Biology and Histopathology of Different

BIOLOGY AND HISTOPATHOLOGY OF DIFFERENT

ISOLATES OF ANGUINA TRITICI ON TRITICUM SPP.

Riadh Falih Al-Sabie B.Sc. (Baghdad),

DIC. (Imperial College), M.Sc. (London)

A THESIS SUBMITTED FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY

IN THE FACULTY OF SCIENCE, UNIVERSITY OF LONDON.

Department of Zoology and Applied Entomology

Imperial College at Silwood Park

Imperial College of Science and Technology

AshursE Lodge

Sunninghill

Ascot

Berkshire:

ENGLAND September, 1980. 11

ABSTRACT

Isolates of Anguina tritici from Australia, England, India, Iraq and U.S.A. were maintained on spring wheat. Measurements of males and females and second stage juveniles (J2) showed consistent morphometric differences between some isolates.

Host range studies on species and varieties of Triticwn and AegiZops indicated an ability to infect a wide range of hosts of which 9 were new records.

Infection studies on naturally infected wheat revealed that the growing point was invaded by the second stage juveniles shortly after flower induction.

Artificial inoculation of wheat plants showed that the infective stage needed an association with the flower primordia for at least 24 days (latent period) for galling to be initiated. Further studies were made on the effect of temperature on invasion and distribution of galls in artificially inoculated heads. The histopathology of infection and subsequent gall development was observed.

J2 invaded the primordia of either one stamen only (outer anther), ovary only or both to form the gall.

The J2 survived in the soil for 250 days in the absence of a host, and some could still infect and form galls after 225 days. J2 from all isolates could not survive for more than 40 days in aerated water. Further studies were made on survival of J2 in the field in either presence or absence of the host.

By monitoring the change in length of J2 in different molar concentration of NaCl, it was found that all isolates had a very limited ability to osmoregulate (0.05-0.1 M NaC1).

Respiration rate of J2 varied with temperature and different isolates had different temperature optima for respiration.

The oxygen consumption of all isolates was totally inhibited by 10-4 M NaC N,followed by death within 15 minutes. ACKNOWLEDGEMENTS

I wish to express my gratitude to Dr. A.A.F. Evans for his valuable guidance, supervision and advice throughout this research, and for his interest and help during the preparation of the manuscript. Without his invaluable encouragement this work would not have been possible.

I should also like to acknowledge with thanks the helpful suggestions given by Dr. W.M. Hominick, D.J. Wright and J. Bridge, and also to F. Awan for reading part of the manuscript.

My thanks to Miss. O.M. Goss, Department of Agriculture, Western

Australia; Dr. G. Swarup, Indian Agricultural Research Institute; Dr.

J.N. Sasser, North Carolina State University, U.S.A.; and the staff of the Department of Plant Pathology, Abu-Ghraib, Iraq, for supplying the wheat galls of Anguina tritici isolates; and also to Dr. V. Chapman,

Plant Breeding Institute, Cambridge, and the staff of Kew Gardens,

London, for supplying the wheat varieties collection.

Thanks are due to Mr. R.G. Davies and Dr. S. Young for their help in statistical analysis, and Mr. P. Nicholas for his help with photo- graphy, and the rest of the staff of the Imperial College, Silwood Park for the various ways in which they assisted.

I am immensely grateful to my wife, Basima, for her tolerance, patience, understanding and moral encouragement throughout the duration of this work, and my special thanks to my parents and all the family.

Finally, I take the opportunity to thank the Iraqi Government for the scholarship from the Ministry of Higher Education and Scientific

Research, which has enabled me to pursue this work. iv

TABLE OF CONTENTS

Page

Title page i

Abstract

Acknowledgements

Table of contents iv

SECTION I INTRODUCTION AND LITERATURE REVIEW 1.

a. Introduction 1.

b. Historical view 2.

c. Classification 4.

d. Identification 5.

e. Morphometrics 7.

f. Distribution and host range ___ 10.

g. Biology and life history --- 11.

h. Development of Anguina tritici --- 13.

i. Biochemistry and survival of Anguina tritici --- 15.

j. Effect of Anguina tritici on biochemistry in wheat ------17.

k. Associated with other pathogens --- 18.

1. Control --- 20.

m. Aims and objective of the present study 22.

SECTION II GENERAL MATERIALS AND METHODS 23.

SECTION III STUDIES ON MORPHOLOGY AND HOST RANGE WITHIN ANGUIIVA TRITICI --- 25.

A. A Comparison of 'Morphological Variation Within

and Between Isolates of A. tritici 25.

1. Introduction --- 25.

2. Materials and methods __- 26.

3. Results --- 29. v

Page

B. Host Range and Host Suitability Within Isolates

of A. tritici 51.

1. Introduction 51.

2. Materials and Methods 51.

3. Results 54.

C. Discussion 66.

SECTION IV HISTOPATHOLOGY AND THE INFECTION PROCESS 73.

1. Introduction 73.

2. Materials and Methods 74.

3. Results 75.

4. Discussion 93.

SECTION V FACTORS AFFECTING THE INFECTION PROCESS OF

A. TRITICI IN WHEAT 96.

1. Introduction 96.

2. Inoculation before the double ridge stage 96.

a. Materials and Methods 96.

b. Results 96.

3. Inoculation during and after flower induction 97.

a. Materials and Methods 97.

b. Results 97.

4. Inoculation throughout the year using successive

generations 97.

a. Materials and Methods 97.

b. Results 98.

5. The density of artificial inoculum 98.

a. Materials and Methods 98.

b. Results 101. vi

Page

6. Transfer of inoculum from one plant to another

during the period of inoculation 105.

a. Materials and Methods 105.

b. Results 105.

7. Wheat c.v. MMaris Dove inoculated with

Anguina spp. from Holcus mollis (creeping soft

grass) and creeping soft grass with

A. tritici 106.

a. Materials and Methods 106.

b. Results 106.

8. Galls produced on naturally infested host under field

conditions 108,

a. Materials and Methods 108.

b. Results 108.

9. Discussion 108.

SECTION VI SURVIVAL OF SECOND STAGE JUVENILES UNDER DIFFERENT

CONDITIONS 115.

1. Introduction 115.

2. Survival of J2 under field conditions in the absence

of the host.

a. Materials and Methods

b. Results

3. Survival of J2 under field conditions in the

presence of the host

a. Materials and Methods

b. Results

4. Survival of J2 in different media with temperatures

and their subsequent infectivity 123.

a. Materials and Methods 123,

b. Results 124. vii

Page

5. Survival of J2 in air saturated water 129.

a. Materials and Methods 129.

b. Results 129.

6. Discussion 131.

SECTION VII OXYGEN CONSUMPTION OF ANGUINA TRITICI OF SECOND

STAGE JUVENILES OF DIFFERENT ISOLATES 135.

1. Introduction 135.

2. Oxygen consumption of J2 from single and mixed

galls of the U.K. and Iraqi isolates 135.

a. Materials and Methods 135.

b. Results 138.

3. Oxygen consumption at different temperatures

of J2 from mixed galls of each isolate 141.

a. Materials and Methods 141.

b. Results 141.

4. The effect of different concentrations of aqueous

NaCN on the oxygen consumption of J2 of A. tritici --- 143.

a. Materials and Methods 143.

b. Results 144.

5. Discussion 144.

SECTION 'VIII EFFECT OF OSMOTIC STRESS ON SECOND STAGE

JUVENILES OF DIFFERENT ISOLATES 147.

1. Introduction 147.

2. Materials and Methods 147.

3. .Results 148.

4. Discussion 156.

SECTION IX GENERAL DISCUSSION ------159.

References 166.

Appendices 179. SECTION I

INTRODUCTION AND LITERATURE REVIEW a. Introduction

Nematodes are probably the most numerous multicellular animals in the world (Stockli, 1946), and are found in nearly every biological niche that will support life (Cobb, 1914).

The majority are non-parasitic and free-living in fresh or salt water or in soil, where they feed on micro-organisms such as bacteria, fungi and algae.

However, nematodes have been found to be parasites on virtually all animal and plant life. They can cause great physical discomfort and debilitation .to man and his domestic animals as well as a variety of plant diseases that take their toll of crop production.

In the past, nematode damage to crops was often ignored or attributed to other causes, such as lack of soil fertility, deficient soil moisture or "soil exhaustion". Nevertheless, they escaped notice because most kinds are too small to be seen without the aid of a microscope. Nematodes feed on the roots, crown, stems, leaves and even on the growing point (buds and root tips), of many crop plants.

Among the plant parasitic nematodes, there are many genera where the response of the plant to invasion is the formation of galls on various parts of the plant. Amongst these gall-forming nematodes, the genus

Anguina (Scopoli, 1777) causes galls on the above-ground parts of the plant. Frequently the form and location of the galls are characteristic of the nematode species. Anguina spp. are obligate plant parasites causing galls on floral structures or leaves of host plants. 2.

