliminary observations was that J. viruliferus fed in a gregarious manner, insomuch as numbers of nematodes aggregated at one particular root-tip shortly (4-20 days) after solitary nematodes had been observed to commence feeding. The fast build-up of nematodes was thought likely to result from the attraction of nematodes to chemicals produced by suitable growing roots. The alternative theory of rapid reproduction at the root-tip was demonstrated to be not possible (Pitcher & McNamara, 1970). It was set against this background that my study was based, to investigate the action of plant-root attraction to nematodes in soil. Soil is a complex medium in which biotic and abiotic factors interact, and where most chemical and physical characteristics are buffered to change. It was considered, therefore, that the action of nematode attractants might be more easily studied on the relatively simple substrate of agar: results from these studies were to be investigated later in Scn\» However, despite many attempts, in which variables were systematically altered, and the application of advice sought from many colleagues, X* viruliferus did not move on plates of agar in a replicable or regular way. This was one of the first indications that J. viruliferus was especially 80 96 sensitive to extraction from soil, physical handling and subjection to atypical conditions. Interestingly, T_. viruliferus adopted a different body shape whilst being subjected to adverse conditions characterized by a short- ening and fattening of the animal, with a corresponding loss of cuti- cular features (see plate 2). This'*s&tt««^what Hooper (pers. comm.) described for Paratrichodorus spp as "cuticular swelling", observed whilst fixing specimens for permanent mounts,WkecL Trichodorus spp were reported as not being susceptible to this. Even though viruliferus did not produce replicable results on agar plates ©we. individual , nevertheless, did produce significant results. nematode apparently demonstrated a positive orient- ation towards a source of carbon dioxide which was being emitted from a hypodermic syringe at a comparable rate to that produced by living plants (see fig. 1). The nematode moved towards the carbon dioxide source by a series of direct movements, corrected at a number of points by slight changes in direction. The tracks made by the nematode were of a different appearance to normal random wanderings on agar plates. These tracks seemed to indicate that rather than a smooth sinusoidal movement ("normal tracks") being adopted, a type of movement was used in which the anterior end continuously moved through the maximum distance in the horizontal plane, to produce "broad tracks". This was suggested as evidence for some form of chemotactic orientation to carbon dioxide and may be of assistance to nematodes moving towards a centre of high metabolic activity e.g. a fast-growing root. The above observation also fits in with Green's (1973 ) notion of a "clutch" in Globodera rostochiensis. In this model, the clutch was situated h to 1/3 of the way down the body, and when moving along a 112 97 gradient and sensing a sudden increase or decrease in stimulus, the clutch was disengaged. The anterior end then moved through wide searching arcs. Once realigned, the clutch was en-engaged and waves were again propagated along the length of the worm. The "disengage- ment of a clutch" and cessation of body waves has been observed in the tracks of a number of other nematode species by Croll (1975). The difficulties in using agar plates could not be resolved and so an alternative study system was sought. The method selected was based on Harrison's (1975) soil-box technique for establishing the rate of spread of corky ring-spot by the Trichodorus spp virus-vector. There were certain limitations to the system, discussed in section 2.B.2, but nevertheless it was considered an ideal laboratory system for this study. Soil-boxes were assayed after the given experimental period; by assuming that nematodes moved in a direct way towards an attractant plant it was possible to quantify the amount of "attraction" within a soi-box by using the parameter of "nematode.cm", i.e. for each centi- metre moved along the soil-box, a nematode was given a score of one. Comparison of the number of "nematode.cms" towards the plant and away from the plant (random wanderings and repulsions) offered a comparable index of "attractiveness" for that plant. The statistical analyses Were also based on this parameter: analyses produced could be directly compared for different treatments and gave as much information as was considered possible from the experimental results; results which nec- essarily gave significant variations between replicates, e.g. variation in attractiveness of different plants within one treatment. Using the soil-box it was possible to show that T_. viruliferus could move up to 7 cm in 10 days. This was necessary to support 80 98 Pitcher's (1967) hypothesis that a nematode aggregation required all of the nematodes up to 8 cm from a plant root to move towards that plant root in order to account for the numbers observed in root-tip aggregations. However, although a high percentage of nematodes did move towards the plant in the soil-box a number of individuals did not appear to orientate towards the plant at all. If this was typical of the reaction adopted by nematodes to plant roots in soil some of the individuals arriving at an aggregation must have covered distances exceeding 8 cm. Was this possible? A different series of soil-boxes were inoculated with nematodes at different distances from the plants to determine whether nematodes could move in excess of 8 cm: they could. However, the most import- ant conclusion from this experiment was that J. viruliferus appeared to exist in two activity states and that the proportion of nematodes in each state varied with distance from the attractant source. The closer the nematodes were to the plant, the higher the proportion of "activ nematodes. The existence of two activity states was interpreted as an energy-conserving mechanism in which nematodes only moved quickly in response to attractant chemicals emanating from plant roots. When no such chemicals were present, a comparable state to a fallow piece of land, activity was reduced thereby minimizing the rate of energy and hence increasing the length of time that a nematode could S^rVtVe. This is true of non-feeding infect- ive stages of many animal parasitic nematodes (Croll, 1972; Elliot, 1954; Haley & Clifford, 1960; Payne, 1923 and Rogers, 1939 and 1940) and for some other plant-parasitic species, such as Meloidogyne spp (Balmer & Cairns, 1963 and Bergeson, 1959) and Heterodera schachtii (Golden & Schafer, 1960). All the examples quoted differ from !