1 Interrelationships of the Cereal Cyst Nematode

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INTERRELATIONSHIPS OF THE CEREAL CYST NEMATODE, HETERODERA AVENAE, AND THE TAKE-ALL FUNGUS, OPHIOBOLUS GRAMINIS ON BARLEY.

Roger Cook

Department of Zoology and Applied Entomology, Imperial College of Science and Technology.

A thesis submitted for the degree of Doctor of Philosophy of the University of London. 2

ABSTRACT

Observations on a number of field populations of the cereal cyst nematode, Heterodera avenae and the take-all fungus, Ophiobolus graminis, on barley, suggested a relationship between the two, in which higher nematode populations were associated with low incidence of take-all. The level of root invasion by H. avenae and multiplication of the nematode showed this relationship with the fungus. Further observations, in which the number of eggs per cyst was negatively correlated with level of take-all infection suggested one explanation of the relationship. The effects of 0. graminis on H. avenae were studied in a series of pot experiments. In the presence of high levels of take-all the multiplication of natural populations of the nematode was depressed, and this was associated with reductions in the number of eggs per cyst. Experiments in sterilised soil showed that production of cysts and their contents were decreased by take-all. Smaller cysts were produced on plants infected with O. graminis. On take-all infected plants the rate of development of the nematode was slower and the ratio of males to females produced was higher than on uninfected plants. Take-all appeared to affect the sex determination of larvae, fewer females developing in infected roots. Invasion of barley roots by H. avenae larvae was reduced by prior infection with O. Araminis. In none of the experiments was the degree or severity of take-all affected by the presence of the nematode. 4

ACKNOWLEDGEMENTS

I wish to thank Professors O.W. Richards and T.R.E. Southwood for permission to work in their department. I owe much to the encouragement and advice of Dr. N.G.M. Hague who supervised the work presented in this thesis. Officers of the National Agricultural Advisory Service, Reading, gave valuable assistance, particularly during the early stages of my field work, To these, and the farmers whose fields I visited,I express my gratitude. Especial thanks go to my wife who typed this thesis and gave me every support. The project was financed by an Agricultural Research Council grant, which I gratefully acknowledge.

5 TABLE OF CONTENTS Page Title Page 1 Abstract 2. Acknowledgements 4 Table of Contents 5 SECTION I Introduction 7 SECTION II A Field Observations, 1967 16 B Field Observations, 1969 37 SECTION III A Pot experiments with field soils 1. Introduction 45 2. Materials and methods 46 3. Experimental details and results a Effect of adding 0. graminis to H. avenae populations in eight field soils 51 b Effect of adding O. graminis to three populations of H. avenae in one field soil ▪ • 59 c Effect of O. graminis on Meloidogve naasi in naturally infested soil 66 d Resistance of oats to O. graminis in the presence of three nematode populations 73 4. Discussion 79 B Pot experiments in sterilised soil 1. Introduction 82 2. Materials and methods 82 3. Experimental details and results a Effects of three levels of O. graminis on final populations of H. avenae 84 b Effect of O. graminis on invasion of roots and development of H. avenae 88 4. Discussion 96

Page SECTION IV Further studies on the effects of 0. graminis on development of H. avenae 1. Introduction 100 2. Experimental details and results a Experiment 1 100 b Experiment 2 106. 3. Discussion 111 SECTION V Further studies on the effects of O. graminis on the invasion of barley roots by H. avenae. 1. Introduction 116 2. Experimental details and results a Effect of level of take-all infection on invasion of roots by. H. avenae 117 b Invasion of barley roots by H. avenae in relation to the position of a take-all lesion • • 120 c Invasion by H. avenae of healthy roots of take-all infected root systems 124 d Attraction of H. avenae larvae to take-all infected barley roots 125 3. Discussion 128 SECTION VI General discussion and conclusions 131

References 149

Appendix tables 162 SECTION I

Introduction

This thesis presents the results of investigations into the interrelationships between Ophiobolus Framinis Saco., the causal fungus of the take-all disease, and Heterodera avenge Woll., the cereal cyst nematode, on their common host, barley. Both the nematode and fungus are soil borne pathogens of cereals and have become of greater economic importance as a result of changes in British agriculture since the Second World War. Increased cereal production has resulted in cereals being grown in shorter rotations and in some regions they are grown continuously on the same land. Large acreages of barley are grown continuously in farming areas relatively close to Imperial College Field Station, so that it was convenient to study the relationship between take-all and the cereal cyst nematode on barley. The fungus 0. graminjs causes the take-all disease of cereals. In the field the disease appears as more or less circular patches of stunted unthrifty plants. Under dry summer conditions severely infected plants may die prematurely and the ears of these plants have a bleached appearance. However roots may be infected with take-all without the plant shavang obvious above ground symptoms. The grain produced on infected plants is small and shrivelled so that yields may be much reduced. At maturity the root systems are blackened and rotted and considerable secondary invasion by weakly parasitic or saprophytic fungi occurs. Infection takes place when roots come into contact with mycelium of the fungus, which has overwintered in the soil on plant debris infected the previous season. Coarse dark hyphae produce a network around the root surface and fine hyaline hyphae penetrate the roots. Garrett (1934) has called these hyphae, runner and infection hyphae, respectively. The infection procedure has been described by Fellows (1928) on wheat and proceeds in a similar fashion on barley. Runner hyphae develop on the epidermis and, from these, infection hyphae arise and grow radially through the cortex and into the stele. Spread of the fungus occurs only by contact with the root, when it spreads longitudinally along the outside and in the stele of the root (Garrett, 1942). Rotting of the cortical tissues produces dark lesions which may girdle the roots. Take-all is considered the most important root rot of cereals and considerable yield losses are attributable to it (Garrett,19423 Lester,1967). Conditions of intensive wheat or barley cultivation favour the build up of high levels of take-all, through reducing the period which the fungus must survive saprophytically in the soil, between its parasitic phases on cereal hosts. Some loss of the overwintering inoculum of take-all occurs during this phase of saprophytic survival, but inevitably in cereal monoculture, higher initial levels of inocula remain to infect successive crops (Garret & Mann,1948). It was feared that the increasing practice of cereal monoculture would lead to serious losses due to take-all, and that the disease might be a serious threat to the future of continuous cereal growing. These fears have been justified to a large extent (Lester,1967). However, considerable evidence has been accumulated to suggest that cereals may be grown continuously in Umny cases without maintaining economically unacceptable levels of take-all. This phenomenon has been described as "take-all decline" (Slope & Cox,1964). Shipton (1967) has established the "decline" phenomenon on a range of fields and also provided experimental proof. It appears that after four or five continuous cereal crops take-all reaches peak levels, and declines under succeeding crops to a level which is acceptable to farmers. It has proved profitable to continue growing cereals through the years of maximum take-all, to take advantage of the higher yields of subsequent crops. Evidence from several sources indicates that under conditions of continuous cereal production, a soil microflora antagonistic to 0. graminis develops 10 and this may be responsible for u1 aecline of the disease (Ehle,1966; Skipsna,1960; Zogg,1960). H. avenae, the cereal cyst nematode, is also parasitic on cereals. The life cycle of the nematode has been described by Franklin (1951). Larvae invade the host roots during spring and become sedentary within the root. In response to infection, the roots produce "giant cells", by division of cells and dissolution of cell walls. These are limited to the stefe and provide the nematode with a feeding site at which it remains throughout its development. The larvae grow and moult three times before reaching maturity. Males emerge from the root to fertilise the females. These remain attached to their feeding site, but exposed to the soil through the rupturing of the cortex, caused by their growth. After fertilisation the female produces eggs, which eventually pack all the space inside the female body wall. This hardens and thickens and it is in this encysted state that the nematodes survive the winter. After a period of inactivity the eggs hatch and the larvae invade the host roots in the spring. Although winter oats have been found infected by the larvae in autumn, there is only one generation of H. avenae produced each year on spring sown cereals (Gair,1965). Symptoms of cereal cyst nematode attack are a slight swelling of the infected root, accompanied by branching. 11

Severely infected plants have shallow, much branched root systems whose efficiency is much impaired. In heavily infested soil, plants may be stunted and die, or produce only poorly filled grain. Early attempts to estimate yield losses of barley caused by H. avenae were not entirely successful. However, since the introduction of barley genotypes resistant to the nematode, Cotten (1967) has demonstrated that average yield losses of 10% occur over a range of initial nematode infestations. Dixon (1969) has attributed a yield reduction of 0.6 cwt/acre (75 kg/ha) of barley for every 10 eggs of H. avenae per g of soil. H. avenae is thought to be indigenous to the British Isles (Gair,1965). It is widely distributed throughout the country, often found in old pastures and grass land. When these are ploughed and sown with cereals, crop failures may occur within a few years. The levels of H. avenae at which crop yields are affected may vary with soil and climatic factors, and these factors also affect population levels of the nematode (Dixon,1963; Fidler & Bevan,1963). Winslow (1960) has postulated that, as a result of repeated cropping with host plants, populations of cyst- forming nematodes will rise to a ceiling level. Above this, crop damage is too severe to allow further increaser and the population will fluctuate around the ceiling, depending on the effects of environmental factors. The level of the 12

ceiling will also depend upon the efficiency and tolerance of the host. My investigations were begun in the light of observations on H. avenae populations that conflicted with the hypothesis of a maintained ceiling level. Oollingwood (1962), and Empson (1965) reported decreases in numbers of H. avenae under continuous cropping with susceptible hosts on a number of field and trial sites. No explanation of this decline was apparent. Observations by Williams (1967) on H. avenae under spring wheat showed unexpected responses of the nematode to the application of a formalin soil drench. He found that a presowing treatment with formalin gave increased final populations of H. avenae, in spite of some nematicidal action of the formalin. Associated with these increases were reductions in the levels of root infection by take-all. From these observations it seemed that take-all might influence H. avenae populations in some way. Observations on another cyst forming nematode, H. rostochiensis on tomato, had indicated that the presence of root rotting fungi could adversely affect development of the nematode (Ketudat, 1968). The possibility that there was a relationship between H. avenae and 0. graminis was therefore investigated and attempts made to establish what effects• each organism may have on the other. 13

There is growing appreciation by plant pathologists and nematologists that, in the complex soil environment, it is likely that there will be interrelationships between plant parasitic nematodes and other plant pathogens. A wide range of interactions have now been reported. Attempts to summarise and classify these relationships have been made by several authors. Powell (1963) surveyed known and suggested nematode- fungus interactions. He observed that soil borne diseasea, which may be of complex nature, frequently involve nematodes. In his review, Powell cites several types of relationship. There are those in which the presence of plant parasitic nematodes incites or increases the severity of a fungus disease, and in some cases nematodes may be essential for the development of a disease. There are also relationships in which fungus infected roots increase the populations of plant parasitic nematodes. Powell concludes that the mechanisms underlying most interactions between nematodes and fungi are so far unknown. He speculates that the physiological and biochemical changes in roots infected by the partners in an interaction, are likely to be of importance in understanding these complexes. This line of thought was developed by Pitcher (1965), who used the type of relationship between the nematode and its host as a basis for classification of fungus-nematode 14

interactions. He also conaidored the ability of each of the partners to become established in the host on its own, as part of his classification. The following types of interactions are considered and illustrated with examples. Nematodes may increase the severity of disease caused by their interaction partner by acting as a vector. The other organism may or may not be capable of self-establishment when in contact with the host. There are organisms, particularly viruses, which cause disease only when introduced below the host epidermis by the nematode. The wounds caused by feeding nematodes serve as entry points for a number of soil organisms. The wound may increase disease merely by providing a point of entry or, through breakdown of cells, may provide a food base. This can serve to increase the inoculum potential of weaker, unspecialised pathogens. The modifications of the host initiated by some parasitic nematodes. in feeding in the root may aid the development of fungus diseases. The physiological and biochemical changes resulting from nematode infection may produce metabolites favourable to its partner in the interaction, or may breakdown chemicals antagonistic to it. The normal defence reaction of a plant may be obstructed by the action of nematodes. These types of interaction include some of the more spectacular cases in which resistance of 15

plants to fungi is broken down by nematodes. Pitcher considers that the inevitability of competition between nematodes and other organisms may affect either or both partners. Like Powell, Pitcher considers that understanding of the basis of the interactions he reviews is likely to lie in biochemical hypotheses. The relevance of other nematode—fungus interactions to that between 0. graminis and H. avenae is considered in this theses in the discussion sections. The nature of the relationship established by my investigations is also considered against the background of other interactions in the discussion. The results of these investigations are presented in four sections. The first of these records the results of two seasons observations of populations of H. avenae and O. graminis in barley fields. The second section contains the results of pot experiments on the interaction in naturally and artifically infested soils. In the next two sections, observations on development of the nematode and invasion of roots as affected by O. graminis are reported. The final section is a discussion of the results of these observations and experiments. 16

SECTION IIA

Field observations 1967

1. Introduction A programme of field sampling was carried out in 1967 in which populations of Heterodera avenae and Ophiobolus graminis were assessed throughout the season on a number of field sites. The levels of both the nematode and fungus at each site were compared to determine whether there was a relationship between the two under field conditions. Observations were made on 24 fields in Berkshire and Hampshire. The locations of the farms and fields are given in Appendix Table la. Selection of the fields was based on the number of cereal crops grown. The sites sampled provided a range of cereal cropping histories, from no previous cereal grown to ten cereals grown in successive years since the last break-crop. Fields on several soil types were included, the majority were on chalk but some were on heavier soils. Appendix Table lb shows the number of cereal crops grown and the soil type at each site. As far as possible pairs of fields were sampled on each farm to give one field at an early, and one at a later, stage in continuous cereal cultivation. 17

In 1967 all fields were sown with spring barley. It was considered that this selection, based on the numbers of susceptible crops grown, would provide sites infested with a range of populations of the cereal cyst nematode and the take-all fungus. The dates on which each field was sampled are given in Appendix Table lb. Five of the fields were not visited until the spring plant sample was taken, so that initial populations of the two pathogens are known for only 19 of the fields. These 19 were sampled in early spring before the crop was sown. Samples taken in spring and at harvest recorded the progress of infection of the crops, and final populations of the nematode were assessed in the winter following the 1967 season.

2. Sampling procedures and assessment methods. Populations of H. avenae and 0. graminis were estimated, from soil and plant samples, by the following methods. All bulk samples were taken from a minimum of 50 points on two diagonal traverses of each field. Initial soil samples. The bulk sample was made up of soil taken to a depth of about 8 in with a hand Crowell, to give about 20 kg soil from each field. These samples were brought from the field and then passed through a in sieve, lumps of soil were broken up and stones 18

discarded. The soil was then thoroughly mixed and two subsamples withdrawn by coning and quartering (Anscombe, 1950). The remainder of the soil was stored for use in pot experiments (Section III). One subsample of 1 kg of soil was used for estimation of the initial 0. &raminis level by a bio-assay based on the tumbler-test method (Thorpe,1966). Each subsample was potted in a 5 in diameter, black polyethylene plant pot and sown with sufficient barley seed (cv.Proctor) to give 50 seedlings. After 21 days growth in the greenhouse these plants were washed free of soil and their roots examined for take-all. The roots were floated, in water, over a white background and the number of lesions caused by 0. graminis recorded. The proportions of plants and roots bearing any take-all lesions were also recorded. Identification of the dark-coloured take-all lesions was confirmed by examining infected roots under a stereoscopic microscope for the presence of the characteristic dark- coloured, ectotrophic, "runner" hyphae of 0. graminis. Further confirmation was made by occasional isolations of the fungus from infected root pieces (see Section III for details of isolation techniques). In this way the take-all inoculum in each field was determined as % plants and % roots bearing any take-all lesion and as the number of lesions produced on the root systems of 50 plants grown 19

in 1 kg of soil. The second sub-sample of 400 g soil was used to estimate the H. avenae population level. The techniques described by Goodey (1963) for extraction of H.avenae cysts from soil were used. The sample was allowed to become dust dry in a heated drying cabinet. After washing through the Fenwick can, the "float", of cysts• and other organic debris, was collected on a 175/u sieve, washed onto muslin and dried. A second flotation was done in a 1 1 conical flask and cysts were hand picked, under a stereoscopic microscope, from this second, dried "float". The cysts so obtained were counted and their contents, of eggs and larvae, determined (Goodey ap.cit.). Thus, initial cereal cyst nematode populations were recorded as cysts per 100 g soil and as eggs and larvae per g soil. Spring plant samples. The degree of infection of the crops by both cereal cyst nematode and take-all was assessed from this sample, taken at 6 to 8 weeks after crop emergence. Fifty 4 in lengths of drill were lifted from each field with a small hand fork. A subsample of 50 individual plants was selected by taking one plant, at random, from each drill length. The root systems of these plants were washed free of soil and examined for take-all and cereal cyst nematode. 20

Take-all assessments were made in the way described for the plants from the tumbler tests. Lesions were counted separately on the seminal roots (those which develop from the seed) and on the whole root systems, (including the crown roots which develop at a later stage than the seminal roots, arising adventitiously from the lower nodes of the stem). The relation of these two root systems to the growth of the'plant and to infection by the fungus and nematode is considered in the discussion of this section. The roots were then blotted dry, stained in acid- fuchsin in lactophenol and kept in plain lactophenol to clear (Goodey a.cit.). Examination for the cereal cyst nematode was carried out by pressing the roots between two sheets of clear glass. This flattened out the cortex of the roots, which were then scanned with a stereoscopic microscope and the presence of H. avenae larvae noted. Counts of the larvae present were made on every fifth root system examined. Thus the measures of take-all and cereal cyst nematode infection were % plants and % roots infected by each and the number of lesions and larvae respectively. Summer plant samples. The final level of take-all developing in each field was determined by examining the 21

root systems of 50 plants, collected at harvest in the same way as the spring plant samples. The seminal roots were again scored separately and as part of the total root system. At this stage root systems were extensive and considerable invasion by secondary pathogens had occurred, so that individual take-all lesions could neither be easily nor accurately counted. Take-all was therefore measured only as % plants and % roots infected. Final soil samples. The soil population of H. avenae after the 1967 crop was estimated, as cysts/100 g soil and eggs and larvae/g soil, by the techniques used for the initial soil samples. This sample was taken early in February, 1968. An outbreak of foot and mouth disease in England made it impossible to visit many of the farm sites until this time. The population levels of the nematode should not have been much affected by this delay since very little hatching of the eggs occurs until the early spring (Duggan, 1961).

3. Results The results of these field samples are presented in Appendix table lc, (0. graminis) and table 1g, (H. avenae), They are considered in this section in three parts, dealing with the fungus, with the nematode and with

22 Figure 1. 0. graminisg relationship of plant and root 75 infection. (spring and summer, 1967)

50 d

te 6;345

fec o 0." in 0... / 25 0 ts * w 1 if.. •/ 0 0° 0 ...... ,.- . ,..-g 0. .,..,- .% % roo 0 8...... 1,..e. ..,...... 1...... 4.....,.

. • • . ' . . y...... 41*4 'NZ' 11..`. '...: • —lbw •••ir '''' —./14.. O

25 •50 75 100 % plants infected Figure 2. 0. graminis; relationship of root infection .. 40 and number of lesions (spring 1967)„- . ..

.•'. ..'seminal roots 30 d te

fec 20 ""' • in ••"" ts ..., ....•-• . all roots 9. ,...• X : 0 ....•

% roo I .0. ... t0 0.*.• ,...• 11)0 :' .... . 'lc' oil %It

100 300 500 lesions on 50 root systems

Figure 3. 0. graminis: Build up of take-all infection 100. during 1967 A III-

Plant infection II

50- -CY ..4 I Co------

0 0 4-) Seminal root infection

rj rA III rj 50- • • - XII

S. - °' I ------" ---- ...... ---

E Total root infection

....co III II

12/3 28/5 13/8 Initial Spring Summer Sampling time I = 0-25% seminal roots infected in summer II = 25-50% n 4t tt II II III = 5 0-75% II II It It I/ 24 the possible relationships between the two. OplIobolus graminis. In the initial tumbler tests and the spring plant samples the levels of take-all were recorded as ' plants and % roots infected and as the number of lesions present on 50 root systems. In the summer plant samples lesions were not counted. The relationship between these measures of infection on all sampling dates is shown in Figures 1 and 2. Figure 1 demonstrates that increased plant infection is accompanied by increased root infection. At high levels of take-all the % total roots infected continues to increase whilst seminal root infection gives a more nearly linear relationship with plant infection. Since the number of seminal roots produced is limited, a higher proportion of these is infected at high levels of plant infection. Crown root production is continuous so that new, uninfected roots are being produced on take-all infected plants. Consequently there is a lower proportion of the total roots infected. Figure 2 shows that the number of take-all lesions present increases with increasing root infection. The build up of take-all during the season is shown in Figure 3. For the purpose of examining this build up, the fields have been divided into 4 groups, according to

Figure 4.0.graminis:Buildupanddeclineunder r-I 0 infection (summer 1967) 100 50 75 25

continuous cereals 1 Q'

..' . •

... .

Number ofcontinuous cerealcrops ... : seminal rootinfection : plantinfection 3 45678

o.... total root infection 25

-- %. 41. c i t

i i

, i .: I

, , , ., -1.

sr'

•

.. •••

N., do % '

, % . % ' b.., . N

. ..,,, ).. ... `4

. . .. • . 10 11

26 Figure 5. H. avenae. Relationship of plant and root .25 infection ispring 1967)

15 H.

d by

te 0 fec

in 0

a 0 ts o 0 % ro

25 50 75 100 % plants infected by H. avenae Figure 6. . avenae. Relationship of root infection to 25 larvae per root system (spring 1967)

0 0

15 H.

by d te

fec •• in 0

ts .0.49 0 0.43 80.• .••.o• % roo

10 2do 3d0 H. avenae larvae/root system 27

the level of seminal root infection in the summer sample (Appendix table 1d). The mean level of take-all infection for each group was calculated (Appendix table le) and these levels are shown in the figure. Group 4 consists of only a single field and is not considered; groups 1,2 and 3 consist of 8,9 and 6 fields respectively. It is apparent that take- all increases more rapidly in the period between spring and summer. Figure 4 represents the effects of continuous cropping with susceptible cereals on the levels of take-all in the fields. The data is presented as the mean take-all infection for fields with the same cereal cropping records. The disease can be seen to build up under continuous cereals, reaching highest levels under the fifth and sixth crops and then declining under the following crops. Heterodera avenae. The relationships between the three measures of nematode infection recorded in the spring (% plants, % roots and number of larvae/root system) are shown in Figures 5 and 6. The relationships between initial soil populations and spring infection, and between spring infection and final soil populations are shown in Figures 7 and 8. The correlation coefficients given for these two figures are both statistically significant at the 1% probability interval. Figure 9 shows the relationship between initial 28 Figure. 7. H. avenae. Relationship of spring root infection 25 to initial eits/e soil

0 0 r == 0.7576 0 0

4 15 0...". da Of r0 / w 4, 0 0 eV .....** ft-t 0 0 a, ... 43 0 / 0 O 0 / O 0 0 .d . • •-

10 20 30 40 initial eggs/g soil

Figure 8. H. avenae. Relationship- of spring root infection 25 to final eggs/g soil

4-, 0 0 F4

10 20 30 40 50 final eggs/g soil 29

Fiure 9. H. avenae. Population changes under 1967 crop 50 0

40 -

30 0 /maintenance level to 0 to ta 20 a) o H .• 0 .r4 CH 10 03Acv o o ..s cc 0 0 0 10 20 30 40 50 initialeggstg soil Figure 10. H. avenae. Population changes under continuous 30 cereals

9\ 20 0 0) b,o final t 0 10 - i1 a-40-1 0 0 initial

C)*” .. . 0 3 4 5 6 7 8 ld 11 number of continuous cereal crops 30

and final nematode populations, that is the multiplication of the nematode. In the majority of fields, populations increased during the 1967 season, but most of these infestations were low (less than 10 eggs/g soil) and increases were small. The effects of continuous cereal cultivation on populations of cereal cyst nematode are shown in Figure 10. The egg counts from which these mean values were calculated show considerable variation (Appendix table 1h) so that apparently lower populations under the fifth to eighth cereal crops may be misleading.

Comparison of H. avenae and 0. Framinis populations The population levels of the nematode and fungus at each site are compared in Figures 11, 12 and 13. The degrees of infection measured on the spring plant samples as % plants, % roots or larvae and lesions all show a similar relationship. This would be expected from the relationship between these three measures of infectiOn demonstrated in Figures 1, 2,5 and 6. Consequently, this comparison is made here by considering root infection (Figure 11). A similar pattern is shown in Figure 12, which plots spring take-all infection against final egg populations of the nematode. These comparisons suggest that there mar be an association in the field of the higher levels of cereal cyst nematode with 31' Figure 11. Relationship between H. avenae and O. graminis Root infection, spring 1967.

X a = - 0.528 b = 0.601 •• ..• •• • • 05 •• 0 x .•• 05 xx x •

graminis: % roots infected Figure 12. Relationship between H. avenae and O. graminis Final eggs/g soil and spring take-all 50 0

r1 0

bi)

tO C) 25 a = 11.71 b = 0.744 0 05 C) 0 x

tI ...... 's. = - 2.554 oo x ...... , X ...... b = 0.160 o 0 X . X ...... ,x.--- ..... x 0 20 4 6 0. graminis: % plants infected

Figure 13. Relationship between H. avenae and 0. ,graminis Cyst contents and spring take-all, 1967

• 1 2

m rl 4-) X 0 ri

0 Pi 60 0

x •ri CH X +7

4 0 X C) 0 0 0 C) X

(1) 00 af, X X 4' 6. e0 1 NJ O. graminis, plants infected 33 lower levels of take-all. In these figures this association appears to separate the fields into two groups which are represented by different symbols. Best fitting lines were calculated and are shown in the figures. A further comparison was made between the number of eggs per cyst after the 1967 crops and the degree of take- all in the spring (Figure 13). Although in this figure the fields do not obviously form two groups, the symbols used represent the same fields as in Figures 11 and 12. In Figure 13, higher cyst contents are associated with lower levels of take-all.