The association of the genus Anguina with gall formation on grasses has been known for just over a century and a half (Needham,

1744), when it was described as galls in the flowers on a Gramin- aceous host. There is only one older record of plant damage by nematodes on wheat due to Anguina tritici (Steinbuch, 1799;

Chitwood, 1935). Galls . were formed in place of seeds, and infection decreased grain yield, sometimes by as much as 60% (Anon.,

1964). As wheat is the most important food crop grown by western man, occupying about 515 million acres, it is easy to appreciate the potential importance of such an organism. The nematode Anguina tritici is well known throughout most of the world as the wheat nematode, wheat gall nematode or ear-cockle nematode (Caveness, 1964). It is the longest known plant nematode and has been spread through infested seed to all wheat - growing regions of the world. The gall is pop- ularly known as "Sehun, Tundu, Mammi and Gegla" amongst the cultivators in India (Singh, Singh and Mathur, 1953); in England as "ear-cockles, purples and peppercorns"; in Germany as "Radenkrankheit"; in France as "Blenielle"; and in the United States as "cockle-wheat, hard smut and nematode galls", (Suryanarayana and Thmkhopadhaya, 1971).

In recent literature, the preferred term for the four inter- moult stages of nematodes is "juveniles" rather than "larvae". The juvenile that hatches from the egg is often called a larva, although this is a misnomer because the juvenile is similar to the young adult nematode but differs from it in size, in the lack of gonads and the absence of copulatory structure and sometimes in mouth parts (Hyman,

1951). Hence, J1, J2,...etc. will be used to refer to juveniles in this thesis.

b. Historical view

The symptoms of this disease were already known in the Middle Ages; 3.

certain authors contend that Shakespeare's words "sow'd cockle reap'd no corn" (Love's Labour's lost, Act. 4, Scene 3, Line 379) is evidence that ear-cockle disease caused by Anguina tritici on wheat might have been known in the 16th century (Goodey, 1933;

Thorne, 1961). However, without doubt, Shakespeare used "cockle" to mean cornfield weeds, e.g. The corn cockle (Agrostemma githago

L.). Cornfield weeds are referred to also in Coriolanus, Act 3, Sc.

1,70. The term was in use in this sense, with various spellings, from Anglo-Saxon times, but "cockles" or "ear-cockles" for Anguina galls does not seem to have been used before the mid-19th century

(Southey, 1972).

It was the first plant parasitic nematode discovered by Needham in 1744. He revived A. tritici second stage juveniles (larvae) by suspending galled wheat in water, and his work became a classic example in phytonematology. Roffredi (1775)- explained the relationship between this nematode and the appearance of the wheat gall formation and reported that he had observed different stages in the development of the worms, while he was investigating their life cycle in growing wheat. Steinbuch (1799) described and designated it by the scientific name Vibrio tritici which Bauer (1823) used again, not knowing of Steinbuch's previous work.

Davaine (1857) was the first to describe the wheat gall disease and the life history of A. tritici, and during the following 50 years numerous references appeared in the literature from Germany, England and France, One of the most outstanding contributions was that of

Marcinowski (1909). She reported the involvement of various tissues of flowers in the formation of galls; she also observed the lignified nature of cells of nature gall tissue.

Seed galls are the main galls caused by A. tritici, but Byars 4.

(1920) reported the first case of leaf galls which, he stated,

were a rarity.

•c. Classification

Anguina tritici is a nematode of the order Tylenchida (Thorne

1949), which contains most of the known plant parasitic nematodes.

The eelworm belongs to the superfamily Tylenchoidea (Orley,

1880), Chitwood and Chitwood (1937); to the Tylenchidae family,

according to Hooper (1978); or to the Anguinidae family,according to

Siddiqi (1971).

It belongs to the subfamily Anguinidae (Paramonov, 1962) and is a

member of the genus Anguina Scopoli (1777).. Originally the genus Anguina

and Ditylenchus were contained in•Tyienchus Bastain (1865), then AnguilluZina Goodey (1932), before being differentiated and accepted

as separate genera by Thorne (1949). Hooper and Southey (1978),

recognized about 23 species as being valid.

Several synonyms of A. tritici are listed by Tarjan and Hopper

1974, as:-

Vibrio tritici Steinbuch, 1799;

Rhabditis tritici (Steinbuch, 1799), Dujardin, 1845;

Anguillula tritici (Steinbuch, 1799), Grube, 1849;

AnguilluZina tritici (Steinbuch, 1799), Gervais and Van Beneden, 1859;

Tylenchus tritici (Steinbuch, 1799), Bastian, 1865;

TyZenchus (AnguiliuZina) tritici (Steinbuch, 1799), Bastian, 1865,

Filipjev, 1934 ,

AnguilluZina (Anguina) tritici (Steinbuch, 1799), Gervais and

Van Beneden, 1859, (W. Schneider, 1939);

Anguillula graminearum Diesing 1851 in part;

Anguillula scandens Schneider, 1866; 5.

Tylenchus scandens (Schneider, 1866); Cobb, 1890;

Anguillulina scandens (Schneider, 1866); Goodey, 1932;

Anguillulina (Anguina) scandens (Schneider, 1866); Goodey,

1932; (W. Schneider, 1939).

According to Filipjev and Schuurmans Stekhoven, 1941, the combination Anguina tritici (Steinbuch, 1799) should be attributed to Chitwood, 1935 rather than to Filipjev, 1936.

Chitwood did not specifically cite this new combination but did claim that Anguillulina (Type A. tritici) and Tzylenchus were synonyms of Anguina. With regard to Anguina, he stated that "the type can only be Tylenchus tritici (Steinbuch, 1799)". Anguina tritici (Steinbuch, 1799) Chitwood, 1935. d. Identification

Southey (1972) drew from other authors work the conclusion that the annules of the cuticle are very fine, and usually visible only in the oesophageal region, and the lateral field, with four or more fine incisures in adults. The annules are visible only on young specimens.

The body in the adult female is obese, generally assuming the shape of a closed spiral when relaxed, sometimes ventrally arcuate or S-shaped, broad in the middle, abruptly tapering towards the extremities. The body in the adult male is straight and vermiform when relaxed, gradually tapering anteriorly and posteriorly.

Swarup and Gupta (1971) stated that the body in the fourth stage female also assumes a closed spiral shape when relaxed, broad in the middle, abruptly tapering at the anterior and posterior ends, more so anteriorly. The body of the fourth stage male is slenderer and 6.

more cylindrical than that of the female, gradually tapering anteriorly

and posteriorly.

The body of the third stage female assumes a ventrally arcuate

position when relaxed, and is cylindrical, gradually tapering at the

extremities. The body of the third stage male is slenderer compared

to that of the female, cylindrical, with a longer tail than in the female; from this stage onwards it is possible to distinguish the female from the male.

The body of the second stage juvenile assumes a slightly ven-

trally arcuate shape when relaxed by heat, cylindrical and tapering gradually at both ends. The alimentary canal is the only visible structure, although Goodey (1933) stated that the genital primordium is present as a small group of cells about midway down the intestine on the ventral surface.

Al-Sabie (1977) mentioned that first stage larvae were different in morphology from second stage juveniles as regards the end of the tail, in all isolates studied.

Head:- The head is shaped like a flattened disc with rounded sides, narrower than the body and separated from the latter by a shallow groove or constriction. The lips are visible as six raised, radial ridges.

Stylet:- The stylet is characteristic of plant parasitic nematodes, and can be protruded from the mouth presumably to penetrate plant tissue during invasion and feeding. The stylet is well developed in second stage juveniles, but weak in the other stages, and possesses basal knobs, measuring between 8 - llum.

Oesophagus:- This consists of an anterior region (Procorpus) about

1/3 to 2 the width of the corresponding region of the body and ending in a muscular median bulb at the centre of which the lumen carries 7.

three crescentric thickenings. The bulb is followed by an isthmus

portion about equal in length to the fore part, encircled posteriorly

by the nerve ring. This part is followed by the swollen glandular

region containing the three oesophageal glands, each of which is a

uninucleate cell.

Excretory pore:- The excretory pore is situated in the vicinity of the

hind part of the oesophagus. The intestine is connected to the anus

by a short rectum.

Ovary:- The ovary usually has two or more flexures with many oocytes

arranged about a rachis, and ends distally in a cap-cell. The sperm-

atheca is pyriform; it is broader and is separated from the oviduct by

a sphincter, its narrower end merging into the uterus.

Eggs:- Eggs are usually ovoid in shape and undergo various shape

changes with juvenile development inside.

Testis:- The testis is anterior .and _reflexed once or twice at the

anterior end; the vas deferens is well developed with cellular walls.

Spicules:- These are paired, broad in the middle with an expanded

head, and short; gubernaculum thin; bursa not enveloping the tail com-

pletely.

Tail:- Tail acuminate. The tip shows variations and tends to bifurcate

in some cases; but in the male it is conoid, tapering to an obtuse or

rounded tip. (Marcinowski, 1909; Byars, 1920; Goodey, 1932; Filipjev

and Schuurmans Stekhoven, 1941; Thorne, 1949; Swarup and. Gupta, 1971;

Al-Sabie, 1977).

e. Morphometrics

The body measurements of the different juveniles stages do not

seem to have been dealt with in literature, most publications dealing

only with the measurements of eggs, J2 and adult'males and females

(Table -1-). Experiments • reported in this thesis investigated the

variation between different isolates, 8.