• viruliferus in one respect because they refer to larval stages that had never fed, i.e. they are living solely on lipids persisting from their embryonic stage and these resources are likely to be very restric- ted in quantity. A number of nematode species can enter a special resistant stage in their life cycle in which the animal can avoid a shortage of food e.g. Caenorhabditis elegans (Klass & Hirsch, 1976) or wait for a suitable host to appear, e.g. the entomophilic species, Pelodera coarcteta (Croll, 1970). This special and often optional stage is termed the "dauer larva" and can be recognized on morphological grounds (Riddle, 1978). However, no dauer larval form has been reported for a dorylaim nematode (this group includes T_. viruliferus) and no permanent morphological differences were ever observed in T. viruliferus to indicate this particular form. It is therefore assumed that the inactive state of this nematode species does not represent a distinct dauer larval form. The two-state activity phenomenon might help to explain the observation of Van Gundy et al. (1967) that in Heloidogyne javanica the movement involved when moving towards a root depleted body food reserves faster than when in soil without roots. Nematodes in root-free soil were perhaps in the sluggish phase whereas nematodes in soil with roots were activated into moving towards those roots and used up energy to do so. It is suggested that the existence of two phases of nematode activity could also explain why nematode tracks appear on agar as two distinct types, broad tracks and sinusoidal tracks (see section 2.AA. K The sinusoidal tracks may correspond to random wander- 80 100 ings through the soil, i.e. non-orientated movement, whereas the broad tracks could be those made by the nematode when in orientated movement, the breadth of the track resulting from frequent head waving. In all experiments it was observed that some nematodes did not appear to move from the inoculation point and these may have been in a state of inactivity or they may possibly have been damaged in the original extraction procedure. The latter seemed less likely because their subsequent recovery involved them actively crawling through fine nylon mesh screen. v (2.6 it) Summarizing the results of this experiment/it would seem that nematodes arriving at an aggregation site were orientating from dis- tances in excess of 8 cm (extrapolation of soil-box experiments suggests up to about 12 cm) and the percentage of nematodes moving towards the plant root was r^AcvVec^w to the distance. It can be argued that it is probably not advantageous for an individual nematode to respond to a stimulus that emanates from a distance greater than the nematode is capable of moving, since nematodes have a finite stored energy reserve. Similarly, it would be ineffic- ient if nematodes remained inactive in the soil until plant roots grew to within a very short distance, as this would reduce the chances of that nematode ever feeding. For any nematode there is therefore a particular threshold level of response (the minimum level of attractant to stimulate a sluggish phased nematode into the activated phase) which will allow that nematode to exploit an individual root most efficiently as a food source. Contrary to the above, T. viruliferus is reported to feed on a diverse range of plants (van Hoof et al., 1966) which probably produce different quantities or types of attractant. For the species to be as 101 112 successful as possible in exploiting new situations it must exhibit a wide range of threshold response levels to enable individual nematodes to feed, and hence reproduce, wherever suitable roots grow. For a population of _T. viruliferus there must therefore be an optimal range of threshold response levels to most successfully utilize its environ- ment. The variability in response levels was suggested by the experi- ment because at any one inoculation distance (same attractant concen- tration) only some, not all, nematodes were attracted. Heterodera spp (Weischer, 1959), Hemicycliophora paradoxa (Luc, 1961), Hoplolaimus indicus and Helicotylenchus indicus (Azmi & 3airaj- puri, 1976) have all been shown to increase their locomotory activity as they approach an attractant source. There is no evidence to show that T_. viruliferus likewise increases its S^^-edtother than a skew in the peak of activated nematodes as they approach the plants, observed in the distribution histograms of figure 5. Studies of time-lapse films were also unable to resolve this point. If T. viruliferus does exist in two activity states then it follows that there will be little apparent movement when nematodes are inoculated into soil-boxes with no plants. This was observed, 93% of all nematodes remained within 3 cm of the inoculation point and move- ment was apparently in all directions. Further work suggested that the attractiveness of plant root systems may be a factor of size, the larger the plant, the more attract- ive it is. However,' when nematodes were inoculated equidistant between two plants of differing root size (measured as dry weight) not all nematodes moved towards the largest root system although most did. 80 102 This is somewhat difficult to interpret and requires further experi- mentation to answer satisfactorily. It has been suggested that attraction to roots could be achieved by orientation towards carbon dioxide (CO^), a chemical produced by all living organisms in proportion to their rate of respiration (metabolic rate). However, others would argue that a chemical which is not specifically produced by plants is an unlikely nematode attractant. Indeed, decaying humus is probably a stronger