4. Discussion The results of this 1967 field sampling programme have been used to compare populations of Heterodera avenae and Ophiobolus ,a*raminis. It has been suggested from this comparison that there is an association of higher levels of H. avenae with low levels of take-all. However from the occurvnce of low populations of the nematode at low levels of take-all, it is clear that factors other than take-all are involved. The effects of continuous cereal cultivation on the incidence of take-all have been demonstrated. These results are similar to those presented by Shipton (1967), 34 in support of the Ivpothesis of take-all "decline" under intensive wheat growing. The difference between the present results and those of Shipton is that the peak of take-all is reached a year later in these barley fields than in Shipton's sites. Changes in cereal cyst nematode populations have also been demonstrated under continuous cereals. The data for the nematode levels show: more variation than that for take-all, and this, together with the relatively few fields sampled, means that too great an emphasis should not be placed on the nematode population changes recorded. It is, however, interesting to note that the apparent depression of H. avenae levels under the fifth to eighth successive cereals corresponds roughly with the peak levels of take-all. The field situation is not a static one as regards. the populations of the nematode and fungus. Take-all levels in particular, change through build up during the season and from season to season. This makes interpretation of results from a range of fields difficult. Ar exanple, the association of high nematode levels with lower incidences of take-all, recorded in the spring may be due to either a competitive relationship between the fungus and nematode during invasion of the host, or could be the result of a depression in nematode numbers caused by high levels of 35

take-all in the preceding year. It seems probable that the level of take-all recorded in spring is of more importance in this association than that found on mature plants at the end of the growing season. The main invasion of the host by H. avenae larvae occurs from the end of March to mid-May, and these larvae take about nine weeks to develop to the white cyst stage (Duggan, 1961). Consequently, a large proportion of the nematode larvae would have completed their development before the summer sample was taken. Take-all infection of the crop begins early in the season but proceeds more rapidly between the spring and summer samples. The majority of H. avenae larvae were found in the seminal root system in the spring sample, whereas take-all infects both seminal and crown roots. Direct competition between nematode and fungus may therefore be confined to the seminal roots, and the level of take-all on these is more accurately reflected by the spring sample. Effects of take-all may also be exerted on the nematode through its effects on the common host plant. The seminal roots contribute in large measure to the yield of cereal plants, at least until mid-season. From this time until harvest the crown roots make the greatest contribution to yield (Simonds and Sallans,1933; Sallans,1942). As 36 development of many H. avenae larvae will be completed by mid-season, the importance of take-all on the seminal roots is again emphasised. However, the effect of take-all on the rate of production of crown roots and on their contribution to plant vigour may also be of importance in this association. This may be particularly so when the production of crown roots, capable of compensating for nematode damage to the seminal root system, is hindered by take-all. In this situation the developing nematodes may be vulnerable to the effects of take-all in reducing the vigour of the host plant. Although interpretation of the relationship between H. avenae and 0. graminis, suggested by these field observations, is difficult, the results did suggest a direction for subsequent investigations. These included a second field sampling programme, limited to a single field in order to avoid, as far as possible, differences in factors other than the levels of take-all and cereal cyst nematode. Greenhouse and laboratory studies, (to determine the effects of take-all on the nematode) were also initiated in the light of these first field results. 37

SECTION IIB

1. Sampling procedures In this second season of field observations information was sought with particular regard to the effects of O. graminis on the multiplication of H. avenae. A single field was sampled to compare populations of the nematode with levels of take-all at a number of points in the field. The field selected, (MW 642), had been sampled in the 1967 programme and in 1969 was growing its sixth consecutive barley crop. Samples were taken from an area of the field measuring 50 by 70 yards. Seven points were sampled on each of 5 traverses of this area, making 35 samples, evenly spaced, ten yards apart. Initial estimates of the nematode populations were made by taking 6 soil cores to a depth of 8 in from a square yard around each sampling point. Cysts were extracted from a 100 g subsample and initial infestation levels determined as eggs/g soil. Progess of infection by take-all and cereal cyst nematode was assessed from individual plants, lifted from each small plot at four weeks after crop emergence. The root systems of these plants were examined. Individual lesions and larvae were not counted, a root being scored as infected by the fungus or nematode if one 38 or more lesions or larva© respectively were present. Final samples were taken at harvest by lifting a four inch drill length from each point. These were allowed to dry in the greenhouse and a single plant randomly selected from each sample. The soil from around the roots of this plant was removed and the final H. avenae population determined as eggs/g soil from cysts extracted from this soil. The root system was examined for take-all. Assessments for take-all and cereal cyst nematode were made by the methods used for the 1967 field samples. In addition to the measurements of populations taken in 1967, the multiplication of H. avenae was calculated from the ratio of final to initial populations.

2. Results The levels of take-all and cereal cyst nematode are recorded in the Appendix Table 2a. The means and ranges of these levels are given in Text table 1. Figures 14 to 17 compare populations of the fungus and nematode. The comparisons between root infection by both organisms in spring (Figure 14), and between take-all and final eggs/g (Figure 15) do not show statistically significant correlation. However, the data relating to spring infection levels shows a relationship not dissimilar Figure 14 39 1001 rid Relationship between H. avenae and -Po o 0 0 0. graminis. Root infection, spring 1969

-1-111

+3 0 0 0 0 0 O 50 o © o 0 0 0 0 0 0 0 co 0 IA I 0 % 0 0 L 00 0 1 0 A 50 0. graminis, % roots infected

Figure 15. Relationship of H. avenae and O. agElni2. Final eggsig soil and summer take-all, 1969 100 0

to to

0 50 8 0 0 0 tzs 0 0 0 041 0 8 0 0 da, 0 000 (9 0 oo

56 106 0. graminis, % roots infected 40 to that found from tho 1967 field observations. In both these figures higher levels of the nematode were found at lower levels of take-all.

Table 1

Mean Range initial eggs/g 14.32 1.3 - 50.9 % roots infected 39.05 5.9 - 90.9 H. avenae final eggs/g 24.61 3.5 - 91.8

% roots (spring) 16.56 0 - 50.0 O. graminis % roots (harvest) 42.74 24.0 - 77.0

The relationships between final take-all and the numbers of eggs/cyst (Figure 16) and multiplication of the nematode 17) both show significant negative correlation between the nematode and take-all. For cyst contents, r = -0.374, is significant at p = 0.05, and for nematode multiplication, r = -0.494 is significant at p = 0.01.

3., Discussion It was not possible in this sampling programme to relate levels of plant infection recorded in spring to the initial or final nematode and fungus populations. This indicates that the single plants taken in spring were not 41

Figure 16. Relationship of H. avenae and 0. graminis ess/cyst and summer take-all, 1969

150

100 0 0 cd 0

(1) 0

c73 r = -0.374

0 0 50 0 "•••... 0 0 ••„ o O 0 Oo o oo o o

0 25 50 75 100 0. graminis: % roots infected 42

Figure 17. Relationship of H. a:ztag4„...aaLa:glaalgLi. Nematode multiplication and summer take-a11,1969

25 50 75 100 0. graminis: roots infected 43 representative of the populations at each point. However, useful comparisons of nematode and fungus can be made, particularly from the final sampling date. In this the nematode populations were determined from soil taken from immediately round the roots on which they developed. The degree of take-all on these roots was also recorded. Consequently accurate comparisons of cereal cyst nematode and 0. graminis can be made from these data. The results show that higher levels of H. avenae are associated with lower levels of take-all. This is so for final eggs/g soil (Figure 15), cyst contents (Figure 16), and multiplication of the nematode (Figure 17). It is particularly interesting to note the relationship between the number of eggs per cyst and take-all. This offers a possible explanation for the other relationships, since this reduction in cyst content could explain the relationships with both final egg populations and multiplication. A feature of these results is the occurrence of low nematode levels and low take-all levels together. This was also noted in 1967 and shows that other factors are involved in determining populations of the two organisms. These combinations of low populations of fungus and nematode probably have no bearing on the relationship suggested by this work. If this is so, the association of 44 high H. avenae populations with low take-all would be more forcibly suggested by these observations. The results of these two season's observations provide a basis for consideration of the relationship of H. avenae and O. graminis, in particular suggesting that the effects of take-all on cyst contents may be an important factor. The contribution of the field observations to an understanding of this relationship is considered in the final section where they are discussed, together with the results of the experimental work. 45 SECTION III POT EXPERIMENTS

The relationship between H. avenae and 0. graminis was: studied in two series of pot experiments. The first of these investigated the effects of take-all on natural populations of the nematode in soil collected from barley fields. The second series (Section IIIB) used steam sterilised soil into which the nematode and fungus were introduced.

A. EXPERIMENTS WITH FIELD SOILS 1. Introduction

The first two experiments described in this section were designed to investigate the effects of take-all on H. avenae, by the addition of fungus inoculum to field soils. The third experiment was primarily concerned with the effect of take-all on another sedentary endoparasitic nematode, Meloidogyat naasi. In a fourth experiment oats, which are resistant to take-all, were grown in soils containing three different nematode populations, to assess the effects of these nematodes on the resistance of oats. The information on the interaction of H. avenae and O. graminis, provided by these experiments, is discussed at the end of the section. 46

2. Materials and methods

The techniques described here are common to many of the experiments in this section. Additional information on materials or methods peculiar to an experiment is given in the description of that experiment. Soils used in these experiments were collected from the fields during the winter by the sampling method described in Section II. Soil from each site was passed through a i in mesh sieve, and stored in black polythene bags in a constant temperature room kept at 8°C. When required for use, soils were potted, selecting a subsample for each pot by coning and quartering the bulk sample. Inoculation of soil with 0. graminis,. The soils were inoculated with take-all by the addition of sand-wheatmeal cultures of the fungus (Lester & Shipton,1967). The preparation of these cultures from isolates of the fungus is described below. Roots infected with take-all were surface sterilised in 0.5% sodium hypochlorite for 5 minutes, followed by washing in five changes of sterile water. Infected root pieces were plated on potato dextrose agar in Petri dishes, and incubated at 2000. These first isolates were subcultured onto fresh P.D.A. plates and incubated for 2 weeks. 47

Sand-wheatmeal culture medium was prepared by mixing 500 g of coarse sand with 15 g of wheatmeal in 500 ml conical flasks. 45 ml of distilled water was added to each flask. These were then plugged with cotton wool and sterilised by autoclaving for 30 minutes at 15 lb p.s.i. One week after autoclaving, the flasks were inoculated with two 5 mm discs cut from the advancing edge of the 2 week old cultures of O. grnminis. The flasks were then shaken to bury the inoculum, and incubated at 20°C for 4 weeks. During this time the cultures were shaken regularly to ensure growth of the fungus throughout the medium. After incubation, sufficient cultures were bulked to give the required amount of inoculum, and passed through a T in mesh sieve. Weighed amounts of inoculum were placed with the soil to be treated in polythene bags. Bags were inflated by mouth, their necks drawn together and shaken vigourously to thoroughly mix the inoculum with the soil. This inoculated soil was then potted. Controls (i.e. uninoculated with take-all) were made by the addition to the soil of autoclaved fungus cultures. To allow dispersal of any heat formed products which might be harmful to either plant or nematode, these autoclaved cultures were allowed to stand for one week before use. The efficiency of this method of inoculation was I18 tested by inoculating steam sterilised soil with five levels of the fungus cultures,Es shown in Text table 2. Five pots of each treatment were sown with five barley seeds each and kept in a greenhouse for 21 days to allow take-all symptoms to develop. The root systems were then washed free of soil and examined for take-all. The results (Appendix table 3a) are shown in Text table 2 as the means of five replicates. These results were used as a guide to determining the inoculum level of 0. graminis used in subsequent experiments.

Table 2. The effect of inoculum level on the degree of take-all after 21 days. (means of 5 replicates)

Inoculum level %w.w. 1 5 10 15 20 wt of soil (g) 400 400 400 400 400 wt of take-all culture 5 25 50 75 100 wt of autoclaved culture 95 75 50 25 0

% roots with take-all 30.3 55.5 81.6 89.7 93.0 lesions/root system 22.4 43.2 93.6 100.2 124.6

Cultural techniques. Pots were sown with single presprouted barley seed (cv.Proctor). These seeds were surface sterilised in 2% sodium hypochlorite for 15 minutes, and washed several times in sterile water. They were then germinated on moist filter paper in Petri dishes. After sowing, pots were watered with a nutrient solution based on that of Robbins (1946), a chelated iron source being substituted for the original iron supply (Hewitt,1966). Pots were watered at weekly intervals with this solution for six weeks, additional waterings being made to keep the soil moist. In the first experiment described below, pots stood on a greenhouse bench, protected from the full heat of the sun. In subsequent experiments pots were plunged into an outdoor pit of washed sand ballast. This was watered daily to keep soil temperatures from rising and to help prevent drying out of the pots. Regular waterings were made in dry weather with a lawn sprinkler. Assessment techniques. The methods of assessing take- all infection and cereal cyst nematode invasion of roots have been described in Section II. The "tumbler test" method was used for determining initial levels of take-all in these field soils. Take-all assessments on mature plants were made after pots had been allowed to dry in a greenhouse. The soil and root ball was removed from the pot and the root system shaken and brushed free of soil, before washing and examination for take-all. Initial H. avenae populations were determined as 50 eggs per g of soil from cysts extracted from a sample of soil taken before potting. Final determinations were made on a 200 g subsample of the dried soil from each pot. Cysts were extracted as described previously, except that they were hand picked from the debris of the first 'float' to avoid possible loss cf new, full cysts in the second flotation. 51

3. Experimental details and results a. Effect of adding p:fmniaLoH.avenae populations in eight field soils. For this experiment soil was collected from eight sites included in the, 1967 field observations. The soils were collected in the winter of 1967/68 and stored until use in spring, 1968. Initial cereal cyst nematode populations were determined as eggs per g of soil. These are given in Table 3, with location of the sites and the levels of take-all on the field crop in summer, 1967.

Table 3. Location of fields and soil infestation levels of H. avenae and 0. grminis

Soil no. Field (see also H. avenae 0. graminis Appendix Table 1) eggs/g soil % roots winter 67/68 summer '67

1 M. Wyke 787 46.4 9.2 2 Hill Firs 5.6 5.9 3 Fox Steep 3.9 14.6 4 Hill Barn 16.2 14.6 5 Surgery 22.2 3.9 6 Castlemans 21.7 2.8 7 Policemans 7.3 5.7 8 Bundu 30.5 0 52

The naturally occurring levels of take-all were increased by the addition of sand-wheatmeal cultures of 0. p4raminis at the rate of 100 g culture/kg pot (10% w.w.). The effects of this increase in take-all on the H. avenae populations of the eight soils was investigated by determining changes in nematode numbers after growing barley. Three pots of each soil were treated by the addition of take-all inoculum, and equal numbers of controls with autoclaved fungus cultures. 16 weeks after sowing, final levels of take-all and final root weights were recorded. Cysts were extracted from the soil and their contents determined. The multiplication of H. avenae populations was calculated from the formula: multiplication = final eggs4 soil x 100 (Hague & Hesling, initial eggs/g soil 1958) These data are presented in Appendix Table 4a. Standard errors of the difference between treatment and control means were calculated and Student's t-test applied to determine statistically significant differences. Text Table 4 contains the means for each observation, together with the standard errors and an indication of the signific- anco of the differences. 53

Table 4. Effect of adding 0. ,graminis to H. avenae populations in eight field soils (means on 3 replicates)

FIELD 1 2 3 4 a. % roots with take-all Control 78.9 76.1 43,8 39.3 Treatment 88.9 85.2 67.3 62.5 S.E. ± 4.36 7.56 8.40 2.89 N.S. N.S. ** b. Root wt,(mg) Catrol 120.3 120.0 115.0 147.0 Treatment 136.3 91.0 157.0 201.0 S.E. ± 21.19 15.33 35.17 38.33 N.S. N.S. N.S. N.S. c. Cysts/200 g soil Control 264.3 24.7 29.3 28.3 Treatment 227.0 21.3 21.7 28.7 S.E. + 9.37 1.20 2.65 0.36 N.S. d. Eggs /cyst Control 26.35 62.23 20.61 48.75 Treatment 35.11 54.22 21.97 23.24 S.E. + 5.465 9.278 6.387 5.829 N.S. N.S. N.S. e. Eggs/g soil Control 34.95 7.87 2.99 6.77 Treatment 39.55 5.86 2.11 3.31 S.E. ± 5.081 1.451 0.192 0.504 N.S. N.S. ** f. Multiplication Control 75.4 140.5 76.9 41.8 Treatment 85.3 104.8 54.7 20.6 S.E. + 11.14 26.12 5.43 2.90 N.S. N.S. ** 54

Table 4. (cont)

FIELD

5 6 7 a.% roots with take-all Control 45.8 38.7 25.2 4.2 Treatment 64.5 64.4 64.7 65.1 S.E. + 4.78 2.74 2.86 4.02 *** *** ***

b.Toot....wt(gg) Control 104.3 104.0 388.7 174.0 Treatment 199.7 120.7 290.7 115.7 S.E. ± 47.85 29.35 65.36 12.90 N.S. N.S. N.S. c.0ists/200 g soil Control 129.7 52.0 94.3 146.0 Treatment 120.7 56.3 102.7 125.7 S.E. + 1.15 5.46 5.55 9.00 ** N S. N.S. N.S. d. on rol 28.68 32.27 26.71 19.14 Treatment 39.18 16.03 20.02 13.34 S.E. + 2.801 1.761 4.183 1.647 *** N.S. e.Eggs/g soil Control 18.81 8.31 12.70 13.94 Treatment 19.56 4.82 10.29 8.58 S.E. ± 2.737 0.341 0.833 10.900 7.p. fit** (. f.Multiplication Control 84.7 38.4 169.4 45.7 Treatment 88.3 20.7 141.1 28.1 S.E. + 12.41 4.34 16.30 3.12 N.S. N.S. ** 55

Results The addition of take-all inoculum to these eight soils gave statistically significant increases in the final levels of take-all in six cases. In the two soils with the highest initial levels of take-all (soils 1 and 2) the increase was not significant. Final root weights were not significantly reduced by the addition of take-all inoculum, except in the case of soil 8. In four of the soils significant reduction of multiplication of H. avenae occurred in the presence of increased take-all (soils 3, 4, 6 & 8). In a further two (soils 2 & 7), multiplication was reduced by the treatment, but not significantly so. The multiplication of the other two populations (soils 1 & 5) was not depressed by the addition of take-all inoculum. The numbers of eggs per g of soil reflect the situation described for multiplication of the nematode, except in soil 7. In this case eggs per g were significantly fewer in the treatment than in the control, whilst the depression of multiplication was not significant. Statistically significant reduction of cyst numbers (soils 1,2,3 & 5) and of eggs per cyst (soils 4,6 & 8) occurred in take-all treated soils. In only one soil (3), where take-all reduced the number of cysts, were 56 final egg populations and multiplication of the nematode also reduced. In the three cases where cyst contents were reduced in the presence of increased take-all, final eggs per g and multiplication were also significantly lower. In one soil (5), the number of eggs per cyst was greater in the treatment than in the control.

Discussion The data relating to change in the H. avenae populations suggest that the take-all fungus can cause reduction in nematode infestations. There are also indications of the ways in which these reductions may occur. Multiplication of the nematode is directly dependant on the numbers of eggs, per g of soil, which, in turn, depends on the number of cysts and on the contents of these cysts. Multiplication will therefore be affected by any factors that result in changes in cyst numbers or cyst contents. It is apparent that the depressions of multiplication caused by take-all are not explained by a consideration of the effects of the fungus on the final root weights. In the case of soil 8, however, where increased take-all is associated with reduced weight of root, this reduction of food and space available to the nematode may be responsible for the depression of multiplication. In this soil 57

the difference between control axed treatment take-all levels was the greatest in the experiment. Further consideration is given, in the discussion following this section, to the relationship between take-all, final root weights and nematode populations. In the two soils with the highest control levels of take-all (1 & 2) and in which the take-all treatment did not cause significant increases of the disease, multiplication and final nematode populations were not affected by the. treatment. The effects of take-all on cyst numbers and cyst contents suggest how multiplication may be depressed. In one case (3), depression of multiplication appears to be duo to reduction in cyst numbers. In the other cases (4,6 & 8) reduction of multiplication was related to a reduction in the numbers of eggs per cyst. However, cyst contents in this experiment were low in both treatments and controls. (A newly matured cyst may contain up to 500 eggs,(Andersen,1961). bean cyst contents in this experiment were 30.5 eggs per cyst.) In counting cysts, newly produced ones were not distinguished from older cysts, except where a cyst was flattened, broken and obviously empty. The figures for numbers of cysts and for cyst contents therefore include old cysts whose egg content would have been very low. Also, if take-all is responsible for reduction in cyst numbers,cyst 58

counts would have included fewer new cysts, and consequently, the contents of such a batch of cysts would be lower. It is also possible that cysts produced on take-all infected plants are unable to produce as many eggs as those from healthy plants. Although take-all caused reduction of H. avenae populations in this experiment, it is important to note that nematode levels fell under both treatments and control in all but two of the soils (2 & 7). (A multiplication factor of 100 indicates maintenance of the initial population.) This decline of cereal cyst nematode populations has been mentioned in the introduction to this thesis; further consideration is given this matter, in the light of the present results, in the final discussion. 59

b. Effect of adding 0. graminis to three populations of H. avenae in one field soil.

Interpretation of the previous experiment designed to examine the effects of take-all on cereal cyst nematode, was complicated by differences in both general soil characteristics and in natural infestations of take-all in the eight soils used. This second experiment was made with standard conditions in the controls; that is, by using soil with similar physical and chemical characteristics, and with similar levels of take-all. The soils used differed only in their cereal cyst nematode populations. To achieve this, soil was taken from three areas of a single field (0.S. no.642, Middle Wyke Farm). Preliminary samples taken from the field had shown that populations of both the nematode and fungus varied within the field. This enabled soil to be collected with different H. avenae infestations but with similar levels of take-all. The areas chosen had H. avenae populations of 1 egg per g (N1), 5 eggs per g (N2), and 47 eggs per g (N3). Tumbler tests with soil from these areas detected no take-all. The soil was potted in spring, 1969, with an inoculum of take-all at 10% w.w. in kg pots. Controls were made with autoclaved cultures. Six pots of each soil were treated, and six controls set up to provide three replicates at each 60 of two times of observation. At time one, three weeks after sowing, the root systems were examined for take-all, weighed, and stained in acid fuchsin/lactophenol. The numbers of H. avenae larvae in the roots were counted. The second observations were made 18 weeks after sowing, when final take-all and root weights were recorded, and cereal cyst nematode populations determined from the soil. The results are presented in full in the appendix, Student's t-test was applied to the differences between treatment and control means to determine statistically significant differences (Appendix Table 5a, time one; Appendix Table 6a, time two). Text tables 5 and 6 give means, standard errors and indicate the statistically significant differences.

Results Time One. No take-all had developed at three weeks on the plants in the uninoculated soil, whilst the roots of those in soil treated with take-all inoculum were infected. No differences in root weight were attributable to the fungus at this early stage of the disease. There was considerable variation in the numbers of cereal cyst nematode larvae in the roots of controls and treatments. There were no significant differences between invasion of take-all infected and healthy roots, whether

Table 5. Effect of adding 0. giwilinis to three H. avenae populations in one field soil (means of 3 replicates) (1st observation)

Initial eggs/g

1 5 47 a.% roots with take-all

Control 0 0 0

Treatment 47.0 22.3 29.0

b.root weight (mg) Control 159.7 102.0 151.0 Treatment 91.3 120.3 133.7 S.E. + 45.17 30.20 33.60 N.S. N.S. N.S.

c.larvae/root system Control 52.0 47.3 53.0 Treatment 38.0 42.0 47.7 S.E. + 13.06 5.08 7.35 N.S. N.S. N.S.

d.larvae/g root Control 327.4 469.0 354.9 Treatment 436.9 400.6 371.7 S.E. + 41.81 57.78 18.59 N.S. N.S. N.S.

Table 6. Effect of .adding 0. graminis to'thrge H. avenae populations.in on field roil. 2nd observation (means of three replicates)

Initial eggs/g soil 1 5 47 a. roots with take-all Control 52.3 49.7 49.7 Treatment 82.0 77.0 80.0 S.E. + 6.08 4.28 1.30

* * * * * * * b.take-all lesions /root system Control 44.0 45.7 42.0 Treatment 87.7 88.3 109.7 S.E. + 3.67 6.48 12.96

* * * * * * * c. root weight (mg) Control 458.3 399.7 450.3 Treatment 434.0 488.3 555.7 S.E. ± 63.93 70.65 68.63 N.S. N.S. N.S. .cysts/200 g Control 66.3 120.7 74.3 Treatment 65.3 100.7 81.7 S.E. ± 0.81 18.50 11.66 N.S. N.S. N.S. e. eggs/cyst Control 19.0 23.2 62.8 Treatment 4.6 14.6 27.2 S.E. + 3.11 2.27 8.92 ** * * .eggs/g Control 6.14 13.63 21.33 Treatment 1.40 6.11 8.96 S.E. + 0.661 1.551 0.695 ** ** *** .multiplication factor Control 614.0 272.5 43.9 Treatment 140.0 122.3 19.1 S.E. + 66.12 31.84 6.47 ** ** * 63 the comparison is made of total numbers of larvae per root system or of number of larvae per g root. Although statistical tests were not made on differences between the three nematode infestation levels, invasion did not reflect the differences between these initial populations.