Table -1- Summary of previously recorded measurements of

Anguina tritici

Measurements of the eggs

Characters of Bauer, Filipjev Goodey, Swarup and measurements 1823 Byars, 1920 et al., 1941 1932 Gupta, 1971

Width 28-31 38.7 U.S.A. 33-63 34.9-53.5 38 u Europe 37.4 China a - 39 rc . 43.8

Length 83 85.1 U.S.A. 73-140 75.6-102.3 u Europe 71.4 China is - 85 85 % • 87.1

Measurements of First stage juveniles

Characters of Filipjev Marcinowski, AL-SABIf, measurements et all 1941 1909 1977

Length 0.53-0.67 0,5-0..6 0.5-0.6 mm ac- 0.63

a ratio 42

b ratio 4.5

Measurements of Second stage juveniles

Characters of Filipjev et al., Goodev, ftarcinowski, Swarup and Byars, 1920 Measurements 1941 1932 1909 Gupta, 1971

Length 0.966-0.77 USA 0.75-0.79 0.8-1.0 0.8-0.95 1.0 mm 0.91-0.658 China it . 0.77

Width 15-20 USA 15-20 p smaller, China

a ratio 47-59 x - 54 4.0-6.3 b ratio 4.49-4.74 • īt • 4.5 23-28 c ratio ik - 26

Stylet 9-11 10 U

Tail 50 u

Continued....

Table -1- (Continued)

Measurements of adult Males

Thorne, Characters of Byars, Filipjev, et al., Goodey, Marcinowski, Swarup and measurements 1920 1941 1932 1909 Gupta, 1971 1961

2.04-2.4 Length 2-2.5 1.9-2.5 2-2.5 1.91-2.5 2.4 ELM x - 2.19 21.2-30.0 a ratio 30 2S-29 25 īc • 26.58 ' . 6.3-11.0 b ratio 13 12-13 13.2 9 ;C- 9.29 17-23.8 . c ratio 14 25-28 x ' 19.7 30

66.7-81.4 ' T ratio 80 TK - 75.4 Tail mm 0.19

Stylet 9-11 9-10 U

Measurements of adult Females

Characters of j Byars. Fllipjev, at al., Goodey, Marcinowski Swarup and Thorne, measurements 1920 1941 1932 1909 Gupta, 1971 1961

Len gt 2.64-4.36 mm " 4 4.1-5.2 3-5 4.10-5.23 x - 3.24 3-8

21.2-30.0 a ratio 21 25-30 ' 20 ic - 26.58

9.8-19.4 13 b ratio 19 20-25 19.3 sc - 13.98

17.0-23.8 c ratio 30 32-50 31 rc - 19.70

v ratio 88-97 90-94 70.4-89.6 91 it - 80.7

stylet 9-11 . U 10.

f. Distribution and Host Range

Anguina tritici is found all aver the world where wheat is cultivated. Formerly, it caused heavy losses but, due to modern seed cleaning methods that separate galls from healthy grains, it has become extinct or rare in most parts of western Europe, North

America, Australasia, New Zealand, Syria, Iraq, Pakistan, Brazil,

Canada, Egypt, U.S.S.R. (Kiryanova and Krall, 1963), and China (Chu,

1945).

In the United Kingdom it has been very rare since 1956, (Marshall,

1960). However; it has been reported as troublesome in India, Ethiopia,

Rumania, Yugoslavia, and the Middle East.

This nematode attacks wheat, Triticum aestivum L. (Needham, 1744); rye, Secale cereale L. (Roffredi, 1775); spelt, Triticum spelta L.

(Roffredi, 1776); emmer, T. dicoccum Schrank, (Byars et al.,1919);

T. ventricosum Ces. (Leukel, 1957); einkorn, T. monococcum L. (Mumford,

1961). It was mentioned in host lists compiled by Filipjev and

Schuurmans Stekhoven, 1941; Goodey, Frankling and Hooper, 1965;

Manolache and Romascu, 1973.

It also invades oats Avena sativa L. (Henslow, 1741) and barley

Horde= vulgare L. (Roffredi, 1775), but galls have not been observed and little or no reproduction occurred (Leukel, 1957; Goodey and Hooper

1958). Henslow (1841) observed slight infestations of barley with

A. tritici. Christie (1959) reported that infection in Hansee Hull — less barley occurred with an artificial inoculum.

Bhatti, Dahiya and Dhawan (1978)..however showed the first record of tundu and ear—cockle incidence in barley. They stated that, on an average, one gall contained two males and fourteen females of

A. tritici, but reproduction was not reported. 11.

Typical vegetative symptoms and galled ears were formed in four varieties of Triticale (Anonymous, 1977). Juveniles could not, or could rarely, penetrate the stem below the first node of potted sweet corn, Zea mays (L) and sorghum, Sorghum vulgare (Pers).

Seedlings were inoculated with second stage juveniles of A. tritici.

When more soil was added to the pots, after the first node had formed

(which occurred at the soil surface) and the inoculum was then applied, heavy invasion occurred in the new leaf tissue above the node, but no evidence of growth of the invading juveniles was apparent, and no galls or juveniles were found in formed ears (Limber, 1976).

A convenient numbering scheme to describe the development of wheat plants at different stages in the growth cycle has been devised by several workers, Bonnett (1936); Large (1954); Tottman (1977);

Tottman, Makepeace and Broad (1979), and others and these stages are adopted in this thesis. g. Biology and life history

Anguina tritici is a plant parasitic nematode causing a disease in which the grain of the wheat is replaced by a nematode gall. The cellular reaction of the plant to the nematode invasion produces specific cells, hypertrophy and hyperplasia,in the gall (Wallace, 1963).

This gall, when mature, contains the infective second stage juveniles usually in a desiccated form. When moistened, the juveniles become active and may invade new wheat plants, feeding ectoparasitically on the tissues of leaves near, and on the growing point (Midha, Chatrath and Swarup, 1971).

The primary leaves become wrinkled, twisted, distorted and in affected shoots the basal stem becomes enlarged, and the plant nay die or suffer a complete loss in yield from a heavy attack (Singh, Singh and Mathur, 1953). 12.

Basal swelling of the stem in the initial stages of infection

is .a diagnostic feature, reported by Gupta and Swarup (1968). Old

plants may be dwarfed, deeper green in colour, and produce more till-

ering. The head stalks may also show some twisting and curling.

After the formation of grains, the heads remain stunted and green

longer than healthy ones. Spikes appear earlier (Benlloch, 1947;

Vasudena and Hingurani, 1952; Becker, 1955). The glumes become more

divergent, with swelling or small pimple formation on the awns, glumes

or staminate tissues (Gupta and Swarup, 1968). The grain is replaced

by hard, light brown to dark coloured galls, smaller and sometimes

plumper than healthy normal wheat grains.

The galls derived from ovary tissue are modified grains of wheat,

black in colour, almost devoid of starch. Mention of these symptoms

has been made by several workers like Byars (1920); Carne (1926);

Goodey (1933); Chaudhuri (1935); Filipjev and Schuurmans Stekhoven

(1941); Christie (1959); Thorne (1961); Jenkins and Taylor (1967);

Swarup and Gupta (1971); Paramonov (1972) and Southey (1972 and 1978).

Most experiments, by different authors, confirm the view that

invasion of the host by second stage _ juveniles of A. tritici is very much favoured by wet and cool weather conditions (e.g. in Europe, Kort,

1972). However, Saxena and Khan (1964) showed that wheat in India

is most heavily infected with A. tritici at 30 - 35°C in susceptible varieties. Also, Joshi, Renfro et al. (1970) reported that when weather

is warm and dry ear-cockle develops, but when it is humid and cool,

tundu develops. This is another indication (in addition to differences

in length) that possibly there may exist biologically different strains

of the organism in different geographical regions. Therefore, investigations

on the infectivity together with some observations on different isolates

are warranted. 13.

Marcinowski (1909) observed horizontal migrations over distances

varying between 5 and 20cm. Most juveniles (96%) failed to reach

their host. She also studied infectivity in relation to the vertical

distribution of the galls in the soil and concluded that J2 may be

active in wet soil for up to seven months.

Midha and Swarup (1972) found that no infection occurred using

103 juveniles per 1000 g soil, but when 2 or 4 galls were used sig-

nificant infection occurred.

The size of the green galls was correlated with the number of

adult nematodes inside (Midha and Swarup, 1974). Mean adults per gall

ranged from 10 - 80, but as many as 283 adult nematodes were recovered

from a single large-sized gall. They also found staminate tissues

were generally preferred and converted into a gall, with the un-

fertilized ovary often attached to the galls.

However, Marcinowski (1909) concluded that galls may arise (1-)

from the undifferentiated flower bud, (2-) at a later stage from staminate

tissues which are first differentiated, (3-) from carpellate tissues which are formed last, and (4-) from tissue lying between the stamens

or between the carpels and stamens.