source of CO2 than plant roots and this could induce needless, energy-burning movements towards non-food sources. In addition to this factor, levels of CO2 in soil are generally higher than those in air making a narrow concentration one gradient more difficult to detect and follow. Even thoughjj^[. viru- liferus was apparently shown to orientate towards a C0^ source on agar plates, other considerations such as pH could also have been involved, a factor which may well be buffered against in the soil. Is CO^ the most important chemical emanating from a plant root or do complex chemicals act as more specific signs? This question was investigated by Peacock (1961) using an ionic exchange resin to absorb C0^ (allowed complex chemicals to remain in soil), and activated charcoal to remove complex chemicals (allowed CO^ to remain in soil). This seemed a suitable technique to use for T. viruliferus and hence soil-boxes were prepared in which activated charcoal (4% w/v) was incorporated into soil. The activated charcoal, however, appeared to have a nematicidal effect on T. viruliferus, killing 98% of the original inoculum. Peacock's original findings must, therefore, be considered in question until it can be demonstrated that activated charcoal does not have a similar effect on Meloidogyne incognita. Although not statistically signifi- cant, the few individuals which survived the experiment did seem to 112 103 have moved towards the plant. This offers some evidence that CO2 does play a part in attracting nematodes to roots, possibly in addition to complex chemicals. Modern interpretation is that CO^ acts as a long-range non-specific attractant and more complex chemicals act as short-range attractants (Croll, 1970). On examination of the data obtained in soil-box experiments (section 6.3) it appeared that not all individual nematodes could approach the plant root-system as fast as others viz, • Adults moved faster than larval forms (section 6.3.i). • Females moved slightly faster, on average, than did males (section 6.3.11). ' • Individuals with the greatest quantity of stored lipid reserves tended to move faster than others (section 6.3.iii). Adults are assumed to move faister than larvae because of differences in body size and strength; females faster than males because of hindrance to males by copulatory muscles in tail, and those .individuals with the greatest food reserves are able to supply muscles with an ample energy source, the implication being that the muscles in other individuals may be working less efficiently. A certain anomaly exists in two of the above categories in that the individual nematodes most requiring to feed, the larvae and the individuals with the lowest reserves, will take the longest time to reach the feeding site. The feeding site is only temporary, until root growth ceases, so the slowest nematodes may sometimes reach the site after such time as feeding is possible. However, it is suggested, in 80 104 light of evidence from other dorylaim species, that feeding may not be necessary for development to subsequent larval stages. If this is true for T. viruliferus there may be few disadvantages for larvae not to feed provided they are not "activated" into using up an excess amount of their food reserves. Soil, which had previously been pasteurized, was used as bands in a series of soil-boxes to investigate the possible role of micro- organisms in amplifying chemical signals for nematodes. A striking effect was observed in which heated soil was apparently repellent to nematodes unless there was a strong level of root attractant present. Reasons for the heated soil to have such an effect were reminiscent of McNamara's (1980) work on survival of Xiphinema diversicaudatum in field and heated soil. |4is work suggested the heat treatment affected a biological entity in the soil which might have been an alternative food source for _X. diversicaudatum or, alternatively, an organism which produced quiesence-inducing chemicals. Experimental treatments had excluded the possibility of any chemical changes occurring in the soil being responsible for the observed results. Evidence had already been gathered in this study on a particular aspect of nematode survival, i.e., existence of two activity states, so it seemed sequential to compare the survival of T. viruliferus in soil with that of X. diversi- caudatum. This formed the second part of my investigation. Although the survival experiments were carried out on T. viru- liferus alone it was considered possible that the results could be relevant to an understanding of how many other species of plant-para- sitic nematode were able to endure plant-free periods in soils. (See Table 7.) Indeed, a temporary reduction in activity as a response to a lack of food could have implications for many other species of animals. Animal-parasitic nematodes, for instance, are known to become more active when they come into contact with physico-chemical signals from their host e.g. hookworm larvae (Lane, 1930 and Fulleborn, 1932), Strongyloses stercoralis larvae (Fulleborn, 1932) and Nippostrongylus brasiliensis (Cunningham, 1956): this has direct consequences for in vitro culture of these animal parasites (Roberts & Fairburn, 1965). Many insect larva become totally inactive (diapause) at times of the year, although often to avoid unfavourable climatic conditions e.g. Stenothrips graminum (Franssen & Mantel, 1965). Other soil inverte- brates such as the worm, Cognettia sphagnetorum (01igocheta:Enchytrae- dia) are also reported as entering on inactive form to help reduce their overwinter respiratory cost (Standen, 1973). Examples of temporary inactivity can also be recognized within the classes of the Chordata as hibernation, aestivation or diurnal torpidity, all utilized to con- serve energy during adverse conditions (Odum, 1971). During the course of storage it was shown that J. viruliferus became increasingly pale and it was considered that translucent nema- todes usually died within 3-4 weeks if given no access to food. Well satiated nematodes appeared very dark in the intestinal region when viewed microscopically with transmitted light. This is generally inter- preted as the intestinal cells being well filled