Time two. At this stage root infection by take-all had developed on control plants but was significantly less than that on the treatments. Numbers of take-all lesions per root system also showed this difference, although at this advanced stage of take-all it was often difficult to decide if a lesion was a single one or the result of fusion between several. Again in this experiment, final root weights were apparently not reduced by the take-all treatment. The data relating to H. avenae show that nematode populations were significantly affected by the increased levels of take-all. Multiplication was reduced by the treatment at all initial nematode populations. At Ni (1 egg per g) and N2 (5 eggs per g) the final number of H. avenae eggs per g soil was greater than the initial level in both treatment and control. At N3 (47 eggs per g), the nematode population fell. In all cases fewer eggs were present in the treatment than in the corresponding controls. Cyst numbers were not significantly reduced by the 64 increased levels of take-all, but there were significant reductions in the numbers of eggs per cyst. As in the previous experiment, the egg content of the cysts was low.

Discussion The results of the first observations (time one) make no positive contribution to the understanding of the relationship between O. graminis and H. avenae. They do, however, suggest that any competition between the two must occur after the initial invasion of their mutual host. The second observations add weight to the suggestion, derived from the field studies and the previous experiment, that the effect of take-all in reducing cereal cyst nematode populations is due to a reduction in cyst contents. The results of this experiment show that cyst numbers were not affected by take-all. Since the cyst populations were composed of older cysts and newly formed ones, there are two possible explanations of the reductions in numbers of eggs per cyst. It is possible that reduction in cyst numbers due to take-all may not have been detected, since large numbers of old cysts were present. These cysts have low numbers of eggs and their presence gives lower contents for the batch of cysts examined. Alternatively, if take-all does not affect cyst production, it may still reduce the egg content of cysts developing on take-all infected root 65

systems. A further point concerning the cyst populations is that numbers of cysts recorded at the end of the experiment were not related to the initial egg populations of the three soils. Final egg numbers were related to these initial levels. This point, which is given further consideration later, suggests that the egg content of cysts may be more important in determining cereal cyst nematode populations than is the number of cysts present in the soil. Thus a thriving H. avenae population may consist of full cysts, whereas a declining population may have the same number of cysts but their contents may be much lower. 66 c. The effect of 0.raminis on Meloidogyne naasi in naturally infested soil

The Gramineae root-knot nematode, Meloidogyne naasi (Franklin), is a relatively recently described, sedentary endoparasite on a wide range of hosts, particularly on cereals and grasses (Gooris,1968). First recorded in Britain by Franklin (1965), this nematode has been shown to build up to high levels on susceptible crops, and considerable yield loss of barley has been reported,(Kuiper,1966). In view of its presence in Britain and of the involvement of other species of Meloidogyne in interactions with fungi, (see General Introduction), a preliminary examination of the relationships of M. naasi and 0. graminis was made. The experiment was made in a similar way to those investigating the relationship of O. graminis and H. avenae. Soil, infested with M. naasi, was collected from a barley field in early spring, 1969. Information about the site, at Upperwood Farm, Reading, was kindly provided by Dr.M.T. Franklin. The soil was sieved and potted with either take- all inoculum or with autoclaved cultures at 10% w.w in pots of 500 g soil. Six replicates were set up and sown with Proctor barley. The initial M. naasi population of the soil was estimated (by extracting the nematodes by the Whitehead tray method (Whitehead & Hemming 1965) as 35 larvae per 67

100 g of soil. After 3 months, a sample of soil was taken from the full depth of each pot with a 1 inch diameter soil auger. This core represented 10% of the total soil volume. Nematodes were extracted from these samples on Whitehead trays and counted. Root systems were then washed free of soil, examined for take-all and weighed. They were then stained in acid fuchsin/lactophenol and the numbers of M. naasi females and galls caused by the nematode were counted. These results are presented in Appendix table 7a. Control and treatment means, with standard errors and significant differences are given in Text table 7.

Results Some take-all was present in the uninoculated field soil, but the level of disease was increased significantly by the treatment. Root weights were reduced, but not significantly so, by the increased take-all. The number of M. naasi females in the roots was significantly lower in the treatment than in the control. There were also significantly fewer females per g root. There was no reduction in the extent of galling of the root systems in the presence of increased take-all. Extraction of nematodes from the soil samples gave no 68

Table 7. Effect of 0. graminis on M. naasi • (means of 6 replicates)

Root wt females/ females/ galls/ galls males roots (g) plant g root plant /g /500g with root soil take- all Control 2.72 175.3 67.15 53.2 21.34 13.3333.50 Treatment 2.47 133.8 51.40 50.7 '420.58 16.67 75.57 S.E. + 0.182 8.82 1.931 5.34 2.350 - 1.80 N.S. *** *** N.S. N.S. ***

Effect of 0. graminis on T. dubius (means of 6 replicates)

larvae/ . females/ males/ all stages pot pot pot /pot 0 - Control 43.3 50.0 11.7 105.0 Treatment 136.7 108.3 43.3 288.3 S.E. + 25.38 12.68 4.86 37.25 ** *** *** ***

** and *** indicates differences significant at p = 0.01 and p = 0.001, respectively. 69

second stage larvae of M. naasi and very few males. However, other plant parasitic nematodes were recovered, the most numerous being Tylenchorhynchus dubius, an ectoparasite. Significantly more of all stages, (larvae, males and females), of this species were recovered from pots treated with take-all inoculum. Other plant parasitic nematodes recovered were present in low numbers and only one, an unidentified species of Pratylenchus, was found in all pots. There were no differences of numbers of this nematode in treatment and control pots.

Discussion The nature of the relationship between M. naasi and 0. Faminis suggested by these results, may be similar to that between take-all and H. avenae. The observation that the number of galls produced is not reduced by the increased take-all suggests that invasion and the early stages of development of M. naasi were not affected by the fungus. The production of mature females may be influenced by take- all in two ways. There may be effects on the sex determination of developing larvae, resulting in the production of more males and fewer females. Other species of Meloidogyne do exhibit "sex ratio" effects in this way, such adverse conditions as overcrowding of larvae in host roots or poor 70

host nutrient status, being responsible (Triantaphyllou, 1960; Davide & Triantaphyllou,1967a,b). A second way in which females may be influenced is through direct effects of the fungus on their development. Females have a longer feeding period than males and consequently require more food from their host to complete their development. It may be that take—all infected roots do not provide sufficient food for the development of the same number of females as healthy root systems. In this experiment only mature females were counted, but some immature third stage larvae were seen in the roots. Further discussion on the effects of 0. graminis on the sex determination and sex ratio of M. naasi and H. avenae is included in the discussion of the results of Section IIIB. The presence of few adult males in the soil indicates that most larvae had completed their development and that males were no longer active. The absence of second stage larvae suggests that the newly formed eggs may require a period of dormancy or maturation before hatching. Results of other work with this nematode (Franklin, personal communication) have established that there is only one generation of M. naasi a year, and that increased larval activity, as measured by extraction from soil, occurs after periods at low temperatures. 7T

The increase in numcerb ur gylenchorhynchus dubius on take-all infected roots is paralleled by a report by Mountain & McKeen (1962), that reproduction of Tylenchorhyn- chus capitatus was increased on tomato roots infected by Verticillium dahliae, a wilt fungus. In their experiments, multiplication of Pratylenchus penetrans also increased on fungus infected roots, but no reason for the interaction was established. It is possible to suggest some explanations for the increased numbers of T. dubius in this experiment, but, without further experimental evidence, these explanations can only be considered as tentative hypotheses. Fungus feeding nematodes have been shown to increase in the presence of root disease fungi, (Klink & Barker, 1963). However, there is no evidence for T. dubius feeding on fungal hyphae. It is generally thougnt that ectoparasitic nematodes, such as T. dubius, feed preferentially on healthy roots. It is possible that the higher levels of take-all may have restricted feeding of the nematode at any one site. Cessation of feeding, as a root becomes unfavourable through the presence of take-all, may be followed by a period of egg laying. Thus the numbers of T. dubius could be increased in the treated pots. There is some evidence from work with multispecific nematode inocula,(Jenkins,1968), of competition between other species of Meloidogyne and some genera of ectoparasitic 72 nematodes. In this experiment, whilst the number of females of M. naasi was lower in the treatment, the number of galls was not affected. This reduction in the number of females per gall may enable T. dubius to feed on galled tissues, and consequently increase in numbers. There are, however, no reports of T. dubius feeding on this tissue. 73

d. Resistance of oats to 0. graminis in the presence of three nematode populations

Oats are an efficient host of H. avenae, but are resistant to the wheat and barley strain of O. graminis, (Turner,1940). In some other nematode-fungus interactions, nematodes have been shown to change the resistance of plants to fungi. This has been reported for several species of Meloidagyne,(Cohn & Minz,1960; Powell & Nusbaum,1960), and for some ectoparasitic nematodes,(Holdeman & Graham, 1954). Relationships in which the severity of fungal disease is increased by the presence of a nematode are also recorded. Nematodes involved in these synergistic relationships include Heterodera rostochiensis,(Dunn & Hughes,1964), Meloidogyne spp.,(Powell & Nusbaum,1960; Schindler, Stewart & Semenik,1961), and some ectoparasites,(Holdeman,1956; Van Gundy & Tsao,1963). In view of these reports, an experiment was designed to test the effects of three nematode populations on the resistance of oats to take-all. Although oats are reported as resistant to M. naasi, some invasion and host plant reaction does occur. Consequently, this nematode was included in the experiment. Field soils containing H. avenae (soil A), M. naasi (soil B), and soil C, containing only ectoparasitic 74

nematodes were potted with either live or autoclaved take-all inoculum at a rate of 10% w.w. in 500 g pots. Three replicates of each treatment were sown with a single pregerminated oat seed (c.v.Sun II). Three months after sowing, plants were removed from the pots and their roots freed of soil. Nematodes present in the soil were extracted from a 100 g subsample from each pot. The root systems were examined for take-all and stained in acid fuchsin/lactophenol. Nematodes present in the roots were dissected out and identified. Quantitative estimations of root invasion were not made.

Results No take-all lesions were present on any of the oat roots from this experiment; occasional r:.nner hyphae of 0. graminis were seen, but there was no further infection. In soil A some H. avenae third and fourth stage larvae were present in the roots. White cysts were seen attached to these root systems. Species of Pratylenchus, Pratylenchoides and Helicotylenchus were also identified from the roots. No larvae or adults of M. naasi were present in the roots of oats grown in soil B; Pratylenchus sp. was the only parasitic nematode recovered from the roots. In soil C, no plant parasitic nematodes were found in the stained roots, but low numbers of Aphelenchus avenae, 75

Table 8 Changes in nematode populations in three soils inoculated with 0. graminis. (nematodes/100 ml soil, means of 3 replicates)

F0 Pi S.E. of diff. Soil A

Tylenchorhynchus sp. 494 410 79.5 Tylenchus sp. 110 60 29.1 Pratylenchus sp. 17 23 7.3 ...- Soil B

Tylenchorhynchus sp 47 90 28.0 Tylenchus sp. 87 ' 73 15.5 Pratylenchus sp. 10 10 4.7 Pratylenchoides sp. 20 20 16.3 lielicotylenchus sp. 27 43 12.2 Heterodera avenae 63 53 9.3 (males) Soil C

Tylenchorhynchus sp. 0 3 2.9 Tylenchus sp. 90 80 14.1 Rotylenchus robustus 27 37 14.8 Aphelenchus avenae 20 230 16.0 76

a fungus feeding nematode, were seen in both treatment and control roots. Nematodes extracted from these soils were counted and these numbers are shown in Text table 8 and in Appendix table 8a. Species of Tylenchorhynchus and Tylenchus were present in all soils. Pratylenchus spp. were recovered from soils A and B, and from soilA, Helicotylenchus sp and Pratylenchoides sp were also extracted. Soil C gave Rotylenchus sp and Aphel.enchus avenae. There were no statistically significant differences in numbers of plant parasitic nematodes extracted from fungus inoculated and control soils. In soil C, A. avenae was present in much greater numbers in the pots to which take-all had been added.

Discussion. The presence of plant parasitic nematodes did not affect the resistance of oats to take-all. Of the nematodes present, only H. avenae is. able to cause biochemical and morphological disturbances in the oat loots. Similar changes are thought, in interactions of other nematodes and fungi, to be responsible for breakdown of host plant resistance to the fungus partner. In some way these changes appear to modify the host substrate making it available for colonis- ation by the fungus,(Pitcher,1963). The resistance of oats 77 to 0. graminis has been attributed to the specific action of an inhibitor produced in oat roots,(Turner)960). The production of this substance is therefore presumably unaffected by H. avenae. The presence of runner hyphae on oat roots has been reported by Turner0960). Robinson & Lucas(1967) have recorded runner hyphae on oats and on maize, a non-host plant. The presence of these runner hyphae does not indicate any loss of resistance. The results of this experiment show no evidence that oats are susceptible to M. naasi. This is in agreement with the observations of Gooris,(1968), who reports that although invasion of oat root occurs, there is no further development of this nematode. The numbers of A. avenae increased tenfold in soil to which live take-all had been added. A. avenae feeds on fungal hyphae, either in soil or on roots; it is clear that the provision of large amounts of fungus food, in the form of the take-all inoculum, is responsible for the observed increases. It has been reported that A. avenae can give control of some other root disease fungi, (Klink & Barker, 1968). To investigate whether this might occur with take- all on a susceptible host the following test was made. Barley, growing in soil to which live take-all or autoclaved cultures had been added, was examined for take- all four weeks after inoculating the pots with A. avenae at 78

0;10,000;50,000 and 100,000 nematodes per kg pot. There was no difference in the number of lesions formed in the presence of increased numbers of A. avenae. However, many more nematodes were recovered from soil inoculated with take-all, suggesting that A. avenae was again feeding on the fungus inoculum. The addition of 10% w.w of the take-all culture may have provided sufficient fungus to infect the plant roots and to feed the nematodes. It remains possible that, with lower inocula of take-all, high numbers of A. avenae could reduce root infection. However, the population of A. avenae required to give control is likely to be far higher than any encountered naturally. 79

4. Discussion These observations, on the effects of increasing the levels of take-all in field soils, provide evidence of a relationship between 0. graminis and H. avenae on'barley. Some conclusions about the nature of this relationship can be drawn from these experiments. Difficulties of interpretation of these results arise from the complexity of the soil ecological situation in which the fungus/nematode relationship occurs. These points which are discussed below, are also considered in the final discussion, where they are related to the results of the field observations and other experiments. The relationship suggested by the results is one in which the nematode suffers from competition with the fungus. The effects of this competition are such that final egg populations and, consequently, multiplication of the nematode are reduced by high levels of take-all. This competition apparently occurs within the roots, since the presence of take-all did not, in these experiments, affect host invasion by H. avenae larvae. ThiJ observation is supported by comparison with the results from the experiment with M. naasi. In this case galling of the roots, a consequence of nematode invasion, was not affected by take- all, whilst final M. naasi populations were depressed. 80

The depression of final H. avenae populations appears in most cases to be due to the effects of take-all in reducing the egg content of cysts. Since in these experiments the relationship of take-all to cyst production is not clearly demonstrated, it remains possible that reductions in cyst numbers may also be responsible for depressions of nematode numbers. Comparison with the M. naasi experiment shows that numbers of adult females were reduced by take- all, and these females may be considered as equivalent to the cysts of H. avenae. A heavy attack of take-all would be expected to reduce root weight, and such an infected root system would support fewer nematode larvae than a healthy one. However, measurements of root weights made in these experiments suggest that changes in H. avenae populations are not related in any simple way to an effect of take-all on root size. At any stage, root weights reflect a balance between growth and decay of the system (Troughton,1962). The effects of the cereal cyst nematode on roots has been mentioned in the General Introduction; generally, nematode infection causes the production of a much branched system, and this may increase root weight. Plants may respond to take-all infection by producing adventitious roots at an increased rate (Garrett,1948). Final root weights are, therefore, influenced by the nematode and fungus, both by stimulation 81;

of root production and probably also by increased rate of decay. In consequence, it is difficult to relate final root weight measurements to the size and, perhaps more important, the vigour and physiological state of the roots at the time when nematodes are developing in the roots. It is not surprising then that it is not possible to relate changes in H. avenae populations to final root weights. Interpretation of the nematode/fungus relationship from these results is further complicated by the observed changes in populations of the ectoparasitic nematode, Tylenchorhynchus dubius and the fungus feeder Aphelenchus avenae. These observations emphasise that the ecological situation in which H. avenae, 0. graminis and barley are being studied is extremely complex. The possibility of interspecific nematode competition has been introduced by Jenkins (1968). Miller & Wihrheim (1968) have reported mutual antagonism between Heterodera tabacum and some other plant parasitic nematodes. Such competition may affect the results obtained from this series of experiments. The complexities introduced by ell these factors may be avoided to a large extent by studying the relationship on plants exposed only to H. avenae and 0. graminis. The value of this section is in establishing the relationship in field soils, thus supporting the field observations and lending validity to studies made under more controlled conditions. 82.

B. EXPERIMENTS IN STERILISED SOIL.

1.Introduction

The two experiments described in this section investigated the effects of different levels of take-all on populations of H. avenae in steam sterilised soil. Barley was inoculated with the fungus or nematode, alone and in combination, and final nematode populations measured in both experiments. Additional observations on nematode invasion and development were made in the second experiment.

2. Materials and Methods

Inoculations with O. graminis were made with sand- wheatmeal cultures of the fungus prepared and mixed with the soil by the methods described in Section III A. Garden loam was used in these experiments. After steam sterilisation, the soil was aired for one month before potting, to allow dispersal of any substances produced during sterilisation that might be harmful to the fungus, nematode or plant. Other cultural procedures were as described for the previous experiments. Nematode inoculations were made with hatched larvae of H. avenae. This method allowed accurate estimation of 83 the infestation level of pots.and avoided the presence of old cysts in the estimation of final nematode populations. Inocula were prepared by placing batches of cysts of H. avenae, extracted from soil,onto nylon sieves in Petri dishes of water (Moriarty,1963). At 2 day intervals, larvae were collected by pouring off the water into which they had emerged. Fresh tap water was added to continue hatching. The larvae were concentrated into small volumes of water by allowing them to settle in a measuring cylinder and siphoning off excess water. These hatched larvae were then stored at 2°C in shallow dishes of water until use Sufficient cysts were extracted before each experiment to ensure that no larvae were used which had been hatched for more than one week. Pots were inoculated by pipetting larvae into depressions made in the soil. The surface of the soil was kept moist by regular spraying with low volumes of water. In the first experiment, pregerminated Proctor barley seedlings were planted in each pot directly before inoculation with the nematode. In the second , nematode inoculations were made 10 days after sowing the seed. One week after the introduction of larvae, pots were removed from the glasshouse and placed in an outdoor plunge. Assessments of root weights, take-all infection, nematode invasion. and final populations were made as described in the previous sections. 84

3. Experimental details and results

a. Effects of three levels of 0. graminis on final populations of H. avenae.

Take-all was added to 1 kg pots of steam sterilised soil at the rate of 0, 10 or 100 g of culture per pot (i.e. 0, 1 or 10% w.w). Pregerminated seedlings of Proctor barley were planted in the soil and the pots then inoculated with freshly hatched H. avenae larvae at 09 1,000 or 5,000 larvae per pot in all combinations with the take-all inocula. Thus there were nine treatments, and the experiment was replicated four times. The pots were randomised in a single block in an outdoor plunge. 16 weeks after sowing, grain weigats were determined by stripping the ears and hand rubbing the grain before weighing. Root weights and final levels of take-all were recorded and H. avenae populations estimated from a 200 g subsample of soil from each pot. The results (Appendix table 9a) were examined by analyses of variance and least significant differences calculated where significant variation was indicated. Means and significant differences are given in Text table 9.

Table 9 Effects of H. avenae and 0. graminis on each other and on barley (means of 4 replicates)

Level of a) Grain wt (mg) b) Root wt (mg) fungus larvae/g soil mean for larvae/g soilpean for % w.w 0 1 5 teungus O 1 5 !fungus

0 402.3 395.0 389.3 395.5 494.5 460.3 485.3 480.0 ** 1 352.5 258.8 333.3 314.9 480.0 420.3 435.3 445.2 *** 10 170.5 214.0 171.00 185 409.5 383.0 350.0 380.8 mean for H. avenae 308.4 289.3 297.9 461.3 421.2 423.5

Level of c) roots with take-all d) Cysts/200 g soil ' fungus larvae/g soil mean for larvae/g soil mean for w. w 0 1 5 fungus 0 1 5 fungus 0 0 0 0 0 O 22.50 39.50 20.67 *** 1 65.4 64.6 64.5 64.97 0 30.00 32.75 20.92 ** 10 74.7 72.5 75.7 74.30 O 33.00 24.25 19.08 mean for 46.70 45.70 46.87 O 28.50 32.17 H. avenae *** L.S.D. for interaction means p 0.05, 7.00 P= 0.01, 9.45 P= 0.001, 12.58 Level of e) eggs/cyst f) final eggs/g soil fungus larvae/g soil mean for larvae/g soil mean for w.w 0 1 5 fungus 0 1 5 fungus 0 0 117.45 145.23 87.56 0 14.18 29.55 14.58 1 0 97.30 122.18 73.16 0 14.48 19.9G 11.46 10 0 78.25 109.18 62.48 0 13.03 13.47 8.83 mean for ' H. avenae 0*** 97.67 125.53 0**43.90*20.97 (*,**, or *** marks differences significant at p = 0.05 0.01 and 0.001 respectively). 86

Results The weight of grain produced was significantly reduced by the increasing levels of take-all. 'Take-all also reduced final root weights, but this reduction was only significant at the 10% fungus inoculum level. Significant increases in root infection by take-all occurred at increasing inoculum levels. The level of H. avenae did not affect the degree of take-all infection. Data on H. avenae final populations show no significant effects of take-all. However, both cyst contents and final eggs per g were lower in the presence of take-all than in the controls. Cyst numbers were reduced at the initial inoculum level of 5 larvae per g but actually increased in the presence of take-all at the lower nematode inoculum level. In the absence of take-all, more cysts ere produced on plants inoculated with 5,000 larvae than at the lower level, whilst at the highest take-all level (10% w.w) significantly fewer cysts were recovered from the 5,000 larvae treatment than from the 1,000 larval inoculum.

Discussion The results of this experiment can be used to support the hypothesis derived from the field work and previous experiments, that is, that 0. graminis depresses populations of the cereal cyst nematode through its effects on cyst 87

production and contents. However, in the absence of statistically significant differences in this experiment, further observations on the relationship in sterilised soil were planned. This second experiment was designed to give information on the effects of take-all on invasion of roots. by the nematode; as well as on nematode development. A previous experiment (Section TII A,b) had suggested that when plants are inoculated simultaneously with the fungus and nematode there is no effect of take-all on larval invasion. In the following experiment H. avenae larvae were added to plants which had already been inoculated with 0. graminis. It was hoped that this would permit higher levels of invasion, since the root systems would be larger, as well as providing information on the effects of take-all on root invasion. 88

b. Effect of O. graminis on invasion of roots and development of H. avenae

Sterilised soil in 1 kg pots was inoculated with O. graminis at a rate of 10% of culture per pot. Autoclaved cultures were added to the controls. The pots were then sown with pregerminated barley seed and 10 days after sowing H. avenae larvae were added at 1000, 5000 and 10,000 larvae/ pot, (1,5 and 10 larvae per g soil). There were 6 treatments and 9 pots of each were set up, to give 3 replicates at each of 3 times of observation. One week after nematode inoculation, the pots were placed in an outdoor plunge in three randomised blacks, one for each time of observation. Three weeks after inoculation, one set of plants was washed out and take-all level, root weight and nematode invasion were determined. These results are given in Appendix table 10a, and in Text table 10 below. The second block was used to assess the production of H. avenae males. These were extracted by the method described by Trudgill (1967). Plants were washed from the soil, four weeks after inoculation with H. avenae, and transferred to mesh baskets containing Cornish grit and standing in trays of water. Males which emerged were washed through the grit and collected and counted every three days by examining the water in the tray. Results are given in Appendix table 11a, 89

Table 10 Effect of 0. graminis on invasion of barley roots by H. avenae (means of 3 replicates) , Level of _ a) root wt (mg) b)% roots with take-all fungus larvae/g soil mean for' larvae/g soil % w.w 1 5 10 fungus 1 5 10 0 325.0 438.0 361.3 374.8 0 0 00 10 398.7 382.3 335.0 372.0 19.0 18.3 18.0 mean for 361.9 410.2 348.2 H. avenae

- _ Level of c) larvae/root system d) larvae/g root fungus larvae/g soil can for- larvae/g soil mean for %w.w 1 5 10 ifungus 1 5 10 fungus 0 154.-3 6bl.b 6n.17- 472.7 ' 432.0 1497.7 1754.7 1228.1 10 140.0 271.0 348.3 279.7 375.0 712.3 1059.3 715.5 mean 'or 137.2 461.0 490.5 403.5 1105.0 1407.0 H. avenae *** N.S. *** ** L.S.D. for interaction t.S.D. for interaction means means @ p = 0.05, = 76.2 @ p = 0.05, = 231.7 p = 0.01 ,=106.9 p . 0.01, = 325.3 p = 0.001,=150.9 p = 0.001, = 459.2

** and *** indicates difference significant at p = 0.011 and p = 0.001 respectively 90

and in Text table 11. Final observations on root weights, take-all and final nematode populations were made 18 weeks after inoculation. Cysts extracted from the soil were measured before squashing to determine egg contents. The measurement taken was "cyst length", (the distance from the neck to the vulval cone), and measurements were made with a micrometer eye piece. These results are in Appendix table 12 a, and Text table 12. Significant variation due to the treatments was determined by analyses of variance and appropriate least significant differences calculated. Means and significant differences are shown in Text tables 10,11 and 12.