The Indian researchers' and Marcinowski's isolates-may show a different response in invasion behaviour and in the histopathological changes which they induce, so these were further investigated in this thesis. h. Development of Anguina tritici

Anguina tritici is diploid and amphimictic and both sexes have

38 somatic chromosomes according to Triantophyllou and Hirschmann

(1966); Krall and Aomets (1973). They suggested an YX (Female) -

XY (Male) mechanism with sex chromosomes indistinguishable in form 14.

and behaviour from autosomes.

The eggs developed in the uterus after fertilization (Al-Sabie

1977). Females in water laid eggs in a continuous manner leaving

eggs stuck end to end. Fertile eggs developed rapidly. Infertile

eggs degenerated. The minimum period until eggs hatched was two

days after laying. The hatched juveniles were first stage juveniles which soon moulted to the resistant resting second stage juveniles.

At host maturity, the galls containing only second stage juveniles fall to the soil or are harvested with grain, and when returned to the soil, soften with the first rains which germinate

the wheat, the wall gradually breaking down to release the second

stage juveniles. Juveniles invade wheat seedlings after 25 days

(Midha and Swarup, 1972). Infested plants are usually first seen at the tillering stage (Al-Sabie, 1977). The juveniles feed ectoparasitically at the growing point (Gupta and Swarup, 1968; Midha,

Chatrath and Swarup, 1971).

With the formation of the embryonic flower tissues (flower induction), the second stage juveniles became endoparasitic and developed rapidly (Al-Sabie, 1977). Within 3 days, it was possible to distinguish the third stage juveniles which willmoult to adults

(males and females); 11 days after invasion of the growing point females began laying eggs, each producing up to 2,500 eggs within a gall

(Filipjev, 1941).

Gupta and Swarup (1968) made the surprising observation of a living adult male and second stage juveniles, alone or-together inside live adult females when the temperature fell below 14°C. Cool conditions are very important for the formation of the gall and the continued production of A. tritici juveniles within. 15.

i. Biochemistry and survival of Anguina tritici

Kostyuk (1965), who measured the total protein, nucleic acids,

lipids and polysaccharides in different stages during development

and anabiosis, concluded that during anabiosis only simple carbo-

hydrates were used, while respiration could not be detected.

Respiration of second stage juveniles was measured relative to

CO2 level and varied according to temperature, osmotic pressure,

humidity, glucose utilization and high ionic concentrations, of sodium

and potassium (Bhatt and Rohde, 1970). A relationship was suggested

between resistance to desiccation and ability to respire at high

osmotic pressures. Respiration reached a maximum rate of 25p1/mg d.wt/hr

about 1 hour after rehydration slowing to 7.2 - 9.0 after 24 hours.

Spurr (1976) however, found that concentrations of Adenosine triphosphate

(ATP) in J2 of A. tritici from galls 3-5 years old were similar to those in active juveniles (about 1 x 10 10 gm/juvenile). Within 40 minutes

of revival the ATP concentration had doubled,but it declined again under

starvation.

Analyses of A. tritici juveniles 24 hours after revival showed

trehalose at only 5% of the level in desiccated juveniles and it had

decreased further after 48 hours (Womersley, 1978). Bound inositol

also decreased and had almost disappeared after 48 hours. Glucose

remained unchanged and glycerol was slightly increased after 24 hours.

However, incubation in 5% inositol solutions improved the survival of

A. tritici during subsequent desiccation and revival (Evans and

Womersley, 1980).

Bird and Buttrose (1974) investigated how A. tritici survived

desiccation, and studied morphological differences in the structure

of the external cortical layer of the cuticle between active and 16.

cryptobiotic stages, which might account for longer survival of second

stage juveniles. They found no difference in width or,cross-sectional

surface area between hydrated and cryptobiotic specimens. The mor-

phology of lipids, in the cuticle and in the droplets, was considered

to be an important factor in the ability of this nematode to survive

in the dry state. They showed that active hydrated juveniles contained

less water than other nematodes. However, they did not report on the

body length.

Anguina tritici is capable of surviving for 28 years in the

dried state (Fielding, 1951). Second stage juveniles of A. tritici

in wheat galls stored at 5°C were able to resume activity after 32

years, and were able to invade wheat seedlings readily (Limber, 1973).

Single second stage juveniles of A. tritici will survive for 7 days

over phosphorus pentoxide in a desiccator (Ellenby, 1969).

Juveniles died in galls kept in moist infested soil for a year

in the absence of the host, but survived indefinitely inside the

gall in dry soil (Leukel, 1957). After 38 years, 60% of a collection

of galls contained viable juveniles (Thorne, 1961), so the maximum

length of survival had not yet been reached then. Once second stage

juveniles were active in the soil, none survived longer than seven months in the absence of host plants (Marcinowski, 1909),

Second stage juveniles in dry galls survived high and low temp-

eratures better than those soaked in water (Bloom, 1963). None survived

in presoaked galls exposed to 55°C for 10 minutes, whereas only 43% in

dry galls were killed after two hours at this temperature. Similarly,

at -16°C all juveniles were killed after 12 days in wet galls and only

31% in dry galls. Thus, dried galls effectively protect juveniles.

Klingler and Lengweiler-Rey (1969) tested the heat susceptibility of '17. active and live desiccated A. tritici by their survival at 50"C in water and at 50% or 100% relative humidity. Womersley (1980) studied the effect of various desiccation regimes on the ability of J2 of

A. tritici to survive drying at 0% relative humidity. He found that both repeated dehydration/rehydration cycles and the length of the desiccation period itself decreased viability of the juveniles.

Vital stains, Chrysoidin R (1 : 20,000) and Phloxine B (0.1%), were used to differentiate between dead and living nematodes (Bloom,

1963).

Mukhopadhyaya, Chand and Suryanarayana (1970) in studies on longevity of A. tritici, buried galls at various depths in the moist soil. They found that many juveniles survived for 90 days at 20cm, but few survived at greater depths or after 120 days. After 300 days,

69% of the juveniles, kept at 5°C to 8°C, were alive and 84% of these kept at 22°C to 37°C survived. Although 30% of the juveniles withstood

7 days at 42°C, none survived a fortnight.

Second stage juveniles of A. tritici were released between platinum electrodes, in sand as a medium, saturated in separate experiments with tap water and nine different salts, each in eight different concentrations. Juveniles exposed to an electrical field in a variety of electrolytes in sand migrated to the cathode, suggesting that the amphids of nematodes contain large negatively charged organic molecules of low water solubility that attract cations in water (Sukul, Das and Ghosh, 1975). j. Effect of Anguina tritici on biochemistry in wheat

Most of this work was carried out on material from a Rumanian infestation of winter wheat. Anguina tritici caused marked changes 18.

in enzyme and respiratory activity in different parts of the host

plant (Horovitz, Romascu and Enescu, 1969). The hydrolytic enzymes,

acid phosphatase and invertase, were active in the, juveniles of

infested plants and especially active in the infested grains (galls),

whereas there were only slight changes of peroxidase and catalase

activity. Invertase activity appeared to be the most important factor

in causing metabolic changes in infected tissues. The great increase

in metabolic activity, including respiration, in the ripening galls

was related to egg hatching and the activity of the resultant mass

of juveniles; but Al-Sabie (1977) found that the eggs could hatch in

tap or distilled water only two days after the females had laid the

eggs in the water.

Brad, Romascu, Ciobanu and Gheorghe (1970) showed that morphological,

physiological and biochemical changes occurred in winter wheat plants

when attacked by A. tritici. Also they found that among the same

substances occurring in larger amounts in the susceptible or resistant

plants, there were respectively attractants and repellents.

Gall extracts at dilutions of (1 : 1000) stimulated wheat plant

growth in the initial stages (Midha and Swarup, 1974). Chemical analysis

of the gall extract showed the presence of amino acids equivalent to 8

micromoles of Leucine, and sugars equivalent to 42 mg of glucose; also

a diet containing galls had no toxic effects when fed to rats. k. Associations with other pathogens

Fahmy and Mikhail (1925) ; Carne (1926);. Cheo (1946) ; Gupta and

Swarup (1968) and Hingorani and Bekele (1969) mentioned that yellow

ear rot disease of wheat caused by Corynebacterium tritici (Hutchinson)

is invariably associated with the presence of A. tritici; it,has

been known for many years as "tundu" disease. The characteristic 19.

symptom is the presence of a. bright yellow slime, consisting of massed bacteria, on the abortive ears and ensheathing juveniles while the ears are still in boot. Typical Anguina symptoms are apparent in the seedling stages, but few or no galls are formed in infected heads. Cheo (1946) and later workers showed that the bacterial disease does not develop in the absence of A. tritici.

Swarup and Singh (1962) found no evidence that A. tritici juveniles were vectors of the bacteria. They suggested that they were carried on the gall surface, though Cheo's work indicated that the bacteria were carried mainly within galls. Gupta and Swarup (1972) found that when bacteria were inoculated with seed-galls or nematode juveniles, with or without further addition of the bacterium, in both, ear-cockle and bacterial symptoms developed. Juveniles surface sterilized with one of five different chemicals (Agrimycin 1% for 45 minutes; streptomycin sulphate 0.1% for 30 minutes; hydrogen peroxide 8% for 30-- minutes; sodium hypochlorite 5% for 30 minutes and mercuric chloride

0.1%for 30 minutes, nematodes afterwards being washed with sterile water)caused only ear-cockle disease; the bacterium alone was not capable of causing disease. However, Midha and Swarup (1972) found the development of ear-cockle and tundu disease of wheat to be dependent on juvenile and bacterial concentrations, temperature, humidity,

age of seedlings and depth of placement of the galls.