with lipid droplets and, because of the refractive properties of these lipid spheres, no light will seem to pass through them, hence their dark appearance. During periods of starvation nematodes utilize these lipid reserves as an energy source; the longer the period without food, the paler the nametode becomes. This decrease in darkness of body colouration with 80 106 starvation time can be utilized for assessing the physiological state of J. viruliferus on extraction from survival experiments. The system was based on that of McNamara (1977) and consists of delegating nema- todes to one of three colour classes with an index score of 1, 2 or 3. This system affords a manner of assessing a "survivability index", by adding up the scores for one experimental pot. If all pots are inoculated with the same number of nematodes then any individuals which die of starvation are effectively included in the survivability index by receiving a score of zero. Survival experiments were carried out in small clay pots main- tained at constant temperature and moisture content. However, the decrease in nematode body contents was considerably faster than that observed in the field, or even in large bags of soil. It is possible to explain this observation by one or more of the following suggestions. Increased oxygen availability. In the field there is a decrease in the availability of oxygen with depth, i.e. a tendency towards anaerobiosis with depth. A lower oxygen level would cause a reduction in the rate of lipid metabolism because lipid, the major constituent of stored food reserves, requires oxygen for its breakdown (Van Gundy et al., 1967). In small pots of soil oxygen is assumed to be more freely available than at the depths in field soil at which X* viruli- ferus normally occurs (15-30 cms). Increased storage temperatures. At increasing temperatures the rate of nematode respiration and activity increases (Thomason et al., 1964) and this might explain the observation of Krusberg (1959) that Tylenchorhynchus claytoni stored at higher temperatures were conspicu- ously devoid of body contents compared with nematodes stored at low temperatures. Pot experiments were carried out in a growth chamber maintained at 17°C, a much higher temperature than were the nematodes stored in a polythene sack (2°C or the varying temperatures (1°C - 19°C) recorded in the field. If the biological activity of the nematode increased when nematodes are stored in pots then the rate of energy depletion must also be increased (Bhatt & Rohde, 1970). Handling of nematodes. Nematodes used in pot experiments were extracted from soil using a Seinhorst Elutriator and were of necessity "handled" during the inoculation into pots. Nematodes in the field or stored in sacks have obviously not been subjected to such a treatment. Since J. viruliferus is notoriously averse to being "handled" (see section 2.B.1) it may be that this in some way interferes with the nematodes' ability to survive. There are obvious advantages in using an experimental system which produces results in the shortest period of time. However, there may be unexpected hazards, as outlined above, so care is needed when basing recommendations for use in the field upon laboratory experiments. X* viruliferus was shown to use up its stored lipid reserves much faster in heated soil than in untreated field soil. On further investigation it was demonstrated that a critical temperature existed to which soil had to be heated before any effect was observed. This was not accurately assessed but considered to be about 70°C. One further variable which was tested was to air-dry a sample of soil before heating above the critical temperature. This latter treatment had the effect of causing the lowest survival rate after a six-week experimental period. Experiments were conducted to determine whether starved (pale) 80 108 nematodes were capable of replenishing lipid reserves in their intesti- nal cells. This was shown possible when pale nematodes were given access to apple roots in soil but no evidence was obtained to show that pale nematodes could assimilate lipids from storage in field soil alone. McNamara (1980) postulated three hypotheses to explain how X.. diversicaudatum might survive for long periods of time in fallow soil, that this nematode may feed directly on microorganisms as an alternative food source; that microorganisms might secrete energy- containing substances which can be utilized by nematodes or secrete substances which could induce nematode quiescence. The evidence to support the three hypotheses above is criti- cally discussed in this thesis. It is considered unlikely that J. viruliferus is obtaining food from an alternative source because pale nematodes were not able to regain body contents when stored in field soil, also because of the complex mouthparts and feeding strategy of the genus Trichodorus. However, that some form of interaction with microorganisms existed which could affect nematode activity did seem possible. McNamara suggested a positive relationship with micro- organisms, i.e. induced quiescence, but I suggest, in light of my own work, that it is more likely that a negative interaction exists. This is interpreted as T. viruliferus naturally becoming less active in fallow soil, i.e. inactivated state, as an energy-conserving mechanism. When soil is heated to 70°C plus, most microorganisms are killed except for the thermoduric, endospore-forming species such as Bacillus spp. These bacteria are known to produce toxins to nematodes and also known to respond to dampening of heated soil which had been pre-dried. It 80 109 is interpreted that in field soil nematodes exist in the inactivated state until stimulated to the activated state by plant root-exudates. However, in heated soil the toxins produced by Bacillus-like species induce the nematode to enter the activated state in an attempt to move away from a chemically unfavourable region. Constant movement in the experimental pot causes rapid starvation of the nematode and hence poor survival in the soil treatment. It is further suggested that storage of nematodes in any medium which is either physically or chemically unsuitable to that nematode species is likely to