Results

Time 1. Invasion At 31 days after exposure to the take-all fungus, infection was present in the inoculated pots. Root weights. at this stage were not significantly affected by either fungus or nematode. Invasion was measured as total larvae present in the roots and as the number of larvae per g root. Significantly more larvae per root system were present at the inoculum levels of 5 and 10 larvae per g than at the lowest inoculum. 911

The numbers present at the higher levels did not differ significantly. Fewer larvae invaded the take-all infected roots at all nematode inocula. Analysis of these results showed significant interaction between the fungus and nematode. Significantly fewer larvae invaded take-all infected roots at inocula of 5 and 10 larvae per g; but at 1 larvae per g there was no difference in invasion between healthy and infected roots. There was no difference in the number of larvae invading roots inoculated with 5000 or 10,000 larvae in the absence of take-all, but on fungus infected roots the difference between invasion at these nematode inocula was significant. Differences in numbers of larvae per g root were similar to those described for the total number of larvae. However, significantly more larvae per g were present at the highest H. avenae inoculum level than at the intermediate level, in both fungus infected and control plants. Signific- ant reduction of the number of larvae per g root occurred on take-all infected plants. There was significant interaction between take-all and nematode inoculum level on their effects on larvae per g. At the lowest nematode inoculum, there was no difference in larvae per g on take-all infected or healthy root systems, but significantly fewer were present in take- all infected plants at the two higher nematode levels. 922

Text table 10a shows the proportion of the inoculum that had invaded roots after 3 weeks (These figures were calculated from the means given in Table 10). These prop- prtions reflect the situation described for total number of larvae and larvae per g root.

Table 10a. Percentage of larval inoculum invading roots at 3 weeks

Level of Initial larvae per pot 0. graminis 1 9000 5,000 10,000 0 13.4 13.0 6.3 10 14.0 5.4 3.5

Time 2. Production of Males Few males were produced and no significant differences were attributable to the treatments. Males were collected over a period of 4 weeks. At the end of this time new roots had been produced by the plants, the older, nematode infected roots having decayed. These older roots were stained and examined. Few larvae were detected and few roots had an intact cortex, most having rotted to the central stele. 93

Table 11 Effect of 0. Framinis on production of males by H. avenae (means of 3 replicates)

Level of Males collected/plant fungus larvae/g soil mean for w. w. 1 5 10 fungus

0 13.7 14.0 6.7 11.5 10 6.3 11.3 4.7 7.4 mean for H.avenae 10.0 12.7 5.7

Time 3. Final H. avenae populations Final root weights were significantly reduced by the take-all infection. Final levels of take-all did not differ significantly at the different nematode inocula. The number of H. avenae cysts produced increased significantly with each increase in initial inoculum. At all nematode levels take-all was responsible for significant reduction of cyst numbers. Cyst contents decreased with increase in the inoculum level and this decrease was significant at 10 larvae per g. Take-all also caused significant reduction in eggs per cyst. Initial nematode levels caused no change in cyst "length" but this was significantly lower for cysts from take-all infected plants. Final egg numbers were significantly higher at higher initial nematode populations and significantly 94

Table 12 Effect of 0. graminis on final populations of H. avenae (means of 3 replicates)

Level of I a) Root wt (mg) b)% roots with take-all fungus--- larvae /g soil mean for larvailg soil mean for 1 5 10 fungus 1 5 10 fungus 0 969.7 630.0 664.0 754.6 0 0 0 0 10 ( 514.3 465.7 519.3 499.E8 55.7 57.3 48.3 53.8 mean for H.avenae 742.0 547.9 591.7 27.9 28.7 24.2

Level of c) Cysts/200 g soil d) Cyst length/2 fungus larvae/g soil mean for larvae/g soil mean for w.w 1 5 10 fungus 1 5 10 fungus 0 13.3'3 25.33 42.67 27.11 367 845 840 856.7 *** 10 8.00 18.33 36.67 21.00 \902 -8T7 733 784.**0 mean for 10.67 20.83 39.67 334.5 831.0 786.5 H.avenae *** *** N.S. N,S.

Level of e) eggs/cyst f) eggs/g soil fungus larvae/g soil mean for larvae/g soil mean for w.w 1 5 10 cCungus 1 5 10 fungus 0 406.8 355.1 312.8 358.2 27.60 44.93 66.83 46.45 *** 10 330.7 323.7 238.5 297.8 13.17 29.83 43.80 28.93 mean for 368.8 339.4 275.7 20.39 37.38 55.32 H. avenae N.S. **

L.S.D. for interaction means p = 0.05, = 11.51 p . 0.01, = 16.16 p . 0.01, = 22.81 or *** marks differences significant at p = 0.05,0.01 and 0.001 respectively)

reduced by take-all. There was significant interaction between take-all and nematode inoculum in their effects on final eggs per g of soil. The relationship between cyst numbers and inoculum levels is shown in Text table 13, where cyst numbers are expressed as % total inoculum and % larvae invaded at 3 weeks. These figures are based on the means in Text tables 10 and 12.

Table 13. Relationship of cyst production to level of H. avenae inoculum and invasion. cysts/inoculum (%) cysts/invaded larvae (%) Level of initial larvae/pot initial larvae/pot fungus w.w. 1000 5000110 000 100015000 10,000 0 6.7 2.5 1 2.1 50.0 19.5 33.7 10 4.0 1.8 1 1.8 28.6 33.9 52.3

The proportion of the inoculum producing cysts was lower on plants infected with take-all, and also decreased with increasing the number of larvae inoculated. The relationship between cyst production and the level of larval invasion at 3 weeks is more complicated. At 1000 larvae per pot, fewer of the larvae invading take-all infected plants developed into cysts than in healthy roots. However, at 5000 and 109 000 larvae per pot higher proportions of the invading larvae produced cysts on the fungus infected roots. 96 This situation is related to the effects of 0. graminis on nematode invasion (Text table 10a) and is considered in the following discussion.

4. Discussion Observations on invasion of take-all infected root systems by H. avenae, made in field soil, had suggested that nematode invasion was not affected by the fungus. However, in the present experiment it is clearly demonstrated that fewer larvae invaded fungus infected roots. The difference in these results can be attributed to the time of inoculation of the host with each organism. In the experiment in field soil, plants were sown in infested soil and therefore exposed to take-all and cereal cyst nematode at the same time. In this experiment, in sterilised soil, nematode inocula were added 10 days after exposure to the fungus and in this period the suitability of roots for nematode invasion appears to have been affected. At three weeks after inoculation with H. avenae, root weights had not been significantly reduced by the take-all, although lesions and runner hyphae were present. These symptoms of the fungus disease must therefore decrease the suitability of either the affected roots or the whole root system, so that nematode invasion is reduced. 97 On take-all free plants in this experiment, nematode invasion was at a maximum at an initial inoculum of 5,000 larvae per pot. Increase in inoculum above this level did not give increases in the number of larvae present in the root 3 weeks after inoculation. However, in the presence of take-all, the maximum level of invasion was not reached even at an inoculum of 10,000 larvae per pot. It is apparent from the number of larvae present in the roots at 3 weeks that a large proportion of the inocula had not invaded. Probably some of these larvae would be capable of invading, and consequently further increases would occur as root space became available to the larvae. The numbers of males produced were too low to permit any conclusions to be drawn about the relationship of take- all and the cereal cyst nematode. (At least as many males as females should have been recovered; in fact, more than ten times as many females were found.) The technique of extracting the males was subsequently repeated and again few males were recovered, and roots quickly became rotten. Development of the larvae and consequently emergence of males is apparently adversely affected by this technique, probably during the washing and transferring of plants to the baskets of gravel. It is likely, too, that the decay of the roots also upsets nematode development. This decay may be a consequence of damage to the roots during transfer. 98

Trudgill (1967) has used this technique for Heterodera rostochiensis on potato, and Ketudat (1968) for the sane nematode on tomato. The roots of both these plants have a considerably thicker cortex than cereal roots. This may protect larvae from damage during the transfer from pots to baskets. The observations on final nematode populations confirm the results of previous experiments and of the field observations. 0. graminis appears to depress H. avenae populations through its effects on cyst production and on the number of eggs produced by each cyst. The reduction in cyst contents by take-all may be related to the observed reduction in cyst size. Hague & Hesling (1958) have reported that increasing initial infestations of H. avenae and H. rostochiensis results in the production of smaller cysts with reduced egg contents. In the present experiment, cyst contents were lower at the higher initial inocula, but cyst sine was not significantly reduced. The proportion of cysts produced from the initial inoculum is affected by competition between nematode larvae for a feeding site within the root and by continued competition after invasion. Both these factors appear to be affected by take-all. Since take-all reduces invasion, it may also reduce inter-nematode competition within the 99 . root. However, competition between the reduced number of larvae in the root and take-all will still occur. Also invasion may well continue after the stage at which it was assessed in this experiment. Consequently, it is difficul4, from the present information to speculate on the precise effects of O. graminis on the development of H. avenae. However, it is clear that take-all adversely affects the nematode. Whether or not invasion is affected appears to depend on the relative activity of each organism. Whatever the effects on invasion it is also apparent that cyst production and contents, and consequently final egg numbers and multiplication of the nematode are reduced. The following sections of this thesis report further studies on the action of take-all on development and invasion of H. avenae. 100

IV FURTHER STUDIES ON THE EFFECTS OF O. GRAMINIS ON DEVELOPMENT OF H. AVENAE

1 Introduction The two experiments included in this section compared the development of H. avenae larvae in healthy and take-all infected roots. Results of previous experiments had shown that O. graminis can affect invasion of roots by H. avenae. Consaquently in these experiments plants were inoculated with larvae befor,?, inoculation with the fungus.

2. Experimental details and results a.Experiment I Seedlings of barley with roots approximately tin long were placed in Petri dishes on a layer of moist, fine sand. The roots were covered with another layer of sand and freshly hatched larvae of H. avenae were pipetted over the roots, in 1 ml of suspension. The dishes were covered and kept at 15°C to allow invasion to occur. Two days after inoculation roots were gently washed free of sand and unpenetrated larvae. These plants were then potted in sterile loam with 10% w.w of take-all inoculum or of autoclaved culture added. Six plants were inoculated with each of three levels of larvae, (500, 2000 or 4000 per plant). and half transferred to take-all inoculated soil, half serving as controls. There 1011 were therefore three replicates of each treatment. 24 days after transplanting (26 after inoculation with H. avenae) root systems were washed free of soil and stained in acid fuchsin in lactophenol. The larvae present within the roots were counted and identified as second stage, third stage, or fourth stage and adults. Those in the latter category were also identified as males or females. These examinations and identifications were made on the basis of shape of the larvae, with a binocular dissecting microscope. The morphological changes of the larvae of Heterodera schactii, described by Raski (1950) were used as an aid to this identification. The results of these observations are presented in Appendix table 13a. The proportions of larvae at each stage of development were calculated and treatment differences examined by analysis of variance. Means and Significance of differences between treatments are given in Text table 16. The proportion of males to each female (sex ratio) was calculated and the mean values for each treatment are shown in Text table 17.

Results Root weights of take-all infected plants were significantly lower than those of control plants. The interaction between 0. graminis and H. avenae on their effects 102

Table 16. Effect of 0. graminis on development and sex ratio of H. avenae. (means of 3 replicates)

Root weight (mg) i Level of Initial larvae. Uifor L.S.D. for interaction fungus fungus means w.w 500 2000 4000 (4) p = 0.05, 81.5 p . 0.01, 114.3 0 421.0 399.3 211.3 p = 0.001,161.4 343.9*** 10 249.7 218.3 194.3 220.8 lii for H.avenae 335.4 308.8 202.8 N.S. **

Larvae/root system Larvae/g root Level of Initial larvae Tri for Initial larvae *for fungus fungus fungus w.w. 500 2000 4000 500 2000 4000 0 27.7 116.3 171.3 105.1 65.6 312.4 826.4401.4 N.S , 10 24.3 122.3 186.0 110.9 100.8 524.5 990.4 538.6N. TT for 26.0 119.3 178.7 83.2 418.4 908.5 H. avenae *** ** ** ***

% 2nd stage larvae % 3rd stage larvae Level of Initial larvae Wfor Initial larvae If for fungus fungus fungus % w.w. 500 2000 4000 500 2000 4000 0 0 0 9.7 3.23 0 6.8 10.5 5.77 * * 10 0 0 21.9 7.30 0 12.9 20.8 11.23 m` for H. avenae 0 0 15.8 9.85 15.65 *** *

103

Table 16 (cont.)

% 4th stage & adult males % 4th stage & adult ._._..f males Level of M. for ff for fungus Initial larvae !fungusg Initial larvae fungus w.w. 500 2000 4000! 500 2000 4000

0 92.0 88.2 78.4 186.20 8.03 5.00 1.43 4.82 10 96.8 85.1 56.8 179.571. S. 3.17 1.97 0.53 1.89 id for 1 H, avenge 94.40 86.65 67.6q 5.60 3.32 0.98 * *** i

L.S.D. for interaction means p = 0.05, 11.60 p = 0.01, 16.28 p = 0.001, 22.98 104 on root weight was significant. On take-all infected plants root weights were not affected by the initial nematode inoculum. However, on healthy plants the highest nematode inoculum significantly reduced root weight and, at this level of H. avenae, take-all had no effect on weight. Mean root infection by O. gaaminis was 67%; there were no differences in take-all levels between nematode treatments. The numbers Of larvae per root system and of larvae per g of root increased significantly with increase of the nematode inoculum. Take-all did not affect the total numbers of larvae present, and although more larvae were present per g of root on take-all infected plants, the differences were not statistically significant. Second stage larvae were present only in those roots inoculated with 4000 larvae, and more were present in those infected with take-all. Third stage larvae were similarly more numerous in take-all infected plants at nematode inocula of 2000 and 4000 larvae. No third stage larvae were found in plants receiving the lowest H. avenae inoculum: The majority of nematodes in the roots were fourth stage or adult males. Increasing the initial larval inoculum resulted in significantly fewer of these stages recovered. Differences in the proportions of the larvae identified as males on take-all infected or control plants were not 105 statistically significant except at a larval inoculum of 4000 larvae per plant. Few females were present but take-all significantly reduced the proportion of the invaded larvae that had become female, at all initial nematode inocula. Although the proportions of females fell with increased larval inoculum, the differences were not statistically significant. Sex ratios were calculated from the numbers of males and females produced on each plant. In three cases, males, but no females were found; these sex ratios (of infinity) were ignored so that three of the means in Text table 17 are based on two replicates, instead of three. The table shows that the ratio of males:females increased with increasing nematode inoculum, and was also higher on plants infected by take-all.

Table 17 Effect of 0. graminis on sex ratio of H. avenae (means of 3 replicates)

ratio of males:females

Level of Initial larvae 0.graminis 500 2000 4000

0 12.67 21.77 33.25 10 20.51 27.21 104.6

(*means of 2 replicates) 106

The information provided by these results is discussed with that from the following experiment at the end of this section.

b. Experiment 2 Seedlings of barley were placed in dishes of fine sand, 5 plants per dish, and their roots covered with a second layer of sand. These dishes were kept at 15°C for 5 deys to allow growth of the root systems before inoculation with H. avenge larvae. Each dish was inoculated with 4000 larvae and after 2 days plants were washed and potted as in experiment 1. 20 plants were potted in take-all inoculated soil and 20 in uninoculated soil to give five replicates at each of four times of observation. Invasion was assessed by staining the roots of 5 plants in acid fuchsin in lactophenol. Subsequent assessments were made at 7, 14, 21 and 28 days after inoculation. Larvae were dissected from the roots of each plant and their stage of development recorded. The sex of third and early fourth stage larvae was determined by examination of the genital primordium under a compound microscope (Raski,1950). These results are given in Appendix table 14a. In Text table 18, the proportion of larvae at each stage of development is shown. These proportions were examined by analysis of variance, and significant differences 107 are given in the table. The sex ratios at the three later assessments were determined and are included in Text table 18. Results

Take-all infection was not apparent at 7 days, (5 days after exposure to the fungus). At 14 days water soaked areas preceding cortical lesion development, were present on roots. After 21 and 28 days take-all lesions were present; at 28 days 50% of all the roots bore lesions and of the roots which had been inoculated with H. avenae, 68% were infected by take-all. Root weights were lower in the take-all inoculated soil at all four times of assessment, significantly so in three cases. Neither the number of larvae per root system nor of larvae per g root in take-all infected plants differed significantly from that in the controls. At 7 days after inoculation, significantly more of the larvae in take-all infected roots were second stage larvae and significantly fewer were third stage than in the controls. Too few of the third stage larvae could be sexually identified to allow male:female sex ratios to be calculated. At the time of the second assessment, 14 days, there were no significant differences in the proportions of second and third stage larvae in treatment and control, although more second and less third stage larvae were present 1 _ I i.ptg cpc. 1-3 (means of 5 replicates) % f rat 0 m Il sex / l T l % % % st st st

0 0 emal g ar ot a 2 3 ad 1-1 A, P 0 4t 5 nd rv rd a a a i vae h ult al r ge ge ge c+ c+ 0 o

ae oot e

0 c+ s Control 176.0 29.0 154.9 j88.86 11.14 7 0.graminis 142.0 24.2 164.3 94.30 5.70 - - - - - S. E. + 13.7 4.49 14.21 2.02 2.05 * N.B. N.S. * * Control 240.0 26.6 123.4 37.3062.70 30.78 4.98 6.2 14 0.graminis 196.0 23.6 117.4 47.74 52.26 32.42 3.49 8.5 a.B. + 21.8 3.91 10.37 8.4502.04 6.71 0.72 0.33 * N.S. N.S. N.S. N.S. N.S. N.S. *** Control 478.0 21.4 46.0 7.6 8 42.26 50.06 57.28 31.18 1.91 21 0.jzraminis 392.0 20.4 59.8 18.48 41.80 39.72 - 66.39 11.78 7.40 S.E. + 62.0 2.14 9.40 1.22 3.32 3.93 9.83 1.69 1.92 N.S. N.S. N.S. *** N.S. * N.S. *** * Control 962.0 18.8 21.64 10.88 15.32 73.80 60.88 39.10 1.77 28 0.graminis 858.0 17.4 21.16 - 11.54 24.10 64.36 83.82 16.18 5.65 U..E. + 33.7 4.85 3.84 0.75 1.94 4.54 5.15 4.36 0.73 * N.B. N.S. N.S. ** N.S. * ** -***

N.S.,*," 9*** indicate non-significance or significance at p = 0.05,0.01,0.001 respectively 109

in take-all infected roots. Nor were there statistically significant effects of the fungus on the proportions of male or female third stage larvae. However the sex ratio was significantly increased by take-all. After 21 days, more of the larvae in take-all infected roots were present as second stage larvae, there was no difference in the proportions of third stage larvae, and significantly fewer larvae had reached the fourth stage than in the control plants. A higher percentage of the larvae had become male but not significantly higher than that in the control; significantly less were female. The ratio of males to females was significantly greater on take-all infected plants. At the final assessment all larvae had passed beyond the second stage and there were no differences in the proportions of third stage larvae in treatment and control. Significantly higher proportions of fourth stage larvae were present in fungus infected roots than in the controls. However at this time most of the larvae had reached the adult stage. Fewer were adults on take-all inoculated roots than in the controls, but this difference was not significant. All nematodes recovered from these roots were sexually identified. It is possible that some males had matured and left the roots, since many mature males were present, although still within the fourth stage cuticle, and fewer 110 nematodes were recovered than in previous examinations. On take-all infected roots the proportion of the total larvae that were male was significantly greater than in the control and significantly fewer of the larvae were female. The sex ratio of H. avenae was increased significantly by the take- all treatment. 3. Discussion The rate of development of H. avenae larvae in these experiments corresponds with that described by Johnson and Fushtey,(1966), for this nematode in oat roots. In their experiments daily examinations were made for the first six days after invasion, with subsequent observations at three day intervals. They reported that many third stage larvae were present after nine days, and fourth stage after 18 days. In my observations, at longer time intervals, more than half the larvae in control plants were in the third stage of development at 14 days, and at 21 days after inoculation half were fourth stage. Johnson and Fushtey found mature males at 24 days and females at 27 days. Most of the nematodes in my experiments were adults at 28 days after inoculation. In these experiments take-all affected development of the nematode, apparently by increasing the period between each moult. A similar effect occurred in the first experiment through increasing the larval inoculum. These observations suggest that the observed delays in develop- meat may be due to competition, between larvae and between larvae and the fungus, for either food, space or some other factor. Although root weights were reduced by both take-all and increased nematode inocula, the effects of these on development does not appear to be simply related to their 112 effects on root weight. In the second experiment, the number of larvae per g root was not affected by take-all and yet nematode development was delayed. This relationship of take-all, H. avenae and root weight is further considered in the final discussion. The results of the first experiment suggest that the maturation of females was delayed by take-all; in the second experiment fewer females and more males developed on fungus infected roots. The possibility that take-all may be responsible for determining the sex of developing larvae is supported by observations in the first experiment. In this, up to 90% of the larvae in the roots were males. The root systems at inoculation were small, and may have been damaged during transplanting. It is possible that th3se factors may have had an adverse effect on the nematodes' environment, and in response the developing larvae became males. Previous observations on parasitic nematodes have given rise to conflicting opinions on the possibility of environmental sex determination, as measured by the ratio of males to females. Mblz (1920) suggested that sex of Heterodera schactii larvae could be affected by the environment. However subsequent observations on this species by Sengsbusch (1927) led him to change his opinion and attribute increased sex ratios to the inability of females to develop 113

in unfavourable conditions,(Molz,1927). Cobb, Steiner and Christie (1927), and Christie (1929) provided conclusive evidence of environmental sex determination in parasitic nematodes. At high levels of infestation all larvae of a mermithid nematod-e, parasitic in grasshoppers, became male. Ellenby (1954) suggested that sex determination of Heterodera rostochiensis was infliEnced by the environment. Winslow (1960) considered that there was no evidence for environmental sex determination in plant parasitic nematodes. Since then however Triantaphyllou (1960) has provided such evidence for Meloidogyne incognita, where increases in invasion level led to increased production of males. Some of .these had two, instead of one, testes, suggesting that they had been initially genetic females before being affected by the environment. Trudgill (1967) has reported studies in which a variety of environmental factors influenced the sex of larvae of H. rostochiensis in potato and tomato roots. He considered that only larvae with giant cells of sufficient size and nutrient content are capable of becoming female. At high larval densities within a root the possibility of such a giant cell developing is reduced and an increased proportion of the larvae become moles. Some recently published observations on sex ratios of H. schactii favour the view that environment does not affect sex determination, but that adverse conditions prevent 114

maturation of female larvae (Johnson & Viglierchio,1969). These authors make the distinction between sex determination and the ability of larvae to develop to maturity, and suggest that false sex ratios may be obtained by not counting immature larvae. These may well be female and account for the preponderance of males in unfavourable conditions. Although there is some conflict over the nature of sex determination mechanisms in plant parasitic nematodes, there is no reason why both sex determination and rate of development or ability to mature should not be affected by the environment. A combination of these effects may explain increased male to female ratios. In the final assessment of the second experiment of this section all larvae in the roots were sexually identified; any that may have been lost are likely to have been adult males, since these were on the point of emergence from the root whilst females were still embedded in the cortex. As the number of larvae intake-all infected and control roots did not differ throughout the experiment, the possible loss of males would not have affected the difference in sex ratio between the treatment and controls. The observed changes in sex ratio of H. avenae on take- all infected plants may then be due to a combination of delayed development and the increased production of males. The results indicate that sex determination must be affected 115 at an early stage in the development of take-all, since third stage larvae could be sexually identified at 14 days after inoculation, (12 days after exposure to the fungus). In addition to these results, work by Williams (personal communication) suggests that take-all might also affect sex ratio of H. avenae on wheat. Ketudat (1968) has shown that other root rotting fungi increase the proportion of males to females produced by H. rostochiensis on tomato. 116

V FURTHER STUDIES ON THE EFFECTS OF 0. GRAMINIS ON THE INVASION OF BARLEY ROOTS BY H. AVENAE

1. Introduction Previous observations on the invasion by H. avenae larvae of roots infected with take-all, had suggested that these were less heavily invaded than healthy roots. This was found only when roots were infected with 0. graminis before inoculation with the larvae; in simultaneous inoculations with the fungus, invasion by H. avenae was not affected. The experiments described in this section further investigated these effects of take-all on invasion. The materials and methods of inoculation and assessments used to these experiments were for the most part similar to those described for previous sections. Addition- al techniques are described where relevant. Four experiments are described, investigating invasion of roots in relation to the degree and position of take-all infection, and the attractiveness of infected and healthy roots to II avenae larvae. 117

2 Experimental details and results

a. Effect of level of take-all infection on invasion of roots by H. avenae In this experiment barley roots were inoculated with O. graminis in such a way as to produce plants with different proportions of their roots infected with take-all. These plants were then inoculated with H. avenae larvae, and the effects of the fungus on nematode invasion determined. Take-all inoculations were made with 1 in diameter discs cut from 2 week old agar plate cultures of the fungus. These discs were placed in glass tubes, 2 in long and 1 in diameter , buried i in deep in pots containing 100 ml of sterile loam, so that the agar disc was in contact w.th the surface of the soil. Five levels of take-all inoculum were established by the addition of either fullj, i or i circles of fungus culture or of no fungus (These inocula were designated F4, F3, F2, Fi and F0 respectively). The sectors of each disc removed to provide the five levels of take-all were replaced with potato dextrose agar cut from a sterile plate. In this way a layer of agar, about 4 mm thick, was formed across the full area of the tubes. The tubes were then filled with sand and each sown with a single pregerminated barley seed; pots and tubes were kept at 15c‘C in an illuminated growth room to allow 118 infection of the roots by take-all. The barley roots grew down the tube, through the agar and into the soil in the pot. In this way the five inoculum levels could be expected to expose 100% (F4), 75% (F3), 50% (F2), 25% (FI) or none (F0) of the root system to infection by take-all. After 10 days growth the soil in the pots was infected with H. avenae by the addition of 1,500 larvae per pot. Three days were allowed for nematode invasion, when root weights, take-all infection and larval invasion were determined. These results are given in Appendix table I5a, and means and significant differences in Text table 19.