Mathur and Misra (1961) observed the simultaneous occurrence

of A. tritici in the top part of the ears and Tilletia foetida in the

lower part of the same ears. Intermediate stages occurred with nema-

tode galls containing bunt spores and bunt galls containing nematodes.

According to Zopf (1888), it has been discovered that Athrobothrys oligospora is capable of capturing, destroying and feeding upon small

free-living nematodes as well as upon the larvae of A. tritici, when 20. these are supplied to the fungus in question under experimental conditions. Atanasoff (1925) found A. tritici acting as a vector of the fungus Dilophopora. alopecuri, and claimed that this fungal disease of wheat only occurred in the presence of the nematodes.

1. Control

The measures to minimize the infection of the disease should be directed to killing the destructive nematodes in the gall or to separating completely the galls from the seeds, thus rendering the seed clean for sowing purposes.

Chu (1945), gave an account of various control measures, including fanning, screening, hot water dip, chemical treatment, sedimentation by salt brine, flotation in water and separation by means of an indented cylinder or "trieur", which have been suggested by workers in different countries. Unfortunately, none of these methods is entirely effective. In fact, the most effective control is by modern mechanical seed cleaning which eliminates the galls.

Crop rotation to reduce the amount of nematode infection was recommended by an anonymous author in a Chinese handbook (1943); Coleman and Regan (1918), Leukel (1924): wheat and rye should not be sown in infested fields. In moist conditions the absence of the host plant for one or two years is sufficient to free the soil from A. tritici and practically to eliminate nematodes remaining in the field after harvest (Leukel, 1924).

Leukel (1924) was the first investigator to test the different varieties of wheat for resistance against the A. tritici. Initially, none of the varieties tested were resistant, but later he found that the wheat c.v. Kanred was more resistant than a number of other varieties 21.

(Leukel, 1957 )•

Anguina tritici attacked all the 50 wheat varieties tested by

Romascu (1969), where higher resistance occurred in three winter and three spring wheat varieties. Motoi (1969) found all of 16 varieties of wheat tested showed variations in susceptibility, and that none was sufficiently resistant to be of use for control. In Yugoslavia,

Tesic (1969) found that 7 out of 319 wheat varieties tested were resistant to A. tritici. Belloni (1954) discovered that Italian wheat varieties were all susceptible to A. tritici. In India, Saxena and

Khan (1964) found a little resistance in only 4 varieties of wheat out of 16 in cultivation, and all the others susceptible. In Iraq,

Al-Baldawi et al. (1975) found that only two had any resistance to

A. tritici out of 13 wheat varieties that were tested in the field and under lath-houses.

Hinfner (1970) showed the resistance and susceptibility of varieties of winter wheat, and he correlated the sensitivity of wheat varieties and the movement of A. tritici population to the plant.

Singh and Prasad (1972) tested the effect of some pesticides and growth regulators on A. tritici in different methods: by treating the soil before it was infested with A. tritici galls; by putting galls into the treated soil, and by spraying the plants grown in infested soil with pesticides and/or growth regulators, 25 and 35. days after germination. They found there was no gall formation in the case of dibromo chloropropane (DBCP), carbofuran and phorate in treated pots.

In the case of aldicarb, thionazin, fensulfothion and disulfoton the gall percentage was nil to very low. And, in spraying, it was found that ethyl-parathion was most effective and registered complete elimination of galls when sprayed 35. days after germination, 22.

Krnjaic (1973) tested the invasion capacity of the juveniles,

after exposure to gamma irradiation (Co60), with 25, 50 or 1QQK -rads;

the juveniles invaded wheat in large numbers and galls with viable

contents were formed at the end of the growing season. The juveniles

irradiated with 200K rads caused symptoms in wheat after tillering

but no galls formed, Irradiation with 300K rads and above was lethal. m. Aims and objectives of the present study,

Anguina tritici is an important parasite of wheat especially

in Iraq and is widely distributed around the world. However, much

information on the biology of this nematode is still lacking, especially

in the intraspecific variability. The aims and objectives of the present

study, therefore are to find out:-

1) The practical and theoretical implications of the host range & morpho10 \'.

ii) The various factors affecting infection and host-parasite

relationship.

iii) The survival ability of the active J2 of A. tritici in soil.

iv) The variability in different isolates of A. tritici as regards

the type of respiration and osmoregulation.

In view of this, it has been thought worthwhile to obtain more

substantial evidence concerning the differences between A. tritici

isolates. 23.

SECTION II

GENERAL MATERIALS AND METHODS

Origin and Maintenance of Nematode Culture

Galls

Five isolates of Anguina tritici from U.K., Iraq, U.S.A.,

India and Australia were used. These were obtained from the following sources:-

Isolate Source U.K. Rothamsted Experimental Station, Harpenden,

Hertfordshire, England.

Iraq. Department of Plant Pathology, Abu-Ghraib,

Baghdad, Iraq.

U.S.A. Department of. Plant Pathology, North Carolina

State University, Raleigh, U.S.A.

India. Division of Nematology, Indian Agricultural

Research Institute, New Delhi, India,

Australia. Department of Agriculture, (Western Australia)

Jarrah Road, South Perth, Western Australia.

These were maintained on 'several varieties of wheat.

Hosts

Two spring wheat varieties, which have a short growing period, were used. Triticum aestivum c.v. Maris Dove and T. aestivum c.v.

Sicco; the latter being less susceptible to powdery mildew.

Seeds of the two varieties were germinated in a sand tray at an average temperature of 20°C in greenhouse. Three days after germination, healthy seedlings were selected and transferred to John Innes 24.

Nd. 1 compost (7 parts loam + 3 parts peat + 2 parts sand + 4oz. John

Innes fertilizer + boz. chalk). Three wheat seedlings were planted in a 3 inch diameter clay pot, for different treatments, unless otherwise stated. In addition to above hosts-, other varieties were also used for host range tests (see below).

Inoculation

Two methods were used to provide galls continuously:- a. Seedlings were inoculated by mixing the galls with the soil every

4 months. b. Seedlings were planted every two weeks throughout the year and, when flower induction had just occurred (at growth stage code number 37,

Tottman and Makepeace, 1979) plants were inoculated artificially with second stage juveniles of A. tritici, see below. The growth of wheat plants varied throughout the season depending on daylight and temperature conditions; and the time of flower induction was determined by dissecting some plants regularly. The J2 were recovered from soaked galls and allowed to revive for 24 hours before use. Active juveniles were obtained by movement through an 841rm sieve overnight and washed several times with sterile tap water. The suspension was concentrated by removing excess water. 1 ml hypodermic syringe and 63 needle (25mm aperture) was used to inoculate the flower prim- 100 ordium (wheat head) with 0.05 ml (five drops) of J2 suspension by carefully inserting the needle down the flag leaf using a rolling movement until the wheat head was reached (Al-Sabie, 1977). 25.

SECTION III

STUDIES ON MORPHOLOGY AND HOST RANGE WITHIN ANGUINA TRITICI

A. A comparison of Morphological Variation within and Between Isolates

of A. tritici

1. Introduction

The literature on the characteristic measurements and host ranges for different geographical isolates of Anguina tritici is full of apparently inconsistent results (see literature review page 7).

Earlier workers described and measured different stages of A. tritici occurring in different parts of the world. Their measurements tend to differ from one worker to another and few comparative studies have been made.

Principal components analysis displays maximized. variation between the groups, thus revealing the mutual relationships of the isolates,

An alternative method of depicting the relationships of'the isolates, by multivariate methods, is that provided by canonical variate analysis,

This analysis assumes the existence of groups and aims at transforming the data so as to present them in a space which maximizes the between- group variance relative to the within-group variance as revealed by a variance ratio test (F, test). However, this significance test does not apply to any two selected isolates, and in practice it may be important to know how far they are distinct and howmuch reliance may be placed on the differences between them. This problem can be dealt with by computing a separate linear discriminant function between each pair of isolates compared.

The first aim of this research was to examine morphological characters by measuring nematodes from different isolates under identical conditions and analysing them by the three methods mentioned above. .26.

2. Material and Methods

Second, stage juveniles

Juveniles from 5 different source of imported galls (Old J2) of each isolate (U.K., U.S.A., Australian, Iraqi and Indian) were revived and killed by gentle heating in water at 50°C. Then the specimens were fixed in TAF (7m1 of formaldehyde, 2m1 of triethanolamine and 91m1 of distilled water). The specimens were transferred to 100% glycerol for processing by glycerol-ethanol rapid method (Seinhorst,

1959), and mounted permanently in glycerol. The cover-slip was supported by glass fibres. Measurements of the body length, oesophagus length, stylet length and gonad primordia length were made on twenty

3.2, selected at random from each isolate.