induce the rapid movement of an "escape response", with the correspondingly reduced capacity for survival. This might include McNamara's (1980) survival of _X. diversicaudatum in sterilized sand where a survival time, comparable to that of heated soil, was obtained yet with the obvious omission of Bacillus-like microorganisms. A "population" of J. viruliferus was stored in a large volume of soil at a lower temperature (2UC) for 25 months. Assessment of the population structure at the beginning and at the end of the storage period demonstrated the following. The proportion of larvae to adults decreased with time and that reproduction is considered unlikely to have occurred. Two theories were suggested for the decrease in percentage larvae. Firstly, that some larvae had moulted into adult forms during the course of storage and secondly, that larvae could not survive in plant-free soil as successfully as adults. From the evidence presented it was not possible to deduce which theory was the most probable. T' viruliferus was shown to be capable of surviving for periods of at least 25 months in laboratory conditions suggesting that survival 112 110 experiments in the field would not be feasible because of a shortage in available time. Unpublished data, however, was available at East Hailing Research Station on the depth distribution of TV viruliferus in fallow soil. This was considered suitable data with which to compare the survivability of J. viruliferus in soil taken from similar depth layers. A high correlation existed between survival and the distri- bution of J. viruliferus with depth. This suggests that some factor was common to both and was not directly related to moisture content, temperature or oxygen availability of the soil. It is to suggest that a positive relationship may have existed between J. viru- liferus and a soil microorganism, the "survival factor", lending support to one of McNamara's (1980) hypotheses. The evidence for this was not conclusive. 1* viruliferus is apparently well adapted to its soil environ- ment and like most successful organisms demonstrates positive inter- actions with other species, in this instance with host plants and possibly soil microorganisms. The ability which J. viruliferus had for obtaining food relie4 upon detection of some chemical produced by that food source; an orientated movement through the complex physical media ' of soil and finally exploitation of a root-tip feeding site. At the feeding site two important physiological events must take place, breed- ing and the storage of food reserves inside the intestinal cells. When the feeding site is no longer viable the nematode responds by, at first, moving away from the decaying root and then it is presumed to remain in an inactivated state in the soil until another root grows close to that nematode. During times of no root growth the nematode is assumed to remain inactive, slowly metabolizing its food reserves. 80 111 This allows the nematode to survive for long periods of time in soil without food. Different nematodes are assumed to respond to different levels of attractant. This may result in some nematodes attempting to locate roots at great distances away from them, if they respond to very low levels of attractant or, alternatively, fail to respond even to roots growing close to them, if they respond only to high levels of attract- ant. In a monocultural system this would not seem to share any advantage to the nematode but it could be important if, in more natural conditions, a wide host range is to be exploited - not all hosts may produce the same level of attractant, or even the same chemicals. Soil is a bcdotficed system in which little change in chemical composition occurs. This allows nematodes to exploit small concen- tration gradients of specific chemicals. However, since X. virulif- erus is considered a pest-species on many crops it may be possible for scientists to formulate nematicides which could operate by interfering with these signals, i.e. "to manipulate the chemicals signals to his (man's) advantage and to the disadvantage of the nematode" (Bone & Shorey, 1977). This might allow control of nematodes in such a way as to be environmentally safe (Baldwin 1977) and, unlike most chemical control methods, produce little selection pressure to favour pesticide- resistant pathogens (Wright, 1981). 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Nematol. _3> 276-279 SUTHERLAND, 3.R. & SLUGGETT (1974) Time, temperature and soil moisture effpets on Xiphinema bakeri nematode survival in fallow soil Phytopathology 64, 507-513 TANIGUCHI, R. (1933) Notes on the chemotactic response of Rhabditis filiformis Butschlii 'Proc. imp. Acad. Japan 9, 432-435 TARJAN, A.C. (1960) Longevity of the burrowing nematode, Radopholus similis, in host-free soil Phytopathology 50, 656-657 TAYLOR, C.E., THOMAS, P.R., ROBERTSON, W.M. & ROBERTS, I.M. (1970) An electron microscope study of the oesaphageal region of Longidorus elongatus Nematologica 16, 6-12 TH0MAS0N, 1.3., VAN GUNDY, S.D. & KIRKPATRICK, 3.D. (1964) Motility and infectivity of Meloidogyne javanica as affected by storage time and temperature in water Phytopathology 54, 192-195 THORPE, W.H., CROMBIE, A.C., HILL, R. & DARRAH, 3.H. (1947) The behaviour of wireworins in response to chemical stimulation 3. exp. Biol. 23, 234-266 VALDEZ, R.B. 