Results The degree of take-all infection shows that the proportion of the roots bearing take-all lesions increased with increasing inoculum level. Although the infection at F1 did not differ significantly from that at F2 theincreases in infection followed the expected pattern. Slightly fewer roots were infected at each inoculum level than had been anticipated. Lesion formation was limited to that part of tht. roots which had been in contact with the layer of inoculum. Although some spread upwards had occurred there was no indication that the fungus had spread on the roots into the soil side ofthe inoculum. Root weights showed considerable variation and were 119 not significantly affected by the fungus treatments.

Table 19 Effect of level of infection by 0. graminis on invasion of roots by H. avenae ,means of 5 replicates)

Levels of % Roots Root Larvae/ Larvae/ fungus infected weight root g root inoculum with take-all (mg) system ____J

Fo 0 188.2 160.8 903.0 FT 16.9 240.8 131.8 580.6 F2 28.6 223.4 101.4 466.4 F3 57.0 223.0 97.6 440.3 F4 81.4 187,4 81.0 439.5 Q.S.D. @ L.S.D.Cu = 0.05 p = 0.05 = 21.44 = 278.08

The differences in the number of larvae per root system were not statistically significant. However, there was a marked trend to lower invasion with increasing levels of take-all: at the highest level of take-all half the number of larvae found in the absence of take-all had invaded, The same trend to reduced invasion was found when the 120

number of larvae per g root was calculated. Invasion was significantly less on take-all infected roots than in the control, but differences in invasion at the different levels of take-all were not significant . Discussion The relevance of these observations to the relationship between take-all and the cereal cyst nematode is considered in the final discussion. They are also discussed at the end of this section together with the results from the other experiments on invasion.

b. Invasion of barley roots by H. avenae in relation to the position of a take-all lesion In this experiment individual roots were inoculated with 0. £raminis and subsequent invasion of the roots by H. avenae was determined. 8 pregerminated barley seedlings were placed one inch apart at one end of a shallow plastic tray of sterilised soil. 8 channels were made in the soil by compressing it with a pencil and the longest root of each seedling placed in one channel. A block of agar cut from the advancing edge of a 2 week old agar culture of O. zraminis, was placed on each root, 2 cm from the proximal end of the root. Controls were made with 8 seedlings set up in a similar way but with a sterile agar block replacing the take-all inoculum. Roots and agar blocks 121, were covered with a layer of sand. A stiff cardboard lid was placed on each tray and these were stood on end with the plumules of the seeds exposed and uppermost. Watering was carried out by removing the lids, with the trays horizontal. After one week hatched H. avenae larvae were pipetted onto the sand in the depression along which the roots were growing. 500 larvae in 1 ml of suspension were inoculated onto each root, 2 cm distal to the position of the agar blocks. After a further two days invasion was assessed by washing and staining the roots. The numbers of larvae in successive 1 cm sections of root were counted and these together with root length, and total invasion are givea in Appendix table 16a. Means and standard errors are in Text table 20, below.

Table 20 Invasion of roots of H. avenae in relation to position of take-all inoctilum (means of 8 replicates)

** or *** indicate significant differences at p = 0.01 & 0.001 root length larvae root larvae/cm root Control 15.25 70.6 4.699 + Take-all 10.40 22.9 + 2.083 S.E. of diff 1.405 2.207 0.227 between 75T ** *** *** L.S.D. @ p = 0.01 4.187 6.643 0.683 0.001 5.817 9.314 0.958 +Id of 7 replicates 122

Pijure 18. Invasion of healthy and take-all infected roots by H. avenge

-* healthy root o---0 take-all infected root

t o ro f o th ng le /cm e larva

0 take-all 5 10 15 lesion on lengths of root 123

Results. Roots inoculated with take-all made significantly less linear growth than the controls. Both the total number of larvae per root, and the mean number of larvae per cm of root were significantly lower in take-all infected roots. The numbers of larvae in successive one cm sections of root are plotted in Figure 18 and show that on fungus infected roots invasion was depressed along the whole length of the root. Highest levels of invasion occurred at and about the point at which the inoculum was applied. 124

c. Invasion by H. avenae of healthy roots of take-all infected root systems The possibility that the presence of take-all on a root system may reduce the suitability for nematode invasion of roots not infected by O. graminis was investigated in this experiment. A "split-root" technique was adopted for this experiment. Pairs of "Jiffy" plant pots, square in cross section and holding 100 ml of soil, were fastened side by side. One pot was filled with soil inoculated with 10% w.w of a sand wheatmeal culture of O. graminis, the second with sterilised soil. Controls were made with both pots containing sterilised soil. Four day old barley seedlings with six roots each were placed on the joined sides and half the roots trained into each pot, The roots were covered with soil and pots kept at 15° C for one week in an illuminated growth room. Inoculations were made with 500 hatched H. avenae larvae being added to the sterile soil pot of the treatment and to one pot of the controls. Two days later these pots were washed out, root length and nematode invasion were recorded from six replicates each of treatment and control. These results are presented in Appendix table 17a, and means and standard errors in Text table 21, below. 125

Results The length of root produced in the nematode inoculation pots by plants with half their roots exposed to take-all was significantly less than that produced by healthy plants. There was no difference in the numbers of larvae invading treatment or control roots, nor in the number of larvae per cm of root.

Table 21 Invasion by H. avenae of healthy roots of take-all infected root systems. (means of 6 replicates)

Root Larvae/root Larvae/cm length (cm) system root Control 9.43 97.0 10.17 Treatment 8.88 104.8 12.22 S.E. of duff 0.13 12.22 1.61 between m ** N.S. N.S. ' L.S.D.@ p = 0.05 0.29 1 0.01 0.41 d. Attraction of H. avenae larvae to take-all infected barley roots This experiment was done to determine whether observed differences in invasion of healthy and take-all infected roots could be attributed to differences in attrcetiveness of these roots. The following simple 126

technique was used to investigate this possibility. 5 ml of water was added to Petri dishes, 5 cm in diameter and 30 ml of sand added, so that the sand was evenly moistened. 1500 larvae in 2 ml of suspension were pipetted evenly over the surface of the sand. In the centre of the dish one of the following test substances was placed; a 5 mm disc of potato dextrose agar, a 5 mm disc cut from an agar culture of 0. graminis, five, 5 mm long, 2 week old pieces of barley root or five similar root pieces infected with take-all. Three replicates of each were set up and kept for 2 days at 15°C. Movement of larvae towards the "attractant" was measured by removing a core of sand from the centre of the dish with a 1 cm diameter cork borer. Each sample was washed onto a nylon mesh sieve, standing in water in a Petri dish. The larvae that passed through the sieve in 24 hours were collected and counted,(Appendix table 18a). Means and standard errors are given in Text table 22.

Results The results indicate that barley roots are attractive to H. avenae larvae; in the absence of attraction the expected number of larvae in the central core would be 4% of the inoculum (= 60 larvae). It is probable that the numbers of larvae recovered from around the agar blocks do 127 not differ from the number that would be expected if the larvae were still evenly distributed.

Table 22 Attraction of H. avenae to take-all infected barley roots (means of 3 replicates)

Larvae attracted Attractant 'hi ± S.E. ( Agar 68.33 8.58 Agar + 0.araminis 41.67 6.94 Healthy roots 842.00 38.31 Infected roots 561.33 67.20

That fewer larvae were recovered from around take-all infected roots may indicate that these are less attractive than healthy roots. However, in these simple observations the roots were not examined for invasion. It is possible that in transferring roots together with the core of sand to the extraction sieve, larvae that had invaded the roots were also extracted. Since invasion of the take-all infected root pieces would be lower than that of the healthy ones this may account for some of the difference in larvae recovered. However, it is unlikely to explain the noted mean difference of over 200 larvae so that there may be reduced attractiveness of take-all infected roots. 128

3 Discussion The results of these experiments further demonstrate that pre-inoculation of barley roots with 0. graminis reduces the number of H. avenae larvae invading the roots, as compared with healthy roots. The invasion is reduced when only a small proportion of the roots are infected with take-all. The effect of the fungus appears to be limited to those roots which are actually infected with take-all, larval invasion of the healthy roots of a take-all infected root system being unaffected. However, some invasion was recorded on roots with take-all lesions, so that the suitability of the roots for nematode invasion is not entirely destroyed by the fungus. It is difficult to explain reduced invasion on the grounds of some decline in suitability of the roots, since some larvae do invade. The effects of take-all on the attractiveness of roots to second stage larvae were investigated and the results suggest that fungus infected roots may be less attractive than healthy ones. Investigation of relationships between other fungi and nematodes has shown that nematode invasion is variously affected by the fungi. McKeen and Mountain (1961) demonstrated that egg plant roots infected with Verticillium albo-atrum supported higher populations of Pratylenchus penetrans, and suggested that .this was_relatud to 129

increased levels of nematode invasion of the fungus infected roots. P. penetrans has also been found to invade alfalfa roots infected with either Fusarium oxysporum or Trichoderma viride more heavily than healthy roots (Edmunds & Mai,1966). These cases obviously differ from that reported here for H. avenae and O. graminis. Support for my observations comes from observations by Ketudat (1968) on Heterodera rostochiensis invasion of tomato roots. Infection of the roots by a number of root rotting fungi reduced nematode invasion. Attractiveness of roots to the nematodes may be an important factor in governing invasion levels. The production of exudates by plant roots has been widely stgges ted to be responsible for the attraction of roots to nematodes (Wieser,1956 ;Viglierchio,1961). The effect of a fungus on the nature and possible attractiveness of root exudates may therefore have considerable effects on nematode invasion. Edmunds and Mai,(1967), have demonstrated that F. oxysporum infected roots were more attractive to P. penetrans. They also showed that the fungus in agar culture was repellent to the nematode. In my experiments it is difficult to estimate the part played by attraction of the roots in determining levels of invasion. It is possible that fewer larvae of 130

H. avenae were attracted to the take-all infected roots, and that consequently fewer penetrated the roots.. In these experiments, the larvae were placed in the immediate vicinity of the roots; it is likely that they were clnse enough to the roots to be influenced by root attraction (Bird,1959, has reported root attraction to nematodes over distances of several centimetres). Whether the stimulus provided by the roots is necessary to activate the larvae, or whether it merely orientates their otherwise random movement, cannot be decided on the basis of my investigations. Further investigation of invasion and attraction of barley roots infected by take-all is required before definite conclusions on this facet of the fungus nematode relationship can be reached. 1311 SECTION VI GENERAL DISCUSSION AND CONCLUSIONS

The results of observations on field populations of H. avenae and 0. graminis suggested a relationship in which higher levels of the nematode were associated with low levels of take-all. Both the level of root invasion by larvae and the multiplication of H. avenae showed this relationship with take-all. Negative correlation between the number of eggs per cyst and the incidence of take-all suggested a possible explanation of tne relationship. In pot experiments with naturally infested soils the effects of take-all on the nematode were investigated. Multiplication of the nematode was reduced in the presence of take-all, and this was associated with reduction of the numbers of eggs per cyst. There was some uncertainty in . the estimation of cyst contents in these observations in field soils. Cysts remaining from previous years could not readily be distinguished from newly produced cysts containing none or very few eggs. However, investigations on the relationship in sterilised soil inoculated with the nematode and fungus alone or in combination established that both cyst production and cyst contents were lower on plants infected with take-all. This reduction in the number of eggs per cyst was related to the production of smaller cysts. 132

The effects of take-all on the development of H. avenae were examined and it was shown that on fungus infected plants the rate of larval development was slower than on healthy plants. The ratio of male to female nematodes produced was affected by take-all, relatively more males being produced on infected roots. This appeared to be due to the effects of 0. graminis on the sex determination of developing larvae, coupled with a differential ability of the sexes to mature on take-all infected roots. Larval root invasion was not affected by simultaneous inoculation with both organisms but it was reduced by infecting roots with the fungus before exposure to larvae. It is possible that take-all may decrease the attractiveness of • roots to nematode larvae. In none of these experiments did the infection or build- up of take-all appear to be affected by H. avenae, final take-all levels being independent of the H. avenae population. The relationship between H. avenae and 0. graminis established by these results is similar to those relationships studied by Ketudat (1968,1969) who showed that Heterodera rostochiensis was adversely affected by three root rotting fungi of tomato, (Rhizoctonia solani, Verticillium albo-atrum and a grey sterile fungus). Some aspects of the relationship between H. avenae and 0. graminis on spring wheat are currently being investigated by Williams (1968,1969 and 133 personal communication). In his investigations take-all has been shown to increase the male:female ratio, altht'ough it has not been possible to attribute these changes in sex ratio to the effects of the fungus on sex determination of the larvae. In both Williams' and my experiments development of the larvae to the adult stages was either delayed or seriously interfered with on take-all infected plants. Throughout the discussions of each section of this work parallels have been drawn with some other fungus nematode interactions. I now wish to consider my results in relation to these other observations. In his review of nematode interrelationships with other plant pathogens, Pitcher (1965) briefly discusses the effects of some of the relationships on the nematode partner. Obligate plant parasitic nematodes are considered to be particularly vulnerable to competition for either root space and/or food. A sedentary nematode, such as H. avenae, would seem to be especially vulnerable, since once established at a feeding site, it quickly loses the ability to move to a fresh site should the original one become unsuitable. General observations by plant nematologists point to the incompatab- ility of plant parasitic nematodes and decay-promoting organisms, (Christie & Perry,1959). In a number of fungus nematode interactions it seems likely that the fungus adversely affects nematode development. Species of Meloidogyne 134

are involved in striking disease complexes with range of fungi in which the fungus partner benefits from the presence of the nematode. The galled tissues and giant cells produced by the plant in response to root knot nematode infection provide particularly good substrates for growth of fungi. In consequence, the severity of fungal disease is often increased and in some cases these interactions enable fungi to attack plants which in the absence of the nematode are resistant (Sasser et.al.,1955;Cohn & Minz,1960;Powell & Nusbaum,1960; Schindler et.al.,1961;Pows11,1963;Porter & Powell,1967; Johnson & Littrell,1969). In all these interrelationships giant cells are extensively colonised by the fungus and this must affect the nematodes' nutrition. Davis and Jenkins (1963) reported that maturation of Meloidogyne spp. females was prevented in these circumstances. Pitcher (1965) refers to work by Davis with a number of pea diseases in which there was, a range of effects on the nematode partner of a relationship, varying from beneficial to detrimental. From the observed effects of 0. graminis on H. avenae1 it seems highly probable that the nema.bode suffers. in competition with take-all and it is worth speculating on the nature of this competition. An observation on interactions between nematodes and fungi made by Powell and Nusbaum (1963) is important in this context. They considered that the host plant is an integral part of these interactions and that the 135

basis of interactions is likely to lie, not in the direct effects of one pathogen on the other, but on the responses of the host. In the present discussion the response of the plant to take-all infection is considered to be the cause of the adverse effects of 0. graminis on H. avenae. The measurements of root weights made in my experiments provide some evidence to suggest that a reduction in size of the root system may play a part in depressing nematode populations. In field soils final root weights were not related to either depression of multiplication of H. avenae or to final take-all levels. The effects of take-all are confounded by the continual production of roots by the plant. At this late growth stage root weight is a balance between growth (which may well have stopped) and decay of the root system. It does not reflect the size and,perhaps more importantI the vigour and physiological state of the roots at the time when H. avenae larvae are feeding within the roots. At this earlier stage, take-all infection may reduce the vigour of the plant and perhaps kill roots in which nematodes are present, or even result in death of the plant. In this situation, the nematode would obviously suffer, but if the take-all infection is less severe, the effects on the nematode may be more subtle. In the field take-all builds up relatively slowly during the time when H. avenae larvae are developing. Rapid build up and peak levels of take-all are reached in 136

late summer, when nematode development is largely completed (Duggan,1961). It has been shown that take-all reduces the efficiency of cereal roots as conducting organs through infection of the tissues of the stele (Simmonds & Sallans,1933;Ludbrook,1942). After penetration of the stele by the fungus, infected cells disintegrate (Fellows,1928). This is likely to interfere with the transport of photosynthetic products from the stem and leaves to the roots which may have considerable influence on H. avenae. In the investigations on the effects of take-all on invasion of roots by H. avenae it was shown that fungus- infected roots were in some way less suitable for larval penetration or less attractive to the larvae. Healthy roots on take-all infected root systems were as heavily invaded as roots on healthy plants. Investigation of other nematode/ fungus relationships has shown effects of the fungus on nematode invasion, for example, the invasion of roots by Heterodera rostochiensis is reduced by root rotting fungi (Ketudat,1968). On the other hand, Edmunds and Mai (1966 and 1367) have shown that alfalfa roots infected by Fusarium oxysporum were more heavily invaded by Pratylenchus penetrans and were more attractive to the nematode. In the H. avenaeJtake-all relationship there may be several explanations of the reduced invasion of fungus infected roots. Take-all may reduce attractiveness of the root 137

to larvae with consequently reduced invasion. There was some evidence for this from the experiments described in this thesis. An alternative explanation may be that take-all affects the response of host roots to invasion by H. avenae. If giant cell production is hindered or prevented by 0. graminis, larvae may penetrate roots but, after failing to stimulate giant cell production, may reemerge from the root. There may be a parallel to this in certain varieties of plants resistant to nematodes. In alfalfa varieties resistant to Meloidogyne incognita acrita there is no response of the roots to penetration by larvae. These roots are as heavily invaded as the roots of susceptible alfalfa, but more of the second stage larvae invading re-emerge from resistant than from susceptible roots (Reynolds & Carter,1969). Cotten (1967) has shown that barley roots resistant to H. avenae are as heavily invaded as those of susceptible barley genotypes. It is likely that there is very little response of resistant roots to invasion by this nematode. Recent investigations that I have made suggest that larvae fail to find a feeding site and that second stage larvae re-emerge from these roots more readily than from susceptible ones (unpublished data). In simultaneous inoculations of H. avenae and 0. gEaminis second stage larvae may become sedentary at feeding sites before the response of the roots to nematode invasion is affected by the fungus. 138

In histological investigations of this relationship Meyer (1968) observed vigorous fungal growth, presumably of O. graminis, in giant cell areas. Mature females were never found in association with these fungus infected giant cells. Comparisons with other fungus/nematode interactions suggests that the invasion of giant cells by a fungus can affect both the nematode and the fungus. In those relationships, previously mentioned in this discussion, the presence of the giant cells apparently benefits the fungus, but the presence of the fungus in its giant cells must be detrimental to the nematode. Roy (1968) found that when giant cells of H. rostochiensis in tomato roots were infected with grey sterile fungus the nematodes failed to mature. There is no evidence however from my studies nor from those of Meyer of an effect of H. avenae on the progress of take-all. Disease severity is apparently unaffected even though the fungus invades the nutrient rich giant cells. According to Fellows (1928), 0, graminis is checked in its extension through the root by the endodermal layer. Whilst this barrier is soon overcome by the fungus it is possible that the giant cells may aid the fungus in its progress into the stele. Careful histological study would be necessary to establish such an effect. It is interesting to note that the response of oat roots to H. avenae did not reduce the resistance of oats to the wheat and barley strain of 139

0. graminis used in the experiments. As a consequence of giant cell invasion by the fungus, or of the reduction in vigour of the giant cells through the presence of take-all on the plant, it is likely that the feeding of H. avenae larvae would be affected. The effects on the larvae will depend on their stage of development when their giant cells become infected. Sex determination may take place within 10 days after penetration, since the moult to the third larval stage occurs at about 10 days after invasion and larvae are then sexually identifiable. If 0. graminis infects the giant cell soon after its formation the larva may obtain so little food that it develops as male. Early attack of giant cells by 0. graminis may also entirely prevent maturation of the larvae whatever their sex. If invasion of nematode feeding sites is delayed umil after sexual differentiation larval development may continue. This situation would perhaps allow males to develop but not females, which probably require much more food. Sengsbusch (1927) has estimated that females of Heterodera schactii require 35 times more food than males and Trudgill (1967) concluded that H. rostochiensis larvae had a minimum giant cell size requirement to become female. The development of females may therefore be affected by take-all attack of their feeding sites. It may be delayed or prevented, the size of the female may be reduced and the 140

number of eggs produced by the female may also be reduced. It is not known whether Heterodera females feed continuously throughout the period of egg production or whether eggs are produced from food stored in the body. Information on this point would be useful in determining what effect disintegra- tion of giant cells might have on the number of eggs produced. It is probable that feeding continues during egg production. In the related genus, Meloidogyne, females produce eggs which pass from the uterus into an extruded eggsac. Several thousand eggs may be produced over a relatively long period (Tyler,1938) and feeding would seem to be essential during this time. If a continual supply of food is necessary for egg production by H. avenael take-all could reduce the number of eggs produced through infection of giant cells, at any stage between fertilisation of the female and completion of egg production. A combination of these possible effects of take-all and responses of the nematode may occur in the field situation. The likelihood of their occurrence is now considered in relation to some environmental effects. Larval invasion may or may not be affected by take-all in the field. The relationship between numbers of larvae in roots in spring and take-all infection has been considered, in the section on field observations, to reflect either depression of initial H. avenae populations by high levels of take-all 1411

on the preceding crop, or to show that take-all reduces invasion. During the winter, when no host is present, both fungus and nematode are inactive. In spring, larvae respond to increasing soil temperatures by hatching and emerging from the cysts. They are able to move through the soil before penetrating host roots. In contrast the fungus does not grow through the soil, infection occurring on contact of a growing root with the take-all inoculum. Initially then nematode invasion should be unaffected by the presence of take-all in the soil. However once root infection by the fungus has begun subsequent invasion by second stage larvae in the field may be reduced. Larval invasion, which normally begins in mid-March, continues until early summer (Duggan, 1961) so it is possible that the adverse effects of take-all on H. avenae may begin with its effects on invasion . In other situations factors which reduce the initial invasion of roots by larvae of cyst-forming nematodes may result in increased final nematode populations, since invasion damage to the roots is reduced and more food and space is therefore available to those larvae which do invade (Jones & Parrot,1969). However, if take-all is responsible for reducing invasion in the field it is unlikely that roots would remain sufficiently vigorous to support large cyst populations, since severe take-all infection would probably develop and adversely affect the 142

development of those larvae that did invade. The rate of development, sex determination and the ability of larvae to mature and produce eggs will be variously affected according to the initial inoculum and rate of build-up of take-all. From season to season this relationship will be affected by any of the soil or other environmental factors that affect the vigour and infective potential of the fungus or nematode, or the resistance or susceptibility of the barley. Included in this seasonally variable situation, is the phenomenon of take-all decline under continuous cereals. When take-all levels begin to decline after several continuous barley crops,H. avenae multiplication may increase. Take-all decline was observed in the 1967 flaeld observations and higher nematode populations were found on fields which had probably passed through the period of peak take-all. This point was discussed in Section II. In spite of the reduction of H. avenae populations by 0. graminis take-all cannot be considered as an exploitable biological control mechanism. The levels of take-all responsible for reductions of nematode numbers are in themselves capable of causing considerable yield loss of the crop. However, the observations in this thesis may contribute to the understanding of the unexplained decline of H. avenae populations under susceptible hosts. These declines have been 143

reported with increasing frequency during the course of my investigations, and in some cases take-all may be involved. Accounts of cereal cyst nematode decline have been published by Gair et al (1969) and Cotten (1970). In both cases the decline has been under continuous cereals and continued for 12 and 4 years respectively. Nematode populations showed no signs of recovering from the decline at the end of these experiments. Other unpublished reports by officers of the National Agricultural Advisory Service indicate that the decline phenomenon is not limited to any particular region of Great Britain. In none of these accounts has take-all been implicated but there are no reports of examinations for take-all in these sites. Jones and Parrot (1969) have suggested that, under continuous cultivation of host crops, cyst nematode populations should eventually reach a stable level. This level would be influenced by host effeciency and subject to variation due to soil and other environmental factors. The stable populations of H. avenae reached in fields showing decline are far below those which the host is capable of supporting, and also appear to be economically acceptable to the farmer. There is therefore interest and possible value in examining the phenomenon. In discussion by plant nematologists of this phenomenon the whole range of soil physical and chemical 144

factors is being implicated. As yet there is no evidence to explain any case of declineylet alone a general explanation. Characters such as soil moisture, and available pore space have been shown to affect population build up of H. avenae (Dixon,1963;Fidler & Bevan,1963) Wallace (1961a,b), has summarised the factors influencing the ability of Heterodera larvae to reach plant roots, and the bionomics of free- living stages of plant parasitic nematodes. In these studies the effects of various soil conditions on Heterodera populations were examined. Although production of continuous cereals may have considerable effects on soil structure, their exact nature has not yet been established. However it is probable that such factors as increased consolidation, which may adversely effect H. avenae, will not explain the phenomenon of H. avenae decline on a wide range of soil types. The stages in its life cycle when H. avenae is most likely to be vulnerable to adverse conditions are the movement'of infective larvae in soil to plant roots, and the emergence of males from roots before fertilisation of the females. In some cases of H. avenae decline it has been noticed that invasion of roots in spring was not related to subsequent decline. Graham (personal communication) has observed that, in a population where the final egg populations declined, large numbers of mature females were attached to the roots. It has been inferred from this that fertilisation 145

may be affected, perhaps by dry conditions. However these are unlikely to be either sufficiently long lasting or widespread to account for the marked reductions in egg populations. In relation to the decline of cereal cyst nematode populations it is worth noting that it is thought that H. avenae is indigenous to the British Isles, commonly occurring under old undisturbed pastures at low levels (Gair,1965). Experiments with several populations of H. avenae have shown that grasses are less efficient hosts than cereals. It is possible that factors other than host efficiency may be responsible for the maintenance of H. avenae populations at low levels under grass (Winslow,19540Cort,1964). There is some evidence to indicate that such a natural balance may exist, and it is possible that under cultivation of cereals the balance is first destroyed and that it may later become reestablished, resulting in the decline. The results of formalin soil sterilisation reported by Williams (1967) may involve more than the control of take-all, also removing other competitors or antagonists of H. avenae. In 1952, Wagner reported that soil treatment with calcium cyanide resulted in increased cyst production by H. avenae. He speculated that in untreated soils nematode multiplication may have been checked by a natural limiting factor. Other evidence suggests that nematodes may be subject 146

in field soils to a wide range of predators, competitors and antagonists. The control of plant parasitic nematodes by nematophagous fungi has been investigated, but whilst some reduction does occur this does not appear to be a practical possibility (Duddington & Duthoit,1960;Duddington et a1,1961; Duthoit & Godfrey01963). Mankau (1962) demonstrated that the addition of organic materials to soils allowed populations of nematophagous fungi to increase. The same author obtained control of Meloidogyne incognita by the addition of organic amendments on field soils. Numbers of larvae were not greatly reduced by the treatments but their infectivity was. Circumstantial evidence from these experiments indicated that bacteria and fungi were involved in the effects of organic amendments on root-knot disease (Mankau,1968). Miller et al (1968) found that decomposing cellulosic soil aaqadments reduced larval emergence and invasion of host roots by Heterodera tabacum. They suggested that the practice of fertilising tobacco with cottonseed meal in some parts of the United States may be responsible for the freedom of these crops from H. tabacum attack. Sayre et al (1965) identified butyric acid as a nematicidal component from decaying plant residues which were toxic to Meloidogyne incognita and Pratylenchus penetrans, but not to saprophagous nematodes. Plant parasitic nematodes have been shown to be vulnerable to competition with other species of nematodes. 147