In the following season J2 in the imported galls of each isolate were used to inoculate the same host separately and the host was grown in the U.K. under identical conditions. Similar measurements as described above were taken of J2 in the galls produced from each isolate

(New J2).

Adults

After the mature galls were carefully dissected in water the adult males and females were released in distilled water and immediately killed by gentle heat.

The males and females were fixed and mounted as for juveniles

(above).' Measurements were made on 20 adult males and females from

-each isolate. Male and female characters of body length, oesophagus, stylet, greatest body width and tail were measured. In addition, length of spicules in males and length from vulva to tail end in females were also measured. The number of males and females in each gall was counted. 27.

Eggs

When the eggs were released from the dissected galls, length and width were measured on 20 eggsfrom each isolate /The results were assessed by computer analysis using three programmes:-principal component analysis, canonical variate analysis for all the above measurements, and a linear discriminant function analysis for two groups between each combination of two different isolates.

All three programmes were written in Fortran IV by Mr. R.G.

Davies of the Department of Zoology, Imperial College, from whom listings may be obtained. A brief summary of the methods used in these programmes are as follows:-

Principal component analysis

This programme carries out a covariance analysis of a correlation matrix. The latter is derived from a primary data -matrix of number of columns (P = characters) and number of rows (n = individual in the group); this follows the normal procedure for an R- modeanalysis. From an n x p data matrix, a p x p - matrix of between characters•(columns) correlation was computed. The first 10 latent-roots and associated vectors of this matrix were then printed out after computation by a Jacobi type sub-routine. The principal components were then computed by post-multiplying the n x p data matrixby the p x 10 matrix, formed by arranging the first 10 latent vectors columnwise. The analysis was then pursued graphically by plotting the first, second and third principal components against each other in pairs so as to detect any evidence of clustering among the isolates.

Canonical variate analysis

These were carried out separately for each development stage.

The pooled within-groups (W) and between-groups (B) sum of squares and 28.

cross-products matrices were computed. The latent-roots and vectors

of the product matrix W-1 B were computed, using a modification of

the Jacobi technique, which can be applied here for dealing with

non-symmetric matrices.

The canonical variate (multiple discriminant functions) were

then computed as linear functions of the original variables, each weighted by the corresponding vector element. As before, the canonical variate axes values for each individual were plotted two at a time to reveal clustering of the individuals and mutual relationships of the isolates.

Linear discriminant functions

These computations for comparisons between two groups followed the usual procedure, as devised by Fisher (1953) and reported in

Davies (1971). The programme is essentially that given by Davies (1971) with only minor amendments.

The linear discriminant functions of two groups there considered as two A. tritici isolates) are plotted in terms of the values of characters, These values may overlap. If, however, the individual points in the first and second isolates are expressed as measurements on a new axis, along which there is no overlap at all between first and second isolates, then the variance within isolates on this new axis is minimized and consequently differences between isolates are maximized. The axis which minimizedthe overlap (and might be called the discriminant axis) was calculated by finding the angle through which the actual axis might be rotated. This in turn gave a linear trans- formation, in which a value was given to a set of coefficients, each of which was used to multiply the original measurement and the results, to give the value of the discriminant function. 29.

3. Results

The mean measurements of the characters were significantly

different for all isolates (Table 2 and Appendix 1, page 180).

The body length of J2• (ōbtained from galls grown in the country

of origin) was longest in the U.K. isolate and shortest in the

Australian isolate. The oesophagus length was greatestin the U.K.

and least in the Indian isolate. The U.K. isolate had the longest

stylet and the U.S.A. isolate the shortest, but the gonad lengths were the exact opposite; the longest was in the U.S.A. and the

smallest in the U.K. isolate.

The body length of J2 (obtained from galls produced in the U.K. under identical conditions) was longest in the U.K. isolate and shortest in the U.S.A. isolate. The oesophagus length was greatest in the U.K. and least in the Australian isolate. The Australian isolate had the "shortest stylet of the isolates.

Body length of the adult males (obtained together with females from galls grown on the same host at the same time) showed the same

trends as the J2, but the oesophagus lengths were longest in the U.S.A. and smallest in the Australian isolate. The stylets of these from the U.K. and U.S.A. were longest and the Iraqi was the smallest.

The body width of that from the U.K. was greatest and of that from

the U.S.A. was smallest (Table 2).

Body length of adult females were longest in the U.K. isolate

and smallest in the U.S.A. isolate. The oesophagus length of the

Australian isolate was smallest and the Iraqi was longest. The stylet

of that from the U.K. was longest and the smallest was the Iraqi. The

greatest body width and tail length were found in the Australian isolate

• 30.

Table 2. Measurements of A. tritici of different isolates.

Stage Character solati Australia O.S.A. Iraq India

..~".i g imm 0.916 0.850 0.860 0.884 0.870 .,.~ Length CD min-max. 0.82-0.97 0.71-0.94 --' 0.75-0.92 0.76-1.00 0.80-1.0 . .4 3 Fi in 221.039 204.284 205.584 214.935 202.856 0 O Oesophagus 4-1 min-sus. 197.4-231.7 189.61-218.18. 189.61-228.57 194.8-231.17 176.62-233.77 O u, 7.4 7.05 3.97 7.203 6.88 .--1 stylet min-max. 6.49-7.79 6.49-7.79 5.19-7.79 5.19-10.39 5.19-7.79

.•~ u • z!t 22.339 25.585 36.431 31.495 34.955 0 - Coned • c° ō mio--aax. 18.18-28.37 20.78-31.17 23.03-51.4 20.78-41.56 15.58-33.24 o u • V :•', b ratio 4.144 4.1609 4.1856 4.1152 4.2927 J2 inside gall 6,680 11,873 9,466 13,620 14,312 2.49 1.99 2.11 2.33 Length min-max. 2.26-2.79 1.43-2.53 1.82-2.8 2.00-2.39

Tot 226.28 206.42 228.14 211.43 oesophagus min-max. 188.57-265.71 183.71-262.84 177.14-262.86 177.14-237.14

Li 10.86 10.36 10.86 9.79 stylet min-sax. 8.57-11.43 8.37-11.43 8.57-11.43 8.57-11.43 width mu 103.00 80.58 83.86 99.86 (body) min-aax. 80.00-128.57 60.0-88.37 62.86-105.71 74.29-120.00 ī21 86.76 74.44 84.14 84.14 min-sax. 80.00-100.00 68.57-81.6 74.29-94.29 68.37-94.29 31.86 32.32 35.0 29.86 spicules min-max. 22.86-37.14 31.43-34.28 28.57-40.0 17.14-34'.29 a ratio 24.1795 26.71 23.108 23.354 b ratio 11.0063 9.646 9.2693 11.124 e ratio 28.705 26.74 5 24.442 27.9474 īmm 4.420 3.01 2.83 4.24 length min-max. 3.89-3.25 2.13-3.63 2.01-3.72 3.44-4.93

K. 721 223.84 204.27 204.36 235.3 oesophagus min-sax. 205.71-265.71 185.71-225.71 162.86-331.43 214.29-262.86 U. iu 11.79 10.40 11.36

he 9.57 stylet

t miarmax. 11.43-12.86 8.65-11.34 10.0-12.86 8.57-11.43

in width iu 214.13 130.82 160.57 198.37 (body) d min-max. 171.43-237.14 114.21-130.00 100.0-242.86 134.29-231.42 :K 86.29 92.01 89.57 89.90

duce Tail 77.14-91.43 89.4-92.3 77.14-108.57 74.21-100.0 ro Length from imm 0.4 0.361 0.343 0.413 p vulva to tail minrmax. 0.3-0.464 0.264-0.469 0.243-0.42 0.322-0.511

lls a a ratio 20.644 23.009 17.13 21.37265

g b ratio 19.748 14.736 13.133 18.037

e ratio 51.23 32.714 31.556 47.2078. From ✓ ratio 91.006 88.002 87.805 90.1473 n1 90.38 96.31 92.58 102.41 Length 69.23-103.83 80.77-121.15 eggs min-max. 86.54-98.08 81.53-104.21 44.13 34.74 39.24 35.77 width aitt-sax. 40.39-46.13 34.74 34.62-46.13 34.62-40.39 x .g Il 3.57 oe Females aim-star- 19-2 2-219 113 2-5 Total 49 66 78 25 ▪1. 4 V 5.71 y 10-7,14, 2.857 w as OW?) 1-5 n • Males min-max. 1- 6 1-13 1-27 e Total 36 40 75 20

Continued... under identical conditions Table 2.(Continued) Length oesophagus gonad stylet Characters min-max. min max. min-max.212.99- min-max-0,84 xp xp xmm xp 217.666 20.78-28.57 24.027 6.49-7.79 0.914 7.53 UK - 0,97 231.17 200-218.18 20.78-31.97 6.49-7.79 Australian 210.907 0.82 -0,94 27.142 0.874 7.07 202.6-223.38 23.36-39.72 5.19-7.79 212.467 0.75 -0.9 33.946 0.851 6.165 USA 202.6-231.17 0.787-0.908 20.78-33.77 216.752 5.19-9.09 Iraqi 29.352 0.868 7.595 31. 32.

and the smallest were in the U.K. isolates The greatest length from vulva to the end of the tail was in the Iraqi isolate and the smallest was in the U.S.A. isolate. Overall the U.K. isolate had the largest measurements and the Australian isolate the smallest.