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EXTRACTION EFFICIENCY AND HANDLING DEATHS OF T. VIRULIFERUS In experiments studying the attraction or survival of J. viru- liferus soils were inoculated with nematodesleft for a period of weeks and then extracted using a modified form of Flegg's (1967) technique (see section 2.B.2.iv). To determine the losses of nematodes result- ing from inefficiencies in the extraction technique and from physical/ osmotic damage during handling or processing, the following experiment was conducted. J. viruliferus were extracted from field soil with a Seinhorst Elutriator (196^ and 175 added to each of two series of five pots. The -SoA inoculated by removing a 0.5 cm diameter soil-core, pouring the nematode suspension (c 1 ml) into the hole and carefully replacing the core (see section 3.2.iii). One series of pots was screened after 3 hours to determine the extracting efficiency of the modified technique of Flegg (1967). The organic debris collected from this extraction technique was placed on to nylon screens and suspended in water for 20 hours. After this time the nematodes which had passed through the screens were counted and the humus remaining on the screen gathered to determine whether any T. viru- liferus had failed to pass through the final screen (this included those nematodes damaged by the handling processes of the extraction technique). Using a sugar centrifugation separation technique (Flegg & McNamara, 1968) it was possible to show that in only two replicates did single X• viruliferus fail to pass through the screen. The number of nema- todes passing through the screen was considered to be a close approxi- mation to the true value of extraction efficiency since any nematodes suffering from handling damage were recovered by the sugar centrifug- ation technique outlined above. The second series of pots was left for 3 days to determine the numbers of ]_. viruliferus which had died in the intervening time between inoculation and extraction. This short time period was selected in order to record those nematodes dying as a result of handling damage and minimizing the additional factor of natural mortality. Nematodes inoculated 175 Nematodes after 3 hours 149.6 - 5.1 Approx. extraction efficiency 86% Nematodes after 3 days 75.2 - 6.8 Total losses 57% Table 12. Experimental findings on extraction efficiency and effect of mortality factors in pot experiments. (Numbers of nematodes presented with their standard errors.) The efficiency of extraction is acceptable at 86% and is higher than that expected for a normal field soil sample. However, in the experimental procedure used above, the soil had previously been sieved and thus the soil was of a homogeneous texture. The value itself, though, may be slightly higher than the true value because it is recog- nized that nematodes recently added to soil are easier to extract than those which have been in situ for some time (Flegg, pers. comm.). This is because the latter have had the opportunity of moving into some of the smaller pore spaces between soil particles, etc., from which it can be difficult to extract. Total losses after 3 days (57%) include nematodes lost in the 101 128 extraction procedure and those dying, probably as a result of physical damage during extraction. In the experiment described in section 3.5 the losses after 4 weeks (same experimental conditions) amounted to 53%, a value similar to that obtained just 3 days after inoculation in this experiment. The inference from this is that few nematodes die after the initial loss, in pot experiments, until, of course, star- vation and/or natural mortality factors become important. 129 95 6.2. TAXONOMIC AUTHORS OF ORGANISMS CITED IN TEXT a. Nematodes Aphelenchoides besseyi Christie A. fragariae (Ritzema-Bos) Christie Aphelenchus avenae Bastian Caenorhabditis elegans (Maupas) Dougherty Criconemoides xenoplax Raski Ditylenchus dipsaci (KUhn) Filipjev [). triformis Hirschmann & Sasser Globodera rostochiensis (Wollelweber) Mulvey & Stone Helicotylenchus dihystera (Cobb) Sher H. digonicus Perry IH. indicus Siddigi Hemicycliophora arenaria Raski HL paradoxa Luc H. similis Thorne Heterodera avenae Wollenweber H. schachtii Schmidt Hoplolaimus indicus Sher Longidorus africanus Merny caespiticola Hooper !L. elongatus (de Man) Thorne & Swanger L_. qoodeyi Hooper I_. leptocephallus Hooper L. martini Merny Meloidogyne hapla Chitwood M. javanica (Treub) Chitwood M. incognita (Kofold & White) Chitwood Nippostrongylus brasiliensis (Travassos) Lane Panaqrellus redivivus (Linnaeus) T. Goodey E.' silusiae (de Man) T. Goodey Pelodera coarctata (Leuckart) Dougherty IP. teres Schneider Pratylenchus minyus Sher & Allen P. penetrans (Cobb) Chitwood & Oteifa P. scribneri Steiner P. thornei Sher & Allen Radopholus similis (Cobb) Thorne Rotylenchus reniformis Linford & Oliveira Strongyloides stercoralis (Bavay) Stiles & Hassall Trichodorus christiei Allen T_. proximus Allen T_. similis Seinhorst X* viruliferus Hooper Tylenchorhynchus claytoni Steiner Tylenchus semipenetrans Cobb Xiphinema americanum Cobb X. bakeri Williams X. diversicaudatum (Micolitzky) Thorne X. index Thorne & Allen Enchytraid worm Coqnettia sphagnetorum (Vejdovsky) Insects Psila rosae (Fabricius) Sinus coeca Schbtt Stenothrips qraminum IJzel Fungi Fusarium oxysporum Schlecht Gaeumannomyces qraminis (Sacc.) Arx & Olivier Phytophthora cinnamoni Rands Pythium aphanidermatum (Edson) Fitzpatrick 26 132 6.3. OTHER ECOLOGICAL DATA RECORDED During some of the experiments, other information was recorded of the nematodes besides numbers in each section, such as counting the ratio of males to females to larvae; whether females were gravid, and colour scoring the nematodes' stored food reserves, on the same basis as McNamara (1980). This is summarized in the following section. Not all criterion were measured of all nematodes, but in all instances a 14-day experimental period and an inoculation distance of 7 cm was used. 99 133 6.3.i SEX RATIO WITH DISTANCE Pitcher & McNamara (1970) showed that root tip feeding sites were also the main breeding sites for J. viruliferus. In view of the necessity to attract both males and females to the same site they speculated that there might be some pheremonal action to ensure that opposite sexes met. This might operate by the females producing a chemical to attract males, as occurs in Heterodera and Globodera spp (Green, 1966a and Green & Plumb, 1970) and Rotylenchus reniformis (Nakasono, 1977) or by both sexes producing different pheremones to attract the opposite sex, as in Panagrellus redivivus (Greet, 1964). The latter seems unlikely because, during orientation towards a plant root there could be conflicting signals, to respond to the plant root ahead or nematodes elsewhere. To test the possibility of pheremonal action occurring during the migration phase, the variation in sex ratio with distance from the inoculation point was studied using information from four soil-boxes. (as) The number of nematodes recovered/at each distance and of each sex was analysed (ANOVA) in log (1+x) transformation