Thus Miller and Wihrheim (1968) showed that Heterodera tabacum was suppressed by Pratylenchus penetrans, another endoparasitel and by Tylenchorhynchus claytoni, an ectoparasite. Jenkins (1968) showed that there was competition between a species of Meloidogyne and other plant parasitic nematodes, and suggested that more competitive relationships between nematodes were likely to exist. In plant pathology there is much evidence for soil_ mycostasis, that is the prevention of growth of fungi with consequently reduced disease levels. Such mycostasis is attributable to deprivation of nutrients, and production of toxins and antibiotics by the soil microflora (Dobbs & Gash, 1965). This: may explain the decline of O. graminis (Zogg,1962;Ehle,1966). The effect of similar phenomena on plant parasitic nematodes has not been investigated but it is clearly conceivable that unematostasis" may occur, and may relate to the effects of organic soil amendments on nematodes. The continuous cultivation of cereals may then lead to changes in the soil environment. These may exert a natural balance on H. avenae populations in a number of possible ways. Some of these possibilities have been considered, in addition to the effects of 0. graminis on the nematode for which this thesis provides experimentdl evidence. The discussion of these results indicates the importance of the 148 whole range of ecological factors in influencing the interrelationships of plant, fungus and nematode. Powell (1968) strikingly illustrated this when he showed the damage done by supposedly non-parasitic fungi to roots which had been infected with Meloidogyne spp. More recently, Powell and Batten (1969) published a report of the effects of nematodes in increasing the severity of foliar diseases of tobacco. It is therefore of considerable importance to consider the whole range o1 biological and ecological factors when investigating the nature of soil-borne diseases. Although it is clearly impracticable to critically examine these factors by including them all in experiments) the complexity of the soil eavironment.and of the interrelationships of organisms in the soil should be borne in mind when drawing conclusions from the results of controlled experiments. 149

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population of the soil, with special reference to the antagonists effective against Ophiobolus Araminis. Z.Pf1Krankh.PflPath.PhSchutz.,73:321-526,326-334(

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KETUDAT,U. (1968) Investigations into the interrelationships of some soil-borne fungi and nematodes on tomatoes. Ph.D. thesis. Univ. of London KETUDAT,U. (1969) The effects of some soil-borne fungi on the sex ratio of Heterodera rostochiensis on tomato. Nematologica,15:229-233 KLINK,J.W. & BARKER,K.R. (1968) Effect of Aphelenchus avenae on the survival and pathogenic activity of root-rotting fungi. Phytopathology,58:228-232. KORT,J. (1964) Grass species as hosts of the cereal root eelworm, Heterodera avenae Wollenweber. Meded. LandbHoogesch.OpzcekStns.Gent,29:783-787 KUIPER,K. (1966) Enige bijzondere aaltjesaantastingen in 1965. Yiidschr.P1Zeikt.,72:210 LESTER,E. (1967) Disease problems in intensive cereal growing. Proc.Er.Insectic.Fungic.Conf.,4:510-516 LESTER,E. & SHIPTON,P.J. (1967) A technique for studying inhibition of the parasitic activity of Ophiobolus graminis (Sacc.) Sacc. in field soils. Pl.Path.,16: 121-123 LUDBROOK,W.V. (1942) Root amputation experiments with wheat under dry conditions, in relation to attack by Ophiobolus graminis Sacc. J.Council Sci.Indust.Res., 15:129-134 RANKAU,R. (1962) Soil fungistasis and nematophagous fungi. Itytopathology,52:611-615 156

MANKAU,R. (1968) Reduction of root-knot disease with organic amendments under semi-field conditions. P1. Dis.Reptr.,52:315-319 MCKEEN,C.D. & MOUNTAIN,W. B. (1960) Synergism between Pratylenchus penetrans (Cobb) and Verticillium alboatrum R. & B. in eggplant wilt. Can.J.Botany, 38:789-794 MEYER,A. (1968) Observations on the histopathology of Heterodera avenae Woll. in barley infected with the fungus, Ophiobolus graminis Sacc. D.I.C. thesis, Imperial College, London. MILLER,P.M.,TAYLOR,G.S. & WIHRHEIM,S.E. (1968) Effects of cellulosic soil amendments and fertilizers on Heterodera tabacum. Pl.Dis.Reptr.,52:441-445 MILLER,P.M. & WIHRHEIM,S.E. (1968) Mutual antagonism between Heterodera tabacum and some other parasitic nematodes. Pl.Dis.Reptr:. 922:57-58 MOLZ,E. (1920) Versuche zur Ermittlung des Einflusses ausserer Faktoren auf das Geschlechtsverhaltnis des; Rubennematoden (Heterodera schactii A. Schmidt.) Landw.Jb.Schweiz.,54:769-791 MOLZ,E. (1927) Zur Frage des Geschlechts verhaltnisses des Rubennematoden Heterodera schactii. S.PflKrankh.,37: 260-266 MORIARTY,F. (1963) A nylon sieve for hatching Heterodera larvae. Nematologica, 9:157-158 157

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162

APPENDIX TABLE Field Samples,1967. Location of sites Field ref. Field Name number Farm Address Chalk Hill tAf 17 Aston Upthorpe Estate Chalk Hill 'B' 18 Aston Tirrold Chalk Hill 'C' 1 Berkshire Star 5 Blackalls Farm Ltd. Hospital 24 Cholsey, Berkshire 0.S. 195 10 Haddon Farm 0.S. 153 2 Didcot, Berkshire Castlemans 7 Hatchgate Farm Manor 11 Hurst Fox Steep 19 Berkshire Hill Barn 14 Manor Farm, West Hagbourne, Hill Firs 3 Berkshire Bundu 23 Derrydown Farm. St. Mary- Surgery 22 bourne, Hampshire Ibworth 13 12 Kingsclere Estates, Ibworth 14 6 Ramsdell, Hampshire M.W.642 . 8 Middle :tyke Farm M.W.779 20 St. Marybourne M.W.787 9 Hampshire Jerry Lane 13 Northington Farms Ltd. Villa 15 Overton, Hampshire Policemans, 4 Southwood Farm Gander Down 21 hummer, Hampshire Rockbourne 9 16 Tenantry Farm Rockbourne, Hampshire 163

TABLE 1 b. Field Samples9 1967. Cropping records, soil type, sampling dates.

Sampling Dates

t h ry

s s u tinuous

brua Marc rop e

. Aug p

l Fe Name f con l c tko er: l

d ty tia •ri a l o i ;-i 68 il P4 erea 19 In Fin Summ c

No. M' So Fie

1 Chalk Hill 'C' light chalk 18 22/5 7 21 1 0.S. 153 greensand 10 23/5 18 23 1 Hill Farm light chalk 22/5 18 23 1 policemans Hint loam over chalk 14 1/6 18 26 3 6tar Thames Terrace 18 22/5 7 21 3 Ibworth 14 chalk 13 2/6 24 26 4 Castlemans London clay 26/5 8 16 4 M.W. 787 clalk loam 5 1/6 9 19 4 M.. 787 chalk loam 5 1/6 9 19 5 0.S. 194 greensand 10 23/5 18 23 5 Manor London Clay 26/5 8 16 5 Ibworth 13 chalk 13 2/6 24 26 5 Jerry Lane chalk 13 2/6 18 26 6 Hill Barn light chalk 22/5 18 23 6 Villa chalk 13 2/6 18 26 6 Rockbourne 9 chalk 17 3/6 25 28 7 Chalk Hill 'A' light chalk 18 22/5 7 21' 7 Chalk Hill 'B' light chalk 18 22/5 7 21 7 Fox Steep London Clay 26/5 8 16 8 M.W.779 chalk loam 5 1/6 7 19 8 Gander Down flint loam over chalk 14 1/6 18 26 10 burgery chalk 5 1/6 18 19 11 Bundu flint loam over chalk 5 1/6 9 19 11 Hospital Thames Terrace 18 22/5 7 21

zi, 9 t'91. L'5L L'Ot C't z*z t'EL 9't 08 9t FE tE 0 0 0 0 0 0 0 0 0 0 0 0 CE L 0 6'C E'L E'CL 5°0 0 5'E 0 EC 8 0 EE te LL 0'6 t't 5*SE 9'E L'O E'LL E*5 09 EC 9L LE LOL 8 5'CL EsC L' Lt e't E'L t'LL E°C t9 9C EL OE OL - 9 -tL I'LL O'Lt 9•0 t°0 0°E - 9L 9 - 6L L L S'L 9'L L'O 5'0 0 O'L t°0 eg 9 EE 9L CE EL 0°9C 0.9E 0'C9 L'C 0 9'2, 8't ooL EC Et LL C6t Cl L'ot 9'5E 6'58 6°9C'6 L'Ot E*5 96 09 OL 9L L6 tC L'LL L°L L' e't 5•0 C'LL 9*LL E6 9t Et 5L 8 - 9*tL 6'6 L' LS 8'0 0 6'L - tL Z - tl. CtC 9t e't9 t'26 8'65 9' LL L'9 9*tC E'5L 00L OL tt CL 9E1 LE 9'LE 'Ll. 9°E9 °L, 5'L 5°Cz 6't 96 9t Oa EL E9 - E'OL S't 0'6E t°E L'O g°L - 99 el. - LL 5e E 5'0E L'OL 5'05 z'a 0 E'C 9*C tL 9L 2 OL 9 L E*6 t'L L'LZ L .0 0 2°L O'C tt OL EL 6 teL LE t'9L 5'5 5'C5 9°L 6•0 5•9e L'9 E6 t9 9E 9 OL - 9°E E'L C'L t'0 E*0 O'L - ta 9 - L 8t 6 C*5 t'C L'CL e'a L'L t*9 5't 9t tE EL 9 9tL OL t'CE E'OE L'6t 5'S 9'E E'91. vt t6 Ot 5L 5 E 0 L'5 9'L L'EE E*0 0 9'0 0 t5 t 0 t 0 - 6'5 O'E 0'81. 0 0 0 - Et 0 - C 0 C°0L 0'5 E'LC L*0 0 C*0 0 89 E 0 E 0 L E*9 E't O'EL 0 0 0 t'0 Ot 0 E L S I 1-3 0 U.i 1-3 a w 1-3 H pri nit 0 1-1 0 0 11 CD 0 ''12 ITT 1-, , 1F -11 c+ 0 0 d 0 0 c+ K 1-1: 1(3, n i CD IS: PJ g al H N 5' ri 0 5* Pl. P P 11 (XI P H H H 1-3

c+ TPT 1-4)CD aemmns PuTads -I.Tui peq.oejui • goon OS poq.oajuT sq.o0H % eq-weT 01, g uoTsarT

-Ere °L96L'seidures piaTa •OI arIgNI

t9L 165

TABTF ld. Field samples41967 Take-all Infection category based on seminal roots infected in summer,1967

Category I ( 0 -C25% seminal roots infected) Chalk Hill 'C' Hill Firs Policemans Ibworth 14 Castlemans Chalk Hill 'B' Surgery Bundu Category II (25 -<50% seminal roots infected) 0.S.153 Star M. W.787 Manor Hill Barn Fox Steep M.W.779 Gander Down Hospital Category III (50 -(75% seminal roots infected) M.W.642. 0.S.194 Ibworth 13 Jerry Lane Villa Chalk Hill 'A' Category IV (75 - 1007; seminal roots infected) Rockbourne 9

166 TABLE le. FIELD SAMPLES,1967. BUILD UP OF TAKE-ALL DURING SEASON

Infection % plants infected INo. of fields category Initial Spring Summer

I 1.01 6.25 36.48 8 II 12.72 21.56 72.00 9 III 27.17 36.00 92.33 6 IV 23.00 80.00 96.00 1

Infection % seminal roots infecte• No. of fields category Initial Spring Summer

I 0.46 1.71 10.88 8 II 3.31 8.08 35.89 9 III 8.03 18.82 57.05 6 IV 5.20 40.10 85.90 1

Infection % all roots infected No, of fields category Initial opring Summer

I 0.46 0.48 4.67 8 II 3.31 2.44 13.69 9 III 8.03 6.27 30.50 6 IV 5.20 18.90 40.70 1 167

TABLE 1f. FIELD SAIIPLES,19670 BUILD UP AND DECLINE OF TAKE-ALL UNDER CONTINUOUS CEREAL CULTIVATION

ds, l

s t iz

ci; seminal A total roots

L n : 1-b 0 c c 0 i c roots infected f fie ls ko infectedp tlo P co O ea ing mer o

r r .1-I

um P 1 ce sp s

Yrs. Or No. M Al4 Ei M M 1 4 0 51 0.28 20.83 0.08 6.53 no data 3 2 32 70 13.30 31.70 1.30 14.35 4 3 27 53 9.77 29.30 2.90 9.37 5 4 38 87 17.50 50.52 5.98 30.78 6 3 43 87 19.77 54.10 8.17 26.50 7 3 15 79 3.53 36.90 1.67 19.37 8 2 34 72 9.53 33.60 3.70 11.25 9 no data. 10 1 8 32 2.50 13.20 0.50 3.90 11 2 23 40 6.20 20.05 2.15 9.20 168

TABLE lg. Field samples,1967. Cereal cyst nematode

Initial Spring Final

no. t, 1 40

f. c) .,_, ._4 Cs -i-n 0 M M P_4 0 M +3 0 \ 0 W re \ 0 40 1 Co -, 0 M \ \ -P M M ld 0 W b0 al W tn 0 0 Fie 0 0 W 'ZP., V. H 0 A- - 1 1.0 0.01 10 1.5 10.0 37 19.7 7.3 CV 3.6 te 2 15.0 0.3 40 5.2 67.2 6 60.0 N 14 40.0 5.6

, 10 1.4 10.1 z1 - 1 5.0 40 6.3 104.0 43 117.0 7.3 .1" 54 9.3 \ 3 2.2 lc 3 56.7 1.7 36 3.3 44.3 73.3 ) Cs 5 36.0 1.8 2 0.1 0.9 4 37.5 1.5 . -

CO 30 4.7 77.5 20 108.5 21.7

61 21.8 13.3 al 70 21.9 15.3 28 3.1 34.0 150 20.9 31.4 90 22.7 46.4

, 0 322.6 152 30.6

,- N- 16 84.4 13.5 70 16.9 241.8 25 70.4 17.6 .

,-- C 4 0.2 2.2 2 20.0 0.4 V - r tr 13 0.7 0.1 4 0.3 1.9 6 5.0 0.3 - \ d

,- 21 90.9 19.1 50 4.7 69.7 7 8.6 0.6 - .

„.- LC 32 7.0 94.3 18 90.0 16.2

\ k 3.3 54.0 ,- 40 9.0 3.6 36 28 23.9 6.7 -C) s C 230 7.8 18.0 70 12.5 232.2 184 8.0 14.7 - - - - - c.-- OD 10 37.0 3.7 30 4.5 98.6 8 53.8 4.3 N-- 0 10 6.0 0.6 42 8.6 80.0 37 17.0 7.3 . 1

0 0 32 4.4 123.3 9 43.3 3.9 .1 C 30 4.0 1.2 11.2 9 46.7 4.2

v- 22 1.4 \I

0 17.7 5.3 26 3.0 42.0 25 25.6 6.4

C 30 . M 4 / 1 1 C' 52 20.5 62 13.7 248.4 60 37.0 trs 39.4 22.2 d C

, 134 8.7 11.7 58 5.8 143.5 86 35.5 30.5 ct- \I

12 35.0 4.2 46 3.0 35.0 1 40.0 0.4

I 1

169

TARI,E 1h. Field samples,1967. Cereal cyst nematode population changes under continuous cereals.

Yrs.cont. No. of Initial Spring infection Final cereals fields eggs/g % plants % roots. eggs/g

1 4 1.77 25.00 3.60 7.10

3 2 1.75 19.00 1.70 1.85 4 3 23.35 49.33 10.20 27.10 5 4 10.90 32.00 5.50 5.20 6 3 10.80 46.00 7.60 12.50 7 3 2.15 34.67 5.80 5.20 8 2 325 24.00 2.20 5.30

10 1 20.50 62.00 13.70 22.20 11 2 7.95 52.00 4.40 15.45 170

TABLE 2a Field samples,1969. Cereal cyst nematode and take-all

•

J n -F - . N I Spring Final i g tio

to o A. l ,--1 0 to lica l no.

ts ts r /100 le —a

n. :.- /g moo` 1 m ts to tip s 0 CD -P M m s c. l ke 40 ta 'Co 'A k0 tat) cy Samp % roo % roo c. fac egg ta V \4' C.) 0 0 mu 1 1 4 2.0 20.0 33.3 29 19 39.0 7.4 3.7 2 32 6.1 9.1 63.7 28 20 165.5 33.1 5.4 3 62 47.0 0 88.5 36 94 97.7 91.8 2.0 4 56 27.0 12.6 63.0 52 74 52.6 38.9 1.4 5 46 5.2 0 70.8 38 60 53.3 32.0 6.2 6 68 22.4 13.4 87.1 34 55 79.3 43.6 2.0 7 98 50.9 9.1 9049 75 34 10.3 3.5 0.1 . 8 77 6.4 38.5 23.1 29 45 42.9 19.3 3.0 9 49 7.8 12.5 44.1 31 74 87.4 64.7 8.3 10 65 28.8 7.1 85.2 32 45 56.7 25.5 0.9 11 62 25.9 16.7 41.5 26 64 20.8 13.3 5.1 12 42 23.0 14.2 56.8 57 71 39.7 28.2 1.2 13 18 11.6 16.7 23.6 41 66 72.6 47.9 4.1 14 5 1.5 16.7 8.3 26 16 55.6 8.9 5.9 15 65 29.5 33.3 20.1 47 10 113.0 11.3 0.4 16 54 14.1 21.3 21.3 35 22 30.9 6.8 0.5 17 89 13.5 35.5 21.3 25 52 97.1 50.5 3.7 18 65 13.1 35.5 14.2 34 90 49.4 44.5 3.5 19 35 4.0 9.1 36.4 24 82 66.5 54.5 13.6 20 11 3.5 23.6 17.7 41 11 31.8 3.5 1.0 21 1 2.0 6.7 60.3 28 62 17.3 1 0.7 5.4 22 59 13.1 0 30.8 56 81 53.8 43.6 3.3 23 58 23.6 50.0 35.5 53 58 12.1 7.0 0.8 24 71 8.4 0 16.6 48 83 24.7 20.5 2.4 25 61 9.6 12.5 31.5 56 87 7.5 6.5 0.7 26 58 7.0 15.4 38.5 48 50 45.6 22.8 3.3 27 39 16.1 28.4 5.9 43 43 27.2 11.7 0.7 28 5 7.0 17.7 5.9 38 11 91.8 10.1 1.4 29 6 4.9 7.7 53.9 62 16 40.6 6.5 1.3 30 22 1.3 0 23.6 28 36 22.5 8.1 6.2 31 34 6.3 36.4 18.2 71 63 31.1 19.6 3.1 32 36 12.4 21.3 35.5 33 65 8.8 5.7 0.5 33 64 11.3 17.7 23.6 50 50 20.6 10.3 0.9 34 98 9.0 6.7 33.3 65 80 31.3 25.0 2.8 35 46 25.8 14.2 42.6 77 100 23.9 23.9 0.9 171

TABU, 3a. Inoculum tests with 0. graminis

Level of Percentage roots infected fungus w.w. I II III IV V in

1 25.0 32.3 27.8 41.2 25.0 151.3 30.26 5 58.8 61.8 31.3 66.7 58.8 277.4 55.48 10 63.9 86.7 88.9 97.3 71.1 407.9 81.58 15 97.3 84.1 85.7 97.3 84.1 448.5 89.70 20 100.0 88.6 100.0 82.4 93.9 464.9 92.98

Level of Lesions/root system fungus w.w. I II III IV V 17

1 16 29 24 23 20 112 22.4 5 59 53 17 67 20 216 43.2 10 97 81 107 103 81 469 93.6 15 151 97 87 82 84 501 100.2 20 158 110 122 106 127 623 124.6 TABLE 4a Effect of adding O. graminis to H. avenae populations in 8 field soils

CD c+ L1 CD • CD 0 a) % roots infected by take-all 6 pu CONTROL TREATMENT I II III I: m I II III Y. m to 1-1)I ' t 1 73.3 80.0 83.3 236.6 78.9 86.7 80.0 100.0 266.7 88.9 4.36 2.294 N.S. 2 70.0 100.0 58.3 228.3 76,1 100.0 84.2 71.4 255.6 85.2 7.56 1.204 N.S. 3 38.5 42.9 50.0 131.4 43.8 50.0 64.3 87.5 201.8 67.3 8.40 2.797 * 4 53.3 35.3 29.4 118.0 39.3 54.2 58.3 75.0 187.5 62.5 2.89 8.039 ** 5 42.9 57.1 37.5 137.5 45.8 62.5 64.3 66.7 193.5 64,5 4.78 3.913 * 6 47.1 33.3 35.7 116.1 38.7 57.1 61.1 75.0 193.2 64.4 2.74 9.365 *** 7 20.0 33.3 22.2 75.5 25.2 57.2 61.9 75.0 194.1 64.7 2.86 13.820 *** 8 12.5 0 0 12.5 4.2 84.2 61.1 50.0 195.3 65.1 4.02 15.150 *** TABLE 4a (cont.)

Final root weights (mg)

CONTROL TREATMENT 0 1inJ., g " 1 II III 27. iii 1 II III 1: TA t

1 220 95 46 361 120.3 99 250 60 409 136.3 21.19 0.755 N.S. 2 103 94 163 360 120.0 111 88 74 273 91.0 15.33 1.904 N.S. 3 101 145 99 345 115.0 247 125 99 471 157.0 35.17 1.194 N.S. 4 142 144 155 441 147.0 280 206 117 603 201.0 38.33 1.409 N.S. 5 223 45 45 313 104.3 219 192 188 599 199.7 47.85 1.994 N.S. 6 174 100 38 312 104.0 128 144 90 362 120.7 29.35 0.569 N.S. 7 415 445 306 1166 388.7 264 459 149 872 290.7 65.36 1.499 N.S. 8 168 212 142 522 174.0 84 167 96 347 115.7 12.90 4.520 * TABLE 4a (cont.)

0'02 ct> • <=+t:cJ :!! • Cysts/200 g soil CD CDO l31-+J sp. r--.- CD ~.