There was a big separation between some of the isolates (Figure 1), when combined values of means of all morphological characters of juveniles in Table 2 ,.were analysed, using principal component analysis

(PCA), and expressed in the three principal component axes. The values of the first components when plotted against the second or third components, showed that the Australian and U.S.A. isolates were well separated from those of the Iraqi and U.K. isolates (Figure 1, a, b).

When principal components two and three were plotted, smaller and less polarized separation was seen (Figure 1, c). The values of the principal components of the 20 individuals for the J2 and adult males and females were also well separated when they were separately analysed

(Figure 2, 3, 4, 5). When the values on the principal component axes one, two and three were plotted one against the other for J2, it was shown that over all isolates there was a strong linear relationship, with a slope of about one (Figure 3). The individuals of the Australian isolate were always in the lowest positions while those of the U.K. isolate were among the highest, This tendency is very clearly seen in plots of the centroids for each isolate (Figure 3, a, b, c).

The values of the same three principal components for all adult male isolates were similar to the J2 results, except that the Indian isolate was not present, as shown by the centroids in Figure 4, a, b, c,

The first three principal components for all adult female isolates showed different locations in the graph (Figure 5). When axis three was plotted against axes one and two (Figure 5, b, c), the U.S.A, and Figure 1.Thefirstthreeprincipalcomponentsrepresentingacom- third component thirdc omponent second component 750 600 550 650 700 550 600 650 700 750 350 400 450 500 550 -

_ Iraqi

' Second component values foreachisolate. bination ofallcharactersfromeachisolate,basedonmean 6600 350 400 450 500550 6600 68007000 X USA X 1 X

1 X Aust.

X Aust. USA 6800 7000 Aust. 1 X I

(b) (a) (c) 1 I

7200 7200 740076007800 i I

USA First component 7400 76007800 First component 1 1

I 8000 8000 1 8200'8400 8200 8400 1

. Iraqi Iraqi X I X 8600 8800 8600 8800 1

XUK X UK 1 9000 I 9000. 33.

First component Figure 2.TheprincipalcomponentsoneandtwoplottedforallJ 640 660 720 680 740 700 760 780 800 820 -250 individuals foreachisolate.

., + °

-260 1 Australian .

0 0

D °♦° -270 + +

+ f 0

Second component 0 o --ō?C+U.S.A. ~~ • 0 ..

~ -280 ~C v07+ • Q

v G a o Indian

k 0 0 ° 0 .. 00 ••• ♦

+ X

v Key

° -290 0 a V

Iragi o Y D .. 0 + • +

• U.K. Indian Iraqi U.S.A. • Australian • •Xt a -300 •

• U.K. m • 2

-310 • a

34. -320 a Figure 3.PrincipalcomponentanalysisonthreeaxesforallJ2 characters foreachisolate;centoidsonly isolate. , U) 0 0 u First component ā —290 —280 41 Second component —270 —300 — 270 — 300 — 280 —290 680 700 720 740 760

Imm 540 550 560 570580590 Aust, 540 550560570580590 680 700720740760 Aust. 'rill X

USIX India (a) Aust. (h) (c) First component Third component Third component USA 'U X SA X . XIndia ī _ Iraqi p Xlraqi Iraqi diaX X X X X X

UK UK 35.

36.

Figure 4. Principal component analysis on three axes for all adult

male characters; centroids only for each isolate.

-1300 (a) X UK -1250

-1200 X Iraqi 0 ō -1150 af 0 0-1100 b G 0 X USA 1a,-1050

-1040 X Aust.

1300 1400 1500 1600 1700 1800 1900 First component

120 (b) XUK

t 110 X Iraqi nen o 100 XUSA d comp 90 X hir

t Aust. 80

1300 1400 1500 1600 1700 1800 1900

First compōnent 120 (c) X UK

t 110 X Iraqi en

on 100

X USA d comp 90 X hir

t Aust. 80

-1000 -1050 -1100-1150-1200 1250 1300 Second component

37.

Figure 5. Principal component analysis on three axes for all adult female characters; centroids Only for each isolate.

(a) — 800 Iraqi X x UK —750

—700

ā —650 a P. —600 59 AuAst.

X USA I —500

2500 3000 3500 4000 4500 5000 First component

- (b) 165

xIragi t

en 160 USA

on XX Au s t . 155 omp x UK d c 150 hir t

2500 3000 3500 4000 4500 5000 First component

t 165 (c) onen )(Iraqi 160 X

d comp X Aust.

ir 155 USA XUK h t 150

1 1 1 1 1 1 -500 -550 -650 -700 -750 -800 Second component 38.

Australian isolates were clustered fairly closely, while the Iraqi

and U.K. isolates were separated from them to form another group.

When axes one and two were plotted there were indications of cluster-

ing like that shown by males and juveniles (Figure 5, a).

The variates on the canonical axis were plotted as above for

principal component analysis and the results are given in Figures 6 s 8 , 9 , 10 , 11 and 12.

The canonical variates for each individual J2, adult males and females showed that the isolates were distributed differently (Figures 6 , 8 , 9 , 10 , 11 and 12). The J2 [from galls grown in the country

of origin (Old J2)]showed a clear separation between the U.K. Australian

and U.S.A. isolates, while the Iraqi and Indian isolates overlapped,

also covering much of the range of variation seen in other isolates

(Figures 6 and 7). The canonical variates for different isolates of

J2 of A. tritici, which was produced in the U.K. under the same host

(New J2), showed a clear separation of the U.S.A. isolate from the

U.K. isolate (Figure 8.). The. U.S.A., Australian and U.K. isolates followed the same pattern as the original isolates grown in the country of origin (see Figure 6), except that the Australian isolate overlapped with the U.K. and U.S.A. isolates. The Iraqi isolate.overlapped with all other isolates but was still within the same pattern as the original isolate. No galls were produced by the Indian isolate.

When the centroids of canonical variates of J2 from different isolates from galls produced in the country of origin (Old J2) and those produced in the U.K. (New were compared (Figure 9), the results showed that the distance between centroids of the "Old J2" produced in the country of origin for the U.S.A., Iraqi and U.K. isolates, is about equal that of the centroids of the "New J2" of the same isolates

39. Figure 6. Canonical variates for second stage Juveniles of A. tritici for different isolates from galls grown in the country of of origin. (Old J2)

110 •

109 0-... P < 0.001 / 108 ...,.., / ...,...... 107 / / __NA 106

105

104

103

102

101

is 100 ....„

l Ax 99 / ica 98 / non < \ 97 • d Ca

on d. AUSTRALIk-, 96 \ • -■ . . Sec x e 95 \ ! i Key \I ) I 94 i --II-- INDIA ... ,/ .II f —41-- IRAQ ...... • i 93 % :I 111.. I. i AUSTRALIA U.S.A. 92 Asi % ___•___ U.K. 91 411.--'''......

1 i I I V I 90 41 1 1 4 1 1 1.1 23 24 . 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

First Canonical axis. 40

I1. K ,

Figure 7. Three dimensional model representative of the three

canonical axes of morphometric variations in "Old JZ„

of Anguina tritici for various countries. 41.

Figure 8. Canonical variates for second stage juveniles of A. tritici

for different isolates which were produced in the U.K. under

the same host•(New J2)

Key Iraq

Australian

--. U.S.A. p<0.001 35 ----•----- U.K.

34 is. 33 l ax

ica 32 n

31 X Aust_ d Cano i 30 Secon 29

-4 -5 -6 -7 -8 -9 -10 -11 -12 -13 -14 -15 -16 First canonical axis 42.

Figure 9. Canonical variates for J2 of A. tritici for different

isolates from gall grown in the country (old J2) and those

produced in the. U.K. under identical conditions(New J2); centroids only.

Y 146 p<0.001

145 USA new •Iraqi old 144 X X Iraq] new 143 • UK old • 142 USA old X UK new is

l ax 141 a

ic Indian old n 140 •

ano Aust• new 139 d c X

Secon 138

137

136

Aust. old 135 •

21 22 23 24 25 26 27 28 29 30

First canonical axis

43.

Figure 10. Canonical variate for adult males of A. tritici for different isolates. Key UK Australia 95 USA Iraq 90 x = Centroid of group

85 m X 80 4 t■ i 1 `

l •UK

ica 75 AustX. 10"-- non 70 P<0.001 d Ca 65 Secon 60

I 1 1 I I 1 I I I 1 60 65 70 75 80 85 90 95 100 105

First Canonical axis Figure 11. Canonical variate for adult females of A. triciti' for different isolates.

2 UK 7 1 .ii x [ USA l 0 ica pst. P<0.001 —1

d Canon 1 —2

Secon —3

—4

t I I I t 1 t t It 10 15 20 25 30 35 40 45 50 55 60 First canonical axis, 44. Figure 12.

Canonical variate for eggs for different isolates; centroid only.