for the different replicates. This method was used after consultation with the Statis- tics Department, East Mailing Research Station. There were signifi- cant differences between the four replicates (P< 0.001), between the sexes (P< 0.001) and among the distances <(P< 0.001), but the sex x distance interaction was not significant (even at the 20% level), indicating that the pattern of distribution of the nematodes was similar OM be kiddeM b^ U mi taJHcns <=>f pcrhcod<*r stoicsKc<4 e suggestfor botsh sexethat s malealHncugs werUe £wanot U being attracte••d XoClsolel^'ha^d'QCujflfley to a pheremona^ j^Thil s j chemical produced by the females moving ahead of the<*\ and that 134 101 both males and females were equally attracted to roots. In a few experiments the nematodes which had travelled the furthest were males: these could not possibly have orientated to a female pheremone. However, although there appeared to be no pheremonal attraction between the sexes, it seemed that male _T. viruliferus were, on average, not able to move towards the plant quite as fast as females. An indi- cation of this can be demonstrated by reference to the value determined in the following equation, 92%. Nematode.cm per male ^QQ Nematode.cm per female This small difference possibly resulted from the development of copulatory muscles in the tail of male Trichodorus spp (sensu stricto) impeding locomotory efficiency. These copulatory muscles, in the latter 20% of the body, allow the tail region to bend and thus intro- duce its spicules in the female's vulva during mating. The extent to which this adaption occurs is clearly observed when a male Trichodorus sp. is "heat relaxed" (Hooper, 1970), since it forms a characteristic "walking-stick" shape whereas males of the closely related genus Paratrichodorus, with less well developed copulatory muscles, remain straight. The soil-box system does not lend itself to investigations to distinguish whether nematodes at a feeding site produce a pheremone or a secondary attractant because of the indirect method of measuring any such response. It may be that chemicals produced in normal nematode excretory material e.g. NH^ were detected by approaching nematodes and used as a basis for a specific orientation towards a nematode aggre- gation. 101 135 6.3.ii RATIO OF LARVAE TO ADULTS WITH DISTANCE Van Gundy (1965), referring to some unpublished work of Black & Van Gundy, suggested that the larvae of Pratylenchus scribneri moved more rapidly than adults, when in the presence of a gradient of root exudates from maize. This contrasted with movement in the absence of root exudate when larval movement was generally less rapid than that of adults: this has also been shown for Panagrellus redivivus (Pollock & Samoiloff, 1976) in response to bacteria. In order to test whether the same was true of _T. viruliferus, the percentage larvae of the total, within each section, was recorded. A line of best fit was calculated for the points (see fig. 10) described by the formula, y = -3.36JC +35.9 The apparent decrease in the percentages of larvae with distance from the inoculation point has been calculated to be statistically significant at the 1% level (P<0.01). The results from this study imply the opposite to P. scribneri in that larval J. viruliferus do not generally move faster than adults, whilst moving towards a plant. The behaviour of P. scribneri might perhaps, therefore, turn out to be atypical for those plant-parasitic species of nematodes in which all cohorts are migratory. The slower migration rate of larvae compared to adults could be predicted as a result of their small size (Wallace, 195?). That larvae move more slowly than adults could explain why some of the aggregations on apple, roots observed by Pitcher & McNamara (1970) contained over 50% adults, a much higher proportion than in random soil samples, because most larvae had not had time to reach the aggregation 136 50 40 o %O LARVAE of total 20 IO • INOC POINT DISTANCE (cm) travelled from inoculation point Figure 10. The variation in percentage of larvae with distance from the inoculation point (P = plant). Results from 3 replicates Numbers of nematodes used in the analysis = 1735 Experimental period =14 days 102 137 sites. Feeding sites are temporary in time (a minimum of 10 days has been recorded) and so the smallest T_. viruliferus larvae might be expected frequently to arrive after the commencement of degeneration and decay of cells in a root tip, i.e. unsuitable as a feeding site. Small larvae would thus seem to be at a disadvantage with regards to successful feeding compared with members of the larger cohorts of J[. viru- liferus. This could cause a very slow, or even a lack of maturation of small larval forms if feeding was necessary in nematode development. However, evidence from other dorylaimid nematodes indicates that small larvae can develop to larger forms without feeding (Griffin & Barker, 1966; Sutherland & Ross, 1971 and Sutherland & Sluggett, 1974). It is possible, therefore, that J. viruliferus can also grow and moult to larger larval forms without feeding. Evidence presented in section 3.2 supports this hypothesis. Presumably, as the larval forms increase in size, they too will become better able to exploit suitable feeding sites. 138 103 6.3.iii NEMATODE MOTILITY IN RELATION TO STORED FOOD RESERVES From work on Meloidoqyne javanica and Tylenchus semipenetrans, it is known that nematodes store food as lipid in gut cells* (Van Gundy et al., 1967) and that this makes the nematode appear dark in trans- mitted light (McNamara, 1980). Using the coding of optical density devised by McNamara (1980) for Xiphinema diversicaudatum, it was poss- ible to categorize individual J[. viruliferus into one of three classes depending on the amount of lipid present (see plate 5) and to give the classes a score of 1, 2 or 3. By calculating an average score per nematode, for each 1 cm section of the box, it was possible to deduce whether there was any relationship between stored food reserves and motility. From nine replicates it was possible to compare mean optical density scores with the distance travelled from the inoculation point (see fig. 11). In J. viruliferus, compared to X.. diversicaudatum, there appears to be only a very short duration of the "pale" state, approximately 3 to 4 weeks (see section 3.4). It is assumed that death follows this condition and hence few pale individuals are encountered (only c. 2% of nematodes from field soil); this explains why most of the mean colour scores fall between 2 and 3. The data from this experiment was tested to a number of mathe- matical models, in consultation with the Statistics Department, East Mailing Research Station, and the model: * Considerable amounts of glycogen occur in some species of nematode and this has been implicated as a further food reserve material (Krusberg, 1971). 