.. ~. ~ §~ .- CONTROL TREATMENT OJ

I II III I iii I II III '> om t

1 281 245 267 793 264.3 209 214 258 681 227.0 9.37 3.984 * 2 23 29 22 74 24.7 26 21 17 64 21. 3 1 .20 2.783 * 3 29 3°1 28 88 29.3 26 24 15 65 21. 7 2.65 2.895 * 4 27 26 32 85 28.3 31 25 30 .86 28.7 0.36 0.942 N.S 5 122 129 138 389 129.7 119 114 129 362 120.7 1 .15 7.832 ** 6 59 60 37 156 52.0 52 54 63 169 56.3 5.46 0.793 N.S. 7 84 99 101 284 94.3 97 101 110 308 102.7 5.55 1.504 N.S. 8 149 136 153 438 146.0 120 149 108 377 125.7 9.00 2.260 N. S. I ; I ! to9•C Lt9'L tC*CL ccrot 9E'SL e0s01 r6L at'LS Lrti. tC"E S 's"N 66S*1. Cert 30'03 SOI09 ereL CrLE woe LL'9z Croe E6°6t. LE*CE W9C L *** CEE*6 1.9L 0 1. Co"91. Wet CC"et. tS'SL E 0EC 08'96 SrtC Ce*tC n.Le 9 etL'C Loe'e er6C SS'LLL 6e 0 CC SC'eC LC*St 9'53 t0'9e St*LC Ls.Le eo-Lz s 9LC't 6Ee'S tE'Ce EL'69 00'5E Ot'Sz SL'et tz"9ti. es.ts t0'65 Ee'ES t 's°N CLE°0 LeC°9 L6•2 06'59 CS*8C CC•EL to'SL L9'oz Ee'L9 oo'EE 9rSt 99- tE C °s°N C9ego eLE'6 E3'tS 99'391. tE•Ct o0°59 Et'tS e"E9 69•9eL 9C*95 LOLL 9e'CL 3 °s'N Co9"L S9t'S LL'SC eCeSoL eroC Wet 9-17 99a SC'9E S0'62, CE•tE EIrSE t9'6E

IRTNIVEM q0UUTOO

q.alco/sPa

tf;

( .q.uo0) Tit a'iav

** L56'g 006'0 egoe tLiE z6*/, zc.'LL 05'9 t6'CL Le*Lt EE'LL oz*CL 6C*LL e * 1763'z 508'0 6e'OL 98'OC 91.'6 z9' 1.L 90°OL 0L'EL OL'LC 90'02 e*L. I. .(:)* SL L *** OCE'OL LtC*0 N't Lt'CL 8t't 6°-t7 to't LC'8 e6'te Lt°9 St'OL 90'8 9 'SIN tLe°0 LSL' 95'6L 89'85 9e*Le 9e*6 96°9e L8'81. tt'95 t8'5e 8L'LL ee'eL g ** 998'9 tO5'0 LC'C 56°6 5L'C 8L'C 00'C LL°9 1C'Oz OS'g 89'L CL'L t * C85't e6L'O LL'e eC'9 68'3 et'L 56'L 66'e 96'8 gO'C 55'e C5'C C 'S'N 98C'L Lgt'L 98'5 65'LL 89°C c2'9 80'L Le'L L9'Ce 00't 81"'LL 5t"8 z °SIN 506'0 00'5 55°6C 59'8LL 58'80 5L'e5 59'Le 56-VC ge -vol, gC'eC 58'O5 59' Lt L

I- cr-41 'i) m —7 III II I M 3 III II I 1-4 CD TS fa mauvaui TOEINOD 0 •.4

'Too 9/s2DE

(.41100) vs, auErvI

TABLE 4a (cont.)

Multiplication factor

P, CONTROL TREATMENT 0CD 1" I II III I: i I II III E. iii E rtb t

1 89.7 66.6 69.8 226.1 75.4 59.7 112.5 83.8 256.0 85.3 11.14 0.889 N.S. 2 150.0 200.0 71.4 421.4 140.5 126.8 121.4 66.1 314.3 104.8 26.12 1.368 N.S. 3 89.7 61.5 79.5 230.7 76.9 51.3 38.5 74.4 164.2 54.7 5.43 4.089 * 4 43.8 47.5 34.0 125.3 41.8 18.5 19.8 23.5 61.8 20.6 2.90 7.310 ** 5 57.7 80.2 116.2 254.1 84.7 121.6 44.6 98.7 264.9 88.3 12.41 0.290 N.S. 6 37.3 48.4 29.5 115.2 38.4 18.4 23.0 20.7 62.1 20.7 4.34 4.079 * 7 212.3 157.5 138.4 508.2 169.4 138.4 158.9 126.0 423.3 141.1 16.30 1.736 N.S. 8 57.1 43.3 36.7 137.1 45.7 21.3 37.1 25.9 84.3 28.1 3.12-5.640 ** TABLE 5a

Effect of adding O. graminis to three H. avenae populations in one field soil. Time 1.

roots with take-al CONTROL TREATMENT I II III II ill I II III Z m N1 0 0 0 43 60 38 141 47.00 N 0 67 22.33 2 0 0 29 0 38 N3 0 0 0 29 15 43 87 29.00

CD • Root wt (mg) CD 0 1-1) TREATMENT CONTROL Fci p, I II III Z lff I II III El M I' t m i-- -1;" Ni 223 42 214 479 159.67 110 51 113 274 91.33 45.17 1.513 N.S. N2 115 79 112 306 102:00 147 44 170 361 120.33 30.20 0.607 N.S. N3 162 139 152 453 151.00 81 206 114 401 133.67 33.60 0.516 N.B. • 0 Ca TABTIF 5a (cont CD • c-F N • Larvae/root system (D 0 Ft) CONTROL I TREATMENT 15 fa+ I II III > I II III I t N1 78 14 64 156 52.00 48 27 39 114 38.00 13.06 0.930 N.S. N2 59 41 42 142 47.33 43 25 58 126 42.00 5.08. 1.409 N.S. k.0 N3 46 59 54 159 53.00 29 62 52 143 47.67 7.35 0.725 N.S.

0" CD CD • c-F N • Larvae/g root CD CD 0 ,' I CONTROL TREATMENT E PJ m w I II III 2: m 1 II III t m Eb, 1,-, t Ni 349.8 333.3 299.1 982.2 327.4 436.3 529.4 345.1 1310.8 436.93 41.81 2.619 N.S. N2 513.0 519.0 375.0 1407.3 469.01 292.5 568.2 341.2 1201.9 400.63 57.78 1.184 N.S. N3 284.0 424.5 355.3 1063.8 354.93 358.0 301.0 456.1 1115.1 371.70 18.59 0.848 N.S. TABLE ea Effect of adding 0. graminis to three H. avenae populations in one field soil Time 2 cr. rn

L. % roots with take-all

5 Pa CONTROL TREATMENT CD 1-6 I II III M I II III E rf E N1 53 55 49 157 52.3 71 97 78 246 82.0 6.08 4.883 ** N2 58 52 39 149 49.7 78 74 79 231 77.0 4.28 6.382 ** N3 43 49 57 149 49.7 74 78 88 240 80.0 1 30 23.254 ***

• W cf. •O . Take-all lesions/root system 0 • 0 CONTROL TREATMENT 0 I II III 57. I II III 2: t CD 1-4) N1 52 45 35 132 44.0 78 101 84 263 87.7 3.67 11.911 *** N2 60 35 42 137 45.7 70 88 107 265 88.3 6.48 6.575 * * N3 43 33 50 126 42.0 86 142 101 329 109.7 12.96 5.224 * * TABLE 6a (cont.)

Final root weight (mg)

fl CONTROL TREATMENT CD I-4 I II III Z id I II III 5- -f,i r4 t

N1 686 287 402 1375 458.3 520 287 495 1302 434.0 63.93 0.380 N.S. N2 462 207 530 1199 399.7 423 578 464 1465 488.3 70.65 1.254 N.S. N3 522 434 395 1351 450.3 457 740 470 1667 555.7 68.63 1.536 N.S. 766°C 616'8 Lra S'1.8 L'e "9112' g'ZI. LL'9 C'e24 vet L'-I 5'1.6 CAT * L6L C 59e'e 09'fl. 9"Ct 1.'1.4 6-CZ 8°8 Oe'CZ 9'69 6'Le 0'9e L'51. en ** 1.59't 601.*C Lget L'el. 6'L e-17 (rt. 50'6L L'L5 5'LL C)*Lz 9.a4 L cH M 7K III II I E 2 III II I rd tH Inaluvalm gOlINOD q_sSoisBa

*S'AT 6e9'0 099'44 L9'1.9 5te gL 9V teL CC"i7L Cee an oi, 45 CN 'S'Al 1.90'1. 005'21. L9'001. e0C 21. 9t M. L9'0F1. e9C c6 M. 9C1. eN 'S°11 Ice't el.Ero CC•59 961. 65 Z9 SL 5C°99 664 cL L5 693 LAT m 4. ri-3 0 m cri d .2 III II I gi _3 III II I •r-I CD rii fel Inouvau TOHIN00

TTos 2 00e/sq-sS0

(*Q-11°°) '09 arialiTI

TABU', 6a (cont.)

Cr) cD • • Eggs/g soil 0 0 I--1) 5 PA CONTROL TREATMENT. CD H• Fb I II III E II III /: CD t N 4.34 7.70 6.39 18.43 6.14 0.38 1.49 2.33 4.20 1.40 0.661 7.170 ** N2 10.64 17.27 12.97 40.88 13.63 5.88 5.74 6.72 18.34 6.11 1.551 4.849 ** N3 23.38 19.14 21.47 63.99 21.33 7.75 10.64 8.50 26.89 8.96 0.695 17.796 **

cDc÷ cn Multiplication o -- 0 CONTROL TREATMENT cD I II III I II III 3: t

N1 434.0 770.0 639.0 1843.0 614.0 38.0 149.0 233.0 420.0 140.0 66.1} 7.169 * * N2 212.8 345.4 259.4 817.6 272.5 117,6 114.8 134.4 366.8 122.3 31.84 4.707 * * N3 49.3 40.7 45.7 135.7 43.9 16.5 22.6 18.1 57.2 19.1 6.47 3.833 .

TABLE 7a CD C/2 et- • Effect of O. graminis on M. naasi and T. dubius • tt CD • CD O 0

I II III IV V VI 1711 S.D. •cD Pi t CO %roots with po 43.3 37.1 35.7 22.2 29.4 33.3 201.0 33.50 6.56 1.80 23.372 *** take-all pi 73.7 75.0 75.0 66,7 82.5 80.0 452.9 75.57 4.75 root 0.8 2.9 FO 4.1 3.4 2.8 2.3 16.3 2.72 1.02 0.182 1.374 N.S. wt (g) p1 3.1 3.6 1.8 1.4 3.4 1.5 14,8 2.47 0.92 . naasi 0 257 220 182 74 230 89 1052 175.33 70.93 8.820 fi?/pot p1 209 195 126 30 180 63 803 133.83 67.57 4.705 *** '7g PO 62.7 64.7 65.0 92.5 79.3 38.7 402.9 67.15 16.48 1.931 8.099 *** 21 67.4 54.7 70.0 21,4 52.9 42.0 308.4 51.40 15.79 galls/ FO 69 71 40 25 52 62 319 53.17 16.40 5.342 0.468 N.S. pot F1 85 68 37 31 56 27 304 50.67 20.98 galls/g Po 16.8 20.9 14.3 31.3 17.9 27.0 128.2 21.34 6.06 0.323 N.S. root F1 27.4 18.9 20.6 22.1 16.5 18.0 123.5 20.58 3.55 2.350 'el FO 0 30 0 10 20 20 80 1.33 - pot 0 30 20 10 0 40 100 1.67 - dubius T. F0 30 50 30 20 70 60 260 43.3 17.9 25.38 3.681 ** larvae Fi 170 100 250 40 140 120 820 136.7 64.7 PO 110 60 30 60 30 10 300 50.0 32.1 12.68 4.599 *** F1 180 90 60 110 150 60 650 108.3 44.7 FO 0 20 30 0 20 0 70 11.7 12.1 6.159 *** 50 20 50 20 60 60 260 43.3 17.0 4.86 all stages PO 140 130 90 80 120 70 630 105.0 26.1 37.25 4.908 *** /pot F1 400 210 360 170 350 240 1730 288.3 94.9

@ 10 d.f. and p = 0.05, t = 2.228, ** I I I I 0.01, t = 3.169, 0.001, t = 4.587, *** 185

TABLE 8a Nematode population changes in three soils inoculated with 0, graminis (nematodes/100 g soil)

• Soil A I II III 3: Ili S.L. of diff.1 Nematode species 1 between meansj Fo 610 470 320 1400 467 66.4 ' Tylenchorhynchus F1 380 340390 1110 370 dubius Fo 60 20 0 80 27 8. 2 Tylenchorhynchus F1 0 60 60 120 40 nannus Fo 180 90 60 330 11029.1 Tylenchus F1 50 70 60 180 60 sp. Fo 30 20 0 50 17 Pratylenchus P1 30 40 0 70 23 7. 3 sp. Soil B Fo 70 30 40 140 47 Tylenchorhynchus F1 160 70 40 270 90 28.0 dubius Fo 110 100 50 260 87 15.5 Tylenchus F1 110 40 70 220 73 sp. Fo 0 20 10 30 10 Pratylenchus F1 10 10 10 30 10 4. 7 sp. Fo 30 30 0 60 Pratylenchoides F1 50 10 0 60 202016.3 sp. Po 20 10 50 80 27 12.2 Helicotylenchus F1 - 20 30 80 130 43 sp. FO 50 70 70 190 63 Heterodera F1 80 40 40 160 53 .3 avenaeImales) Soil C

Fo 0 0 0 0 0 2.9 Tylenchorhynchus F1 10 0 0 10 3 sp. Po 80 90 100 270 Tylenchus Fl 60 60 120 240 9014.180 sp. Fo 10 30 40 80 27 Rotylenchus F1 40 70 0 110 37 14.8 robustus Fo 30 20 10 60 20 Aphelenchus F1 260 180 250 690 230 16.0 avenae 186

TABLE 9a Effect of three inoculum levels of 0.graminis and three levels of H.avenae on each other and on barley

. Grain weight per plant (mg.) i I II III IV 5:. iii FoNo 375 384 478 372 1609 402.3 FON1 399 409 405 367 1580 395.0 FoN2 381 346 381 449 1557 389.3 F1No 364 381 287 378 1410 352.5 F1N1 169 241 402 223 1035 258.8 F1N2 263 266 397 407 1333 333.3 F2No 153 221 172 136 682 170.5 F2N1 183 186 273 214 856 214.0 F2N2 161 195 217 113 684 171.0 mean No Ni N2 7 of F m of F Fo 402.3 395.0 389.3 1186.6 395.5 Fi 352.5 258.8 333.3 944.6 314.9 F2 170.5 214.0 171.0 555.5 185.2 !;`of N 925.3 867.8 893.6 ;m of N 308.4 289.3 297.9

Analysis of Variance defj S of S M.Sq. • V.R. -;---____ F 2 70,297 135,149 45.37 ** N 2,17219 (3N.S. FN 26,7326 223 2.24 Error 27 80,426 2,979 Total 35 79,627 S.E. of difference between 2 means of F = x 2,979 12 22.28 @ 27d.f. and p = 0.05, t = 2.05 L.S.D. = 45.67 p = 0.01, t . 2.77 L.S.D. 61.62 p = 0.0011,t = 3.69 L.S.D. = 82.21

187

TABLE 9a (cont.)

Root weight (mg)

I II III IV I: FOND 485 537 528 428 1978 494.5 FoNI 481 473 464 423 1841 460.3 FoN2 465 477 521 478 1941 485.3 FiNo 500 463 433 524 1920 480.0 FiNl 377 397 482 425 1681 420.3 F1N2 473 397 439 432 1741 435.3 F2NO 417 325 409 487 1638 409.5 F2N1 327 375 450 380 1532 383.0 F2N2 305 342 386 367 1400 350.0

(means) NO N1 N2 Eof F m of F PO 494.5 460.3 485.3 1440.1 480.0 480.0 420.3 435.3 1335.6 445.2 P2 409.5 383.0 350.0 1142.5 380.8 of N 1384.0 1263.6 1270.6 mof N 461.3 421.2 423.5 Analysis of Variance d f. S. of S M.Sq V.R. F 2 60,676 30,338 16.43 ** N 2 12,225 6,113 3.31 N.S. FN 4 4,963 2,482 0.68 N.S. Error 27 49,866 1,847 I Total 35 127,730

S.E. of difference between means of F =it2 x 1847

17.54 12 @ 27d.f. and p = 0.05, t = 2.05, L.S.D. = 35.96 p = 0.01, t = 2.77, L.S.D. = 48.59 p = 0.001, t = 3.69, L.S.D. = 64.72 188 TABLE 9a (cont.) % roots with take-all

I II III IV 12 t FoNo 0 FoN1 0 FoN2 0 FlNo 62.5 68.8 46.9 83.3 261.5 65.4 F1N1 73.0 76.9 46.9 61.6 258.4 64.6 F1N2 62.5 61.1 66.6 69.2 259.4 64.9 F2No 76.4 71.4 68.8 82.3 298.9 74.7 F2N1 69.2 69.2 69.2 82.3 289.9 72.5 F2N2 76.7 74.3 74.9 76.7 302.6 75.7 (mega4 I No Ni N2 Zof F fa- of FT FO 0 0 0 0 0 Fi 65.4 64.6 64.9 194.9 64.97 P2 74.7 72.5 75.7 222.9 74.30 27of N 140.1 137.1 140.6 Tr of N 46.70 45.70 46.87 I Analysis of varian d.f. S.ofS . M.Sq. V.R. ---1 F 2 39,291 19646 351.4 *** N 2 10 5.0 N.S. FN 4 13 3.25 N.S. Error 25 1,508 55.90 Total 37 40,822

411.0011•4•••••••••••••••• S.E. of diff between means of F =`/2 x 55.90 = 3.05 12 @ 27 d.f., p = 0.05, t = 2.05, L.S.D. = 6.25 p = 0.01, t = 2.77, L.S.D. = 8.45 p . 0.001, t = 3.69, L.S.D. =11.26 189

TABLE 9a (cont.)

Cysts/200 g soil I 11 III IV X FONO 0 FoNi 13 25 33 19 90 22.50 FoN2 50 33 38 37 158 39.50 F1 NO 0 F1N1 24 33 37 26 120 30.00 F1N2 32 37 30 32 131 32.75 F2NO 0 F2N1 32 34 35 31 132 33.00 ,F2N2 29 22 17 29 97 24.25

NO N1 N2 Zof F m of F FO - 22.50 39.50 62.00 20.67 Fl - 30.00 32.75 62.75 20.92 112 - 33.00 24.25 57.25 19.08 1E of N - 85.50 96.50 m of N 0 28.50 32.17

Analysis of Variance d.f. S.of S. M.Sq. V.R. F 2 24 12 0.52 N.S. N 2 7441 3720.5 159.7 *** FN 4 678 169.5 7.28 ** Error 27 629 23.3 Total 35 8772 _ S.E. of Jiffs between means of N = /2 x 23.3 = 1.97 12 © 27 d.f. and p = 0.05, t = 2.05, L.S.D. = 3.04 p = 0.01, t = 2.77, L.S.D. = 5.46 p = 0.001, t = 3.69, L.S.D. = 7.27 S.E. of difference between means of FN = I-2-x 23.3 = 3.41 © 27 d,f. and p = 0.05 t = 2.05,L.S.D. = 7.04 p = 0.01, t = 2.77, " = 9.45 p = 0.001, t = 3.69, 11 = 12.58 190

TABLE ga (cont.) Eggs/cyst

II III IV FOND 0 FON1 90.3 166.7 140.1 72.7 469.8 117.45 FoN2 193.4 126.9 142.1 118.5 580.9 145.23 F1NO 0 FiNi 111.3 103.5 88.0 86.4 389.2 97.30 F1N2 70.9 87.9 77.7 252.2 488.7 122.18 F2N0 0 F2N1 74.7 56.3 119.1 62.9 313.0 78.25 F2N2 107.8 128.8 73.9 126.2 436.7 109.18

(means) No Ni N2 z of F Ira of F 110 117.45 145.23 262.68 87.56 Fi 97.30 122.18 219.48 73.16 F2 78.25 109.18 187.43 62.48 E of N 293.00 376.59 m of N 0 97.67 125.53

Analysis of Variance d.f. 0.0i S . V.R. F 2 3,207 1,603.5 1.19 N.S. N 2 103,690 51,845.0 38.4 ** FN 4 2,533 633.3 0.5 N.S. Error 27 36,507 1,352.0 Total 35 145,937

S.E. of difference between means of N =4/2 x 1352 = 15.01 12 @ 27 d.f. and p = 0.05, t = 2.05, L.S.D. . 30.77 p = 0.01, t = 2.77, L.S.D. = 41.58 p = 0.001, t = 3.69, = 55.39 191 TABLE 9a (cont.) eggs/g soil

I II III IV FONO 0 FoN1 5.87 20.84 23.11 6.91 56.73 14.18 FoN2 48.35 20.94 27.00 21.92 118.21 29.55 PIN() 0 FiNi 13.35 17.07 16.28 11.23 57.93 14.48 F1N2 11.34 16.27 11.65 40.35 79.61 19.90 F2N0 0 F2N1 11.95 9.57 20.85 9.76 52.13 13.03 F2N2 15.64 14.17 6.28 17.80 53.89 13.47

(means) NO Ni N2 Hof F ITI of F FO 14.18 29.55 43.73 14.58 F1 14.48 19.90 34.38 11.46 F2 13.03 13.47 26.50 8.83 Xof N 41.69 62.92 II of N 0 13.90 20.97

Analysis of Variance

d.f. S.of S. M.Sq. V.R. F 2 198 99.0 1.77 N.S. N 2 2732 1366.0 24.4 *** FN \ 4 331 82.8 1.48 N.S. Error 27 1509 55.9 Total 35 4770

S.E. of differences between means of N =// 2 x 55.9 = 3.05 12 @ 27 d.f. and p = 0.05, t = 2.05, L.S.D. = 6.25 p = 0.01, t = 2.77, Z.S.D. = 8.45 p = 0.001, t = 3.69, L.S.D. = 11.26 192'

TABLE 10a Effect of 0, graminis on invasion and development of H. avenae Time 1.

Root weight (mg) I II III ::: Tii FoN1 367 251 357 975 325.0 FON2 490 426 398 1314 438.0 FON3 345 366 373 1084 361.3 F1N1 472 226 498 1196 398.7 F1N2 311 411 425 1147 382.3 F1 N3 375 222 410 1005 335.0

N1 N2 N3 E of F ff. of F Fo 325.0 438.0 361.3 1124.3 374.8 F1 398.7 382.3 335.0 1116.0 372.0 of N 723.7 820.3 696.3 I of N 361.9 410.2 348.2

Analysis of Variance d.f. S.of S M.Sq V.R. Treatment 5 27,248 5,4 50 0.99 N.S. Error 12 66,371 5,531 Total 17 93,619

b) % roots with take-all

II FoNi 0 FON2 0 FON3 0 F1N1 17 20 20 57 19.0 F1N2 21 19 15 55 18.3 F1 N3 15 19 20 54 18.0 193

Table 10a (cont.)

Larvae/root system

i I II III T Er FoNi 124 149 130 403 134.3 F0N2 664 615 674 1953 651.0 F0N3 662 599 637 1898 632.7 F1 N1 145 110 165 420 140.0 F1N2 235 258 320 813 271.0 FiN3 360 265 420 1045 348.3 means N1 N2 N3 of F m of F F0 134.3 651.0 632.7 1418.0 472.7 F1 140.0 271.0 348.3 759.3 279.7 of N 274.3 922.0 981.0 El of N 137.2 461.0 490.5

Analysis of variance d.f. S of Sq. M.Sq. V.R. F 1 216,921 216,921 118.4 *** N 2 461,166 230,583 125.9 *** FN 2 120,995 60,498 33.0 *** Error 12 21,980 1,832 Total 17 821,062

S.E. of difference between means of F /2 x 1832 =- 20.17 9 L.S.D. @ 12 d.f. and p = 0.05, t = 2.18, L.S.D. = 43.97 p = 0.01, t = 3.06, it 61.72 p = 0.001, t = 4.32, it 87.13

S.E. of difference between means of N =v/2 x 1832 = 24.71 6 @ 12 d.f. and p = 0.05, t = 2.1 8, L.S.D. = 53.87 p = 0.01, t = 3.06, u 75.61 p = 0.01, t = 4.32, 106.75 S.E. of difference between means of FN a/2 x 1832 =34.94 @ 12 d.f. and p = 0.05, t = 2.18,L.S.D.=76.2 5 - p = 0.01, t = 3.06 - 106.9 p = 0.001„t = 4.32 . 150.9 194

Table 10a (cont.) Larvae/g root

I II III I fi FoNi 338 594 364 1296 432.0 N2 1355 1444 1694 4493 1497.7 N3 1919 1637 1708 5264 1754.7 FiNi 307 487 331 1125 375.0 N2 756 628 753 2137 712.3 N3 960 1194 1024 3178 1059.3

, Ni N2 N3 zfor F E for F Fol 432.0 1497.7 1754.7 3684.4 1228.1 F1 378.0 712.3 1059.3 2146.6 715.5 X of N 807.0 2210.0 2814.0 E of N 403.5 1105.0 1407.0

Analysis of variance d.f. S of Sq. M.Sq. V.R. F 1 1,182,209 1,182,209 69.69 *** N 2 3,180,637 1,5901 319 93.74 *** FN 2 472,979 236,489 13.94 *** Error 12 203,581 16,965 Total 17 5,039,406

S.E. of difference between means of F ./2 x 16,965 = 61.4 9 © 12 d.f. and p = 0.01, t = 2.18, L.S.D. = 133.9 p = 0.05, t = 3.06 " =187.9 p = 0.001,t = 4.32, = 265.3 S.E. of difference between means of N =,/2 x 169 965 = 75.2 el, 12 d.f. and p = 0.05, t = 2.18,L.S.D. = 6163.9 p = 0.01, t = 3.06,L.S.D. 230.1 p = 0.001,t = 4.32,L.S.D. = 324.9 S.E. of difference liEtween means of FN 7/2 x 16,965 .106.3 3 @ 12 d.f. and p 0.05, t = 231.7 p = 0.01, t = 3.06, 11 = 325.3 p = 0.001,t = 4.32, 11 = 459.2 195

TABVR 11a Effect of 0. graminis on invasion and development of H. avenae Time 2.