139 DISTANCE (cm.) travelled from inoculation point Figure 11. Mean colour scores of _T. viruliferus plotted against distance from inoculation point. Number of nematodes used in analysis = 1286 Experimental period = 14 days 104 140 y = a + cdc was found to be most satisfactory. Here, x equals the distance from the inoculation point and y equals the mean optical density score. This model accounted for 82% of the variance. The derived values of the parameters, with standard errors, were: a = 2.025 - 0.018 b = 0.0208 ± 0.0202 c = 0.0108 t 0.0037 This analysis indicates that dark nematodes move faster than nematodes with less food reserves, which effectively means that, in one sense, the nematodes most needing to feed are the slowest ones. Since the feeding site is temporary, lasting only as long as it takes an aggregation of nematodes to kill the particular root, it must sometimes be that the paler nematodes use up already declining food reserves in moving towards an obsolescent feeding site. It might have been expected that the nematodes most requiring food reserves would be the fastest nematodes to reach the food source but these experiments indicate that the motility of these nematodes, and hence the chance of feeding, is reduced with decreasing amounts of stored energy (lipids). To test whether dark nematodes, those with most food reserves, were generally more active than paler nematodes or whether this was only so whilst under the influence of host-plant root exudates, a further experiment was devised. The time taken to pass through sieves in the final stage of the normal extraction procedure was used as an index of motility. This is an active process whereby nematodes crawl through the apertures (53jj diameter) in a sieve suspended in a Baerman Funnel (Flegg, 1967), probably in response to a positive geotropism. By 102 141 recording the number of pale, dark and intermediate coloured nematodes which had passed through the sieve after a given extraction time it was possible to show whether dark nematodes crawled through the sieves faster than the others. Six replicates of the experiment were set up using sampling times after 2, 4, 16 hours and "infinity" (infinity = 48 hours, the time after which no more nematodes were recovered). See Table 13. Time Interval Mean cumulative Mean cumulative Level of sig. (hours) % of dark % of others between colour classes 0-2 69 46 P< 0.001 2-4 90 72 P<0.02 4-16 99 96 P<0.01 16 - £>o 100 100 ut i Table 13. The mean cumulative percentages of dark and paler nematodes for different time periods and the levels of significance between the emergence of the two classes. Number of nematodes used in analysis = 3468 Percentages of nematodes passing through the sieves after 2, 4 and 16 hours were analysed in angular transformation and the values quoted in Table 13 are back transformed from the mean. The analysis showed that dark nematodes are more active than others (pale and intermediate nematodes) in the extraction process and so suggests that the activity of T. viruliferus is somehow related to its level of stored food reserves. K. J. DERRETi? Ph.D 1983 2. B. 3' Movement of T. viruliferus in response to an apple plant. (Numbers of nematodes per 1cm section) Section Replicates Number 1 2 3 4 5 6 7 8 6 1 -4 2 4 4 3 1 6 5 1 1 1 -2 3 3 10 3 4 3 2 2 -1 2 14 64 6 4 22 3 27 N INOC PT. 3^ 39 53 18 3^ 124 36 15^ 1 12 20 20 9 51 85 214 75 2 5 19 31 7 30 38 37 29 3 1 12 43 16 27 22 25 16 4 1 6 17 16 23 23 12 19 5 2 9 7 9 31 12 3 14 6 1 16 63 17 37 12 7 42 PLANT 5 1 K. J. DERRETi? Ph.D 1983 2.B»4 Effect of inoculation distance on the movement of T. viruliferus towards an apple plant ~ (Numbers of nematodes per 1cm section) Section Replicates Number 1 2 3 1 2 3 1 2 3 3 3 1 -3 3 2 1 1 3 1 2 1 -2 8 9 2 6 3 6 3 5 4 -1 11 13 9 12 16 12 19 18 12 XNOCoPT 24 27 33 41 50 k? 62 52 k? 1 41 30 17 28 42 26 23 23 15 2 59 42 25 23 22 18 22 17 16 3 ^3 39 19 25 17 14 2 0 15 12 39 37 17 12 17 7 9 10 5 P L A N T 19 8 4 9 8 6 6 17 7 4 7 6 5 7 6 8 33 5 2 3 8 P LAN T 2 2 2 9 0 1 3 PLANT K. J. DERRETT Ph.D 198.3 2.B.4. Effect of inoculation distance on the movement of T. viruliferus towards an apple plant. (Numbers of nematodes per lcrn section) Section Distance from inoc. pt to plant Number 4cm 7cm 10cm 1 3 -2 2 3 4 -1 8 4 11 inocn.ft. 10 12 23 1 9 5 16 2 9 6 17 3 26 2 5 4 10 3 11 5 PLANT k 3 5 6 10 4 7 5 4 8 PLANT 1 1 9 0 PLANT 0 K. J. DERRETi? Ph.D 1983 2 0 B 0 5 • Movement of To viruliferus in the absenc; e of a plar it. (Numbers of nematodes per 1cm section) Section Replicates Number 1 2 3 k 5 2 1 1 1 4 5 2 1 10 3 5 5 0 11 2 12 15 k 12 1 14 14 2 40 INOC.PT • 61 56 6 3k 1 20 k3 13 13 2 18 20 6 9 3 13 6 1 3 4 2 1 1 k 5 1 0 0 3 K. J. DERRETi? Ph.D 1983 2.B.6. Effect of inoculating between two plants in a soil-box, (Numbers of nematodes per 1cm section) Section Replicates Number 1 2 1 2 PLANT 7 2 3 6 3 1 5 PLANT 3 k k 3 3 3 3 9 7 k 0 2 10 22 3 3 1 20 10 9 6 INOCePT 12 29 27 1 8 7 5. 10 2 0 6 3 19 3 0 7 k 12 k 0 19 6 9 3 PLANT 3 3 6 0 5 7 2 k PLANT K. J. DERRETi? Ph.D 1983 20B.8. Movement of nematodes when bands of heated soil were set into soil - boxes of unheated soil. (Numbers of nematodes per 1cm section) Section Replicates Number 1 2 3 1 2 3 1 2 3 Plant j 1 J 6 0 8 rO 0 8 31 126 31 I 3 0 9 46 ^ 0 0 65 75 59 ° 9 8 42 cO 0 oJ 39' 52 22 3 37 14 84 1 4 7 27 70 42 2 79 26 66 7 3 17 33 12.2 29 1 r44 50 11 23 80 ;;6 73 35 15 ? INOC.PT < Si 26 10 > 22 53 68 27 15 26 -1 -17 53 20 J 14 3k 81 44 ? 13 -2 14 15 6 26 14 k9 30 11 16 -3 22 15 3 21 20 27 31 0 12 -4 3 3 1 <0 5 5 28 0 4 ^ =band of heated soil