Males emerging/plant

1 11 III E a FoNi 4 20 17 41 13.7 N2 13 13 16 42 14.0 N3 1 9 10 20 6.7 F1N1 2 11 6 19 6.3 N2 6 16 12 34 11.3 N3 1 10 3 14 4.7

means N1 N2 N3 Yof F 5 of F FO 13.7 14.0 6.7 54.4 1i.5 Fi 6.3 11.3 4.7 22.3 7.4 of N 20.0 25.3 11.4 la of N 10,0 12.7 5.7

Analysis of variance

d.f. S of Sq M.Sq. V.R. ireatment 5 247 49.4 1.79 N.S. • Error 12 335 27.5 Total 17 582 196

TABLE 12a Effect of 0. graminis on invasion and development of H. avenae. Time 3.

Root weight (mg)

I II III 5- II FoNi 728 135 846 2909 969.7 N2 538 537 785 1860 630.0 N3 745 570 677 1992 664.0 F1 N1 430 657 456 1543 514.3 N2 595 375 427 1397 465.7 N3 431 495 632 1558 519.3

N1 N2 N3 Z of F iii of F F0 969.7 630.0 664.0 2263.7 754.6 F1 514.3 465.7 519.3 1499.3 499.8 21 of N 1484.0 1095.7 1183.3 M of N 742.0 547.9 591.7

Analysis of Variance d.f. S of S. M.Sq. V.R. 1 284,509 284,509 9.98 ** N 2 129,304 64 652 2.27 N.S. FN 2 93,604 46,802 1.64 N.S. Error 12 341,969 28,497 Total 17 849,386

S.E. of difference between means of F =J2 x.289497 = 79.58 9 @ 12 d.f.,p = 0.05, t = 2.18, L.S.D. p = 0.01, t = 3.06, u = 243.5 p = 0.001,t = 4.32, u = 344.0 197

TABLE 12a (cont.)

% roots with take-all

FoNi 0 N2 0 N3 0 F1N1 45 65 57 167 55.7 N2 65 51 56 172 57.3 N3 49 41 55 145 48.3

means

N1 N2 N3 T of F It of F FO 0 0 0 0 0 F1 55.7 57.3 48.3 161.3 53.8 2: of N 55.7 57.3 48.3 M of N 27.9 28.7 24.2 198

TABLE 12a (cont.)

Cysts/200 g soil

1 II III x II FoN1 16 13 11 40 13.33 N2 22 28 26 76 25.33 N3 45 44 39 128 42.67 Fill) 9 7 8 24 8.00 N2 20 18 17 55 18.33 N3 34 36 40 110 36.67

Ni N2 N3 I of F in of P PO 13.33 25.33 42.67 81.33 27.11 Fl, 8.00 18.33 36.67 63.00 21.00 of N 21.33 41.66 79.34 ffi of N 10.67 20.83 39.67

Analysis of Variance S of Sq. M.Sq. V.R. d. f. , F 1 168 168 26.2 *** N 2 2567 1283.5 199.9 *** FN 2 3 1.5 0.23 N.S. Error ' 12 77 6.42 Total 17 2815

S.E. of difference between means of F x 6.42 = 1.195 9 @ 12 d.f.,p = 0.05, t = 2.18,L.S.D.= 2.61 • p = 0.01, t = 3.06, ,, = 3.66 p = 0.001,t = 4.32, = 5.16

S.E. of difference between means of N = x 6.42 = 1.463 6 @ 12 d.f.,p = 0.05, t = 3.19 p = 0.01, t = 3.06, = 4.48 p = 0;001,t = 4.32, = 6.32 199

TABLE 12a (cont.)

Eggs/cyst I II III Z In" F0N1 438.8 432.3 356.2 1227,3 406.8 N2 361.2 357.2 346.9 1065.3 355.1 N3 320.0 317.9 300.5 938.4 312.8 FiNi 288.9 335.6 367.5 992.0 330.7 N2 350.1 336.5 284.4 971.0 323.7 N3 198.4 284.0 233.0 715.4 238.5 means N1 N2 N3 E of F i of F Fo 406.8 355.11 312.8 1074.7 358.2 Fl 330.7 323.7 238.5 892.9 297.6 5:of N 737.5 678.8 551.3 M of N 368.8 339.4 275.7

Analysis of Variance

d.f. S of Sq. M. Sq. V.R. F 1 16,965 16,965 14.8 ** N 2 27,755 13,878 12.1 ** FN 2 2,033 1,017 0.9 N.S. Error 12 13,796 1,150 Total 17 60,549

S.E. of difference between means of F = [2 x 1150 = 15.99 9 @ 12 d.f.,p = 0.05, t = 2.18,L.S.D. = 34.86 p = 0.01, t = 3.06, tt = 48.93 p . 0.001,t = 4.32, ti = 60.08

S.E. of difference between means of N = i2 x 1150 = 19.58 6 @ 12 d.f.,p = 0.05, t = 2.18,L.S.D. = 42.68 p = 0.01, t = 3.06, II = 59.92 p =_ 0.001 ,t = 4.32, = 84.59 200:

TABLE 12a (cont.) Eggs/g soil

I II III Z ili P0N1 35.1 28.1 19.6 82.8 27.60 N2 39.7 50.0 45.1 134.8 44.93 N3 72.0 69.9 58.6 200.5 66.83 F19 N1 13.0 11.8 14.7 39.5 13.17 N2 35.0 30.3 24.2 89.5 29.83 N3 33.7 51.1 46.6 131.4 43.80

means N1 N2 N3 of F ira of F Fo 27.60 44.93 66.83 139.36 46.45 F1 13.17 29.83 43.80 86.80 28.93 X of N 40.77 74.76 110.63 1E of N 20.39 37.38 55.32

Analysis of Variance

d.f. S of Sa. M.Sc. V.R. F 1 1381.6 1381.6 33.05 *** N 2 3661.8 1830.9 43.80 *** FN 2 1068.8 534.4 12.79 *** Error ,12 503.5 41.8 Total 17 5615.7

S.E. of difference between means of F =,j2 x 41.8 = 3.05 9 @ 12 d.f.,p = 0.05, t = 2.18,L.S.D. = 6.65 p = 0.01, t = 3.05, " = 9.33 p = 0.001,t = 4.32, " =13.18 S.E. of difference between means of N = 12 x 41.8 = 3.73 6 © 12 d.f.,p = 0.05,t = 2.18 L.S.D. = 8.14 p = 0.01,t = 3.06 " =11.42 p = 0.001,t = 4.32, " =1602 S.E. of difference between means of FN =;12 x 41.8 = 5.28 3 12 d.f.,p = 0.05, t = 2.18,L.S.D. =11.51 p = 0.01, t = 3.06, " = 16.16 p = 0.001,t = 4.32, n = 22.81 201

TABLE 12a (cont.) Length of cysts(/a)

t I II III 2: bi- FoNi 839 866 895 2600 867 N2 845 876 814 2535 845 N3 798 887 835 2520 840 FiNi 780 825 800 2405 802 N2 843 813 795 2451 817 N3 659 789 750 2198 733

means N N2 N3 Yof F iii of F F0 867 845 840 2552 850.7 F1 802 817 733 2352 784.0 of N 1669 1662 1573 ra- of N 834.5 831.0 786.5

Analysis of Variance

d.f. S of Sq. M.Sq. V.R. F 1 20,066 20,066 12.95 ** N 2 8,586 4,293 2.77 N.S. FN 2 4,728 2,364 1.53 N.S. Error 12 18,582 1,549 Total 17 51,962

S.E. of difference between means of F = 12 x 1549 = 18.58 9 @ 12 d.f.,p = 0.05, t = 2.18, L.S.D.= 40.50 p ==0.01, t = 3.06, u = 56.89 p = 0.001,t = 4.32, = 80.27

L9't04 0"t45 0'404 O'C0I0'044 CET 0'4C444 CK 017'22 8't5 - 5't3 5'05 2N C°3LOCt aN 05'03 0'4t - creL ooe 1-04 L0 eoLLLNLa 53*55 5'99 - 8*55 L'35 CET Coe L 0 t C 41 LL'43 5'59 0'94 O'LC 5'34 i\I„ 0°9 84 t C 44 3N L9'34 O'85 0'64 0'6 0'04 t'ViE CoeLLCCI-Noa III II I m X III II I o.p.ua xas se tee; q.-Enpv pu'a avq.s

L't01. ti.0 404 COL 044 N 0°65 L41. 05 gC ec. N 0'504 SLC oat. U eeL eN clogL gt t 33 64 EN L' S3 LL OC et_ Ce 140-d 0 La 0o554 5ot eLL 55L 86 5N L'LL 55 54 ee 94 coCoL oLc 179 444 554 3K, 0°L Le 8 L 9 ex 5' S39L 6L Le OC Liva 0 Lea I, x sarema.ZnpupTX avq.s 1144, auAreT asq.s pac

(Yet 934 LS tC gC N 5.066 toLL6e 6°LLe45°L3L 0 0 8L6 .1 tq 0 ex 5'te5 t'CL94 3'385 8'545 t°5L5 cli o LiaLa .8"001. 1/•305 5°534 e°L9 L•6o1 4.04 5*9L 6t C LC 54 CK t'939 eo6Lt2 3'69L 6'98L L'536 41 0 3A1 3'345 5'956 6'CLL 6'59E L'96t.11, 0 1110g 9'59 L'964 9*Ct L'LL to5L 4NO3 " 1 - III II I m m III II 1 asAasT 9Peq.S pu 4.00; 2/asAasi

0'984 855 6oe LLL eLL CH C'1764 585 t94 1,e 384 511 C'334 L95 t31, 86 517 4 3N C•eLe 559 543 064 353 Z;N: Cote CL oC 6L te 4N4 1 L' 6173 6tL 653 083 033 1-10-3 54 LLL 1715 064 e61 ECL Cm 5'L43 1759 Lte tte Ctl, CR 5'944 6tC 9L 434 354 3N 5o665 86L4 LS' 55t 905 eNn L' L3 58 oe OC a Lei 0'43t C604 65t 985 ett LN-L1 . , , - 0 1 I" Z III II 'I

maq.sics qmoa/ssAaug (ft) qApTem q_ooa

eVUOAB qi ;o so-p:sa xes uo sTuTtauaV •o" jo q_oe;;a

vC4 aaan

203 203

TABLE 13a (cont.)

Analyses of Variance

Root weight S.E. of difference d.f. S of Sq. M._Sq. V.R. between means F 1 71947 71947 34.4 *** 21.56 N 2 62269 31134.5 14.9 *** 26.41 FN 2 26951 13475.5 6.4 * 37.36 Error 12 25113 2093 Total 17 186280 L.S.D. p 0.05 0.01 0.001 between Si of F 47.00 65.97 93.14 it n u N 57.57 80.82 114.09 FN 81.45 114.32 161.40

Larvae/root system d.f. S of Sq M.Sq. V.R. S.E. of difference between means F 1 150 150 0.24 N.S. N 2 71077 35539 58.2 *** 14.26 FN 2 244 122 0.20 N.S. Error 12 7323 610.3 Total 17 78794 L.S.D. p = 0.05 0.01 0.001 between TT of N 31.09 43.64 61.60

Larvae/g root S.E. of difference d.f. S of Sq M.Sq. V.R. between means Fl 1 98556 98556 5.22 * 64.77 N 2 2057556 1028778 54.49 **-x 79.32 FN 2 34144 17072 0.90 N.S. Error 12 226719 18877 Total 17 2416975 L.S.D. @ p = 0.05 0.01 0.001 between ffl of F 141.20 198.20 279.81 u. It n N 172.92 242.72 342.66

204

TABLE 13a (cont.) % second stage larvae S.E. of diff d.f. S of Sq. M.Sq. V.R. between means 1 74.02 74.02 5.73 * 1.69 2 1000.67 500.33 38.73 *** 2.08 FN 2 148.02 74.01 5.73 * 2.94 Error 12 154.99 12.92 Total 17 1377.70 L.S.D. (4) p = 0.05 0.01 0.001 between fri of F 3.69 5.18 7.32 N 4.53 6.37 8.99 " FN 6.40 9.00 12.67

% third stage larvae S.E. of diff d.f. S of Sq. M.Sq. V.R. between means P 1 135.6 135.6 6.88 * 2.09 N 2 751.2 375.6 19.07 ** 2.56 FN 2 80.7 40.4 2.05 N.S. Error 12 236.5 19.7 Total 17 1104.0 L.S.D. @ p = 0.05 0.01 0.001 between IT of F 4.56 6.40 9.03 ,, ,, N 5.58 7.83 11.06

% males S.E of diff d.f. S of Sq. M.Sq. V.R. between means F 1 197 197 4.65 N.S. N 2 2286 1143 27.00 ** 3.07 FN 2 563 281.5 6.64 * 5.32 Error 12 509 42.4 Total 17 3555 L.S.D. @ p = 0.05 0.01 0.001 between. T11 of N 6.69 9.39 13.26 FN 11.60 16.28 22.98 205 TABLE 13a (cont.)

% females S.E. of diff d.f. S of Sq. M.Sq. V.R. between means F 1 38.7 38.7 9.28 * 0.96 N 2 61.1 30.56 7.69 * 1.18 FN 2 11.8 5.90 1.42 N.S. Error 12 50.0 4.17 Total 17 164.6

L.S.D. @ p = 0.05 0.01 0.001 between Eof F 2.10 2.95 4.15 it II N 2.57 3.61 5.10

Sex Ratios I II III 2: II F0N1 10.0 9.0 19.0 38.0 12.67 N2 12.3 37.0 16.0 65.3 21.77 N 32.7 33.8 (C6 ) 66.5 33.25 In of 2 3 20.50 F1 N1 23.0 18.0 ( c ) 41.0 27.27 N2 30.5 24.3 ( C---) ) 54.8 I t It It N3 110.0 103.0 101.0 314.0 104.67 TABLE 14a Effect of O. graminis on development of H. aIena3

V DI Time 1.(2 days) I II III IV c+ Total larvae 40 15 26 46 42 16.9 33.80 Time 2.(7 days) root F0 200 170 200 180 130 880 176.0 13.71 2.48 weight F1 180 150 150 140 90 710 142.0 Total larvae FO 32 35 19 28 31 145 29.0 4.49 1.07 N.S. Ill 37 12 30 33 9 121 24.2 1;;;;:e7g FO 160.0 70.6 150.0 155.6 238.5 774.7 154.94 14.21 0.72 N.S. root F1 205.6 80.0 200.0 235.7 100.0 821.3 164.26 2nd stage F0 26 30 18 24 30 128 25.0 larvae F1 33 11 29 31 9 113 a.6 3rd stage F 0 4 1 2 1 8 1.5 (males) F10 4 1 1 2 0 8 1.6 3rd stage F0 0 1 0 0 0 1 0.2 (female) Fi 0 0 0 0 0 0 0 3rd stage PO 6 0 0 0 0 6 1.2 (unsexed) Fi 0 0 0 0 0 0 J TABLE 14a (cont.) 0" CO CD • txi tm • CD ID 0 1-1) Ei a) r 33. Time 3 (14 days) II III IV V E root weight lo 250 270 290 130 260 1200 240.0 ri 200 150 200 190 240 980 196.0 21.82 2.48 total larvae Po 37 36 18 25 17 133 26.6 3.91 0.77 U. S. 21 15 39 7 36 118 23.6 larvae/g 148.0 133.3 62.7 193.8 79.2 617.0 123.40 10.37 0.58 'U.S. root p1 105.0 100.0 195.0 36.8 150.0 586.8 117.36 2nd stage Po 11 11 7 13 6 48 9.6 larvae F1 5 7 17 6 14 49 9.8 3rd stage 10 14 10 7 5 5 41 8.2 (male) 11 9 7 14 0 14 44 8.8 3rd stage 1 3 0 2 1 7 1.4 (famale) 2 0 2 0 1 5 1.2 3rd stage 11 12 4 5 5 37 7.4 (unsexed) 5 1 6 1 6 19 3.8 ce TABLE 14a (cont.) 0 • c+ • (D (D 0 0 14) 1-6 11 CD t-'• FI) 0 0 Ft) cF CD Time 4.(21 days) I II III IV V Po 470 510 350 550 510 2390 478.0 62.00 r 1 . 39 L S . root weight p i 500 340 140 410 570 1960 392.0 Total PO 18 16 23 26 24 107 21.4 2.14 0.47 N.S. larvae P1 20 31 12 20 19 102 20.4 larvae/g PO 38.3 31.4 65.7 47.3 47.1 229.8 45.96 N . S . root F1 40.0 91.2 85.7 48.8 33.3 299.0 59.80 9.40 1.46 2nd stage F0 2 1 3 1 1 8 1.6 larvae P1 5 5 0 5 5 20 4.0 3rd stage F0 3 4 4 4 8 23 4.6 male 5 13 4 5 5 32 6.4 3rd stage FO 4 2 4 4 4 18 3.6 female 2 2 1 0 3 8 1.6 3rd stage FO 0 1 3 0 0 4 0.8 unsexed P1 1 2 0 0 1 4 0.8 4th stage FO 7 6 7 10 8 38 7.6 male 6 9 6 9 5 35 7.0 4th stage FO 2 2 2 7 3 16 3.2 female P1 1 0 1 0 3 0.6

TABTR 14a (cont.) cn ?

1Time 5.(28 days) I II III IV V ffi

PO 730 1380 820 1040 840 4810 962.0 33.65 3.09 root weight 11 750 680 1290 810 760 4290 858.0 17 22 18 94 18.80 28 9 4.85 0.29 . N.S. Total larvae0F1 16 19 23 11 18 87 17.40 larvae/g Po 38.4 6.5 20.7 21.2 21.4 108.2 21.64 3.84 0.13 N.S. root F1 21.3 29.4 17.8 13.6 23.7 105.8 21.16 3rd stage Po O 0 0 0 0 0 0 male p1 2 0 0 1 1 4 0.8 3rd stage 0 2 2 1 3 1 9 1.8 female Pt_ 1 1 1 1 1 5 1.0 4th stage Po O 0 1 1 1 3 0.6 male Pi 4 4 3 2 3 16 3.2

4th stage 110 5 1 1 2 3 12 2.4 female F1 1 1 0 1 1 4 0.8 adult FO 13 4 12 13 9 56 11.2 male F1 8 13 16 6 10 53 10.6 adult 0 3 2 2 3 4 14 2.8 female P1 O 0 3 0 2 5 1.0 210

TABLE 14a (cont.)

% roots with take-all lesions at 28 days

I II III IV V M ff All roots 45.5 40.0 60.0 50.0 54.6 250.1 50.02 Roots with H. avenae 80.0 60.0 66.7 66.7 66.7 340.1 68.02 TABLE 15a Effect of level of take-all infection on invasion of roots by H. avenae

root weight (mg) Analysis of Variance I 11 III IV V I. 1 d.f. S of Sq. M.Sq V.R. F0 199 090 167 188 297 941 188.2 Treatment 4 11252 2813 0.82 N.S. F1 155 299 153 323 274 1204 240.8 Residual 20 68438 3422 F2 253 240 251 172 201 1117 223.4 Total 24 79690 F3 236 186 230 231 232 1115 223.0 F 215 206 088 235 193 937 187.4

roots with take-all Analysis of Variance I II III IV V Y. 1 d.f S of Sq M.Sq V.R. * * FO 0 0 0 0 0 0 0 Treatment 4 21086 5272 20.05 F1 16.7 12.5 14.3 28.6 12.5 84.6 16.9 Residual 20 5258 262.9 F2 42.9 42.9 14.3 28.6 14.3 143.0 28.6 Total 24 26344 F3 33.3 100.0 71.5 42.9 37.5 285.2 57.0 F4 85.8 100.0 80.0 55....) 85.8 407.1 81. S.E. of cliff. between 2E1 of 5 = 10.26

@ p = 0.05 t = 2.09, L.S.D. = 21.44 0.01, t = 2.84, = 29.14 0.001, t = 3.83, = 39.50

TABLE 15a (cont.)

Larvae/root system Analysis of Variance I II III IV V Z a Id.f. S of Sq M.Sq V.R. FO 184 129 79 153 259 804 160.8 Treatment 4 20112 5028 2.60 N.S. F1 105 98 120 204 132 659 131.8 Residual 20 38648 1932 P2 62 137 123 126 59 507 101.4 Total 24 58760 F3 63 89 161 94 81 488 97.6 F4 102 97 42 81 83 405 81.0

Larvae/g root Analysis of Variance 1 II III IV V Z Er 3 d.f S of Sq M.Sq V.R. Fo 924.6 1433.3 473.1 813.8 870.1 4514.9 903.0 Treatment 4 777490 194373 4.39 * F1 677.4 327.8 784.3 631.6 481.8 2902.9 580.6 Residual 20 885049 44252 F2 245.1 570.8 490.0 732.6 293.5 2332.0 466.4 Total 24 1662539 F3 267.0 478.5 700.0 406.9 349.1 2201.5 440.3 P4 474.4 470.9 477.3 344.7 430.1 2197.4 439.5 S.E. of diff. between ra of 5 , _ 12% fl

p = 0.05, t = 2.0% L.S.D. = 278.08 p = 0.01, t = 2.84; 377.86 p = 0.001, t = 3.85, “ = 512.24 TABLE 16a Invasion of barley roots by H. avenae in relation to position of take-all infection. (larvae/cm root) cm secuions of root 0 11 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 I 0 0 9 13 13 18 8 1 5 1 1 1 0 0 0 0 - - II 0 0 2 11 21 18 11 21 8 2 0 0 0 0 0- -- 11100 7 10 13 13 3 4 2 7 1 1 0 0 0 0 - - IV 00 2 15 6 12 6 4 3 1 0 0 00 0- - - V 0 0 3 17 10 6 14 14 11 0 1 6 0 6 0 3 0 0 VI 00 2 10 5 12 11 12 7 4 2 10 ------VII 0 3 2 14 5 11 5 6 3 0 2 0 00 0- - - VIII 0 6 9 6 9 8 6 2 6 6 0 0 0 0 0- --

0 9 36 96 98 98 64 64 45 21 7 18 0 6 0 3 0 0 IT 0 1.1 4.5 12.0 12.3 12.3 8.0 8.0 5.6 2.6 0.9 2.3 0 0.9 0 1.0 0 0 TABLE 16a (cont.) Invasion of barley roots by H. avenae in relation to position of take-all infection. (larvae/cm of root)

L cm sections of root FT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1 0 0 0 5 6 0 6 II 0 0 7 10 2 3 III 0 2 0 2 18 9 6 2 0 1 1 0 3 IV 0 0 3 12 6 5 6 5 5 1 0 0 1 0 0 0 V 0 0 3 3 1 4 4 0 0 0 0 0 2 VI 0 0 0 7 0 1 2 3 0 0 0 0 0 VII 0 0 0 13 4 3 2 2 0 0 0 0 0 2 VIII 0 0 0*

17 0 2 13 52 37 25 26 12 5 2 1 0 6 2 0 0 Y. 0 0.3 1.6 7.4 5.3 3.6 4.3 2.4 1.0 0.4 0.2 0 1.2 1.0 0 0 ---I (* =position of take-all lesion) TABLE 16a (cont.)

20 p1 Root Larvae Larvae Root Larvae Larvae length /root /cm root length /root /cm root

I 16.0 70 4.38 6.8 17 2.50 II 14.4 94 6.53 6.0 22 3.67 III 16.0 69 4.31 12.8 44 3.44 IV 14.4 56 3.89 16.0 44 2.75 V 18.0 87 4.83 12.4 17 1.37 VI 12.0 80 6.67 12.8 13 1.02 VII 15.6 51 3.26 13.6 26 1.91 VIII 15.6 58 3.72 2.8 0 0

M 122.0 565 37.59 83.2 183 16.66 Ya- 15.25 70.6 4.699 10.40 22.9 2.083 216

TABLE 17a Invasion of healthy roots by H. avenae, on a take-all infected root system

Root length Larvae/root Larvae/cm (cms) system root

Rep. F0 F1 F0 F1 F0 F1 I 7.00 4.50 78 46 11.14 10.22 II 11.50 9.25 104 127 9.05 13.73 III 7.50 10.25 70 167 9.33 16.29 IV 10.75 9.25 143 55 13.29 5.95 V 6.75 6.00 61 109 9.04 18.17 VI 13.75 14.00 126 125 9.15 8.93

II 56.55 53.25 582 629 61.00 73.29 21 9.43 8.88 97.00 104.83 10.17 12.22 217

TAB-17, 18a Attraction of H. avenae larvae to roots of barley infected by take-all

Larvae recovered I II III Agar 74 83 48 205 68.3 Agar & fungus 47 53 25 125 41.7 Healthy roots 872 904 750 2526 842.0 Infected roots 417 702 565 1684 561.3