(a)

35 X UK 34

is p<0.001

l ax 31 ica 30

non X USA a 29 d c n

co 28 Se 27

26 x Iraq 25 X Aust. t t I 1 t t 24 I 92 93 94 95 96 97 98 First canonical axis- Canonical variate for adult males and females inside the gall of each isolate; centroid only.

(b) 2.5 X USA • is

x 2

l a 1.5 0.01>p>0.001 ica anon

d c 0.5 on Iraq X X UK Sec 0

—0.5 X Aust.

1 2 .3 4 First canonical axis. 45.

produced in the U.K.. The centroid of the Australian isolate produced

in U.K. was closer to the centroid of other isolates (old/new), while

the centroid of the same isolate produced in the country of origin

was far away from other isolates. This possibly accounts for the

significant difference (P<0.01) observed with the Australian isolate

when the linear discriminant function for the two groups (old/new)

of J2 of each isolate were compared (Figure 13). No significant diff-

erences were observed among other isolates when similar comparisons

were made..

The pattern of the canonical variates for adult males was less

clear than J2(Figure 10), but the adult females showed a clear separation

between the U.K., Iraqi and the U.S.A., Australian isolates. There was little overlap between the U.K. and Iraqi isolates, the U.S.A. and Australian isolates, respectively. (Figure 11).

A similar separation was shown for mean egg characters and mean number of adults inside the galls (Figure 12, a, b). In all cases a variance ratio (F-test) for the significance of differences between group means gave F<0.01.

The linear discriminant functions of J2 showed that the U.K. isolate did not overlap with either the Australian or U.S.A. isolates

(Figure 14, a, b). However, the Australian isolate showed a smaller separation from the U.S.A. isolate than did the others (Figure 14, e).

The Iraqi/Indian and U.S.A./Indian comparisons (Figure 14, i, j) showed lower significance and the values overlapped, for the differences between them (P<0.01 as against P<0.001). The Iraqi and Indian isolates overlapped the most but despite these overlaps the mean values (centroid) of the discriminant function (indicated by arrows in the diagram) were significantly different.

46.

Figure 13. The linear discriminant function for two groups (old/new)

of J2 of each isolate. The significance value of F—test

is above each comparison.

UK old II I I I IIll I II UK new 231 11 II 11 I28 I 1 1

P <0.01

Aust. old Aust. new 1 1 1 1 ➢ 11I I I II

USA old -18 1Ifl I III I -12 USA new 11 9Ii

Iraqi old 5 i II III I I1 Iraqi new u ' I' ii? e~ 1 I I i

47.

Figure 14. The linear discriminant function for two groups of J2 of each isolate. The significance value of F-test is above'each

comparison. p.<0.001 a) +150 1 111111111111111111 +190 UK I i I II 11 1pl I I I I Il Aust.

P, x0.001

b) +145 i iii u i w 1 u1ai i III +200 UK I II I I 111 111 III III { USA

P <0.001 c) -7 UK +4 Iraq II III1II II

P<0.001♦ UK d) +60 +90 India II I II I I11 I I I 111 l

P<0.001 Aust. e) 0 1 T i n a rnti mi i 11 +30 USA II II I 111! Iill I III II

P,<0.001 USA Jin ~~~ ~ ~ ~ -25 h) -36 Iraq 11 r ' -I

pK0.01 USA i) -8 1111J I I II I I I i II I I 0 India II I , 1 I 14r I I~ 1 I ti l

PK0.01 Iraq nit I I 111 I I III 1 III J) +26 +33 India III 1111 1 48.

The linear discriminant function of adult males showed that

the U.K. isolate was separated most from the Australian isolate

(Figure 15, a). The U.S.A. isolate had less overlap with either the

U.K. or Australian isolates while the Iraqi isolate was overlapped

by both the Australian and U.S.A. isolates (Figure 15, e, f). The

U.K./Iraqi comparison showed the most overlap and the mean values

(centroids) were of lower significance (Figure 15, c), though even

these have P<0.01.

The linear discriminant function of adult females again showed

that the U.K. and Iraqi isolates did not overlap with either the

Australian or the U.S.A. isolates respectively (Figure 16, a, b, e, f), but some overlap was shown in the U.K./Iraqi comparison (Figure 16, c).

The Australian isolate showed a large overlap with the U.S.A. isolate, and therefore a slight (less significant) difference (Figure 16, d).

The number of J2 inside the gall was largest with the Indian isolate followed by the Iraqi, Australian and U.S.A. isolates, while the U.K. isolate had the fewest (Table 2). The data for ratios of measurements and for egg dimensions and number of adults in galls showed much variation (Table 2 and Figure 12, a, b).

The significance of the results in this section is discussed

together with the results of host-range and host-suitability studies which follow.

49.

Figure 15. The linear discriminant function for two groups of adult males

of each isolate , the significance value of F test is above

each comparison. p<0.001 K a) +95 II 111111111 +136 . U 11 I Aust. i III III 111 1111

P<0.001 UK b) +35 II 1111111 111 1 11111 1 I +62 USA i i iii if f ii i u II i

P<0.01 UK c) +39 111 J. 11 11111111 1 1 11 +51 Iraq 1 1111 11i1 111 1 1 111

p Aust d) -81 <0.001 II ll MI 111 IIII II ll -62 USA I I I I I I I 111111II1 I Il

P<0.001 e) -44 (II I 11111 11 III I I 23 Aust 1 1 1 111 1 111 111 1 III Iraq

50.

Figure 16. The linear discriminant function for two groups of adult females of each isolate. The significance value of F-test is above each comparison. p<0.001 UK a) +48 111 111 III 111 1111 I +125 I Ault' I I IIII IIII IIII I I I

p<0.o01 UK c) -6 +16 Iraq 111 1 I II III 111 . 1 1111 11 i

p<0.01 d) -5 II IIIItIIII) II I +7 Aust USA I I I II ll ll 1 I l l l I

p<0.001

1 1 11 Aust e) -82 1 11 111 111 11 11 1 j.43 lull i ii i 111 1 11 1 1u 1 Iraq

P<0.001

USA f) -46 I II I 11111 1111111 I I +2 Iraq I I III (~IIII~III II 51.

B. Host Range and Host_ Suitability Within Isolates of A. tritici

1. Introduction

The wheat gall nematode A. tritici has been reported as a pest

of nine cereals other than wheat. The host range may provide insight

into the geographical origins and interrelationships of isolates of

A. tritici and can be a factor of considerable importance in the breed-

ing of resistant cereals and, therefore, control of the nematodes.

The purpose of the present study is to investigate the host

range within modern cultivars and the wild ancestors of wheat, and

to study the effect of A. tritici isolates on them.

2. Materials and Methods.

A range of plants were tested for their ability to act as host

for A. tritici. Where these can be referred to collectively they

will be termed the host range.

Eleven Triticum and AegiZops species were tested for susceptibility

to invasion and parasitism by J2 of different isolates of A. tritici

(U.K., U.S.A., Iraqi, Australian and Indian). These Triticum and Aegiiops

species were selected by Dr. Victor Chapman, Plant Breeding Institute,

Maris Lane, Trumpington, Cambridge, England, to give a range of

characteristics.

Five galls of each isolate were mixed with 5 seeds of each member of the host range, germinated in John Innes No. 1 compost, in 3 inch diameter clay pots sealed at the bottom with wax. The wax seal was used to prevent J2 escaping from the pot. They were kept in a greenhouse for one month. In the following season Hordelymits europaeus was also tested as a host for all isolates of A. tritici using the same 52.

method. The Hordelymus europaeus was donated by Kew Botanical Gardens,

London.

The species and varieties (host range) were as follows:-

Species Variety Notes

1. Triticum boeoticum aegilopoides 11 Diploids?

it 11 12 Wild Einkorn,

tt tt 13 small, one

n n 15 row seeded

it It n 18 race.

2. Triticum boeotieum thaoudar 2 Diploids,

tt 11 It 3 Wild Einkorn,

11 t it 4 Larger, two row

t tt it 6 seeded race.

3. Triticum monococcum 6 Diploids, cultivated

it t 7 Einkorn.

it t 9

It t 10 18

4. Triticum dicoccoides 9 Tetraploids, Wild

tt n 10 Emmer.

11 1 IB 76

11 1 IB 110

tt 1 IB 113

5. Triticum dicoccum E24 Tetraploids,

It rt E26 Cultivated Emmer,

tt rt E27 two-grained wheat.

It tf E28

11 E29

53.

Species Variety Notes 6. Triticum durum Indian Runner Tetraploids,

~ n Kubanka Cultivated Emmen

• 1, Stewart

7. Triticum timopheevi Tetraploids, Cultivated Emmet

8. Triticum araraticum Tetraploids, Cultivated Emmet

9. Triticum polonicum Tetraploids,

Cultivated Emmer.

10.Aegilops cylindrica A Diploid, Wildgrass•

11.AegiZops squarrosa A Diploid., B Wildgrass•

,1 C

,1 It G

,1 It L

12.Hordelymus europaeus Diploid , Wildgrass.

After germination in a cool greenhouse all the plants were moved

to the field. Wax seals were removed and the pots were plunged in

gravel.

Each host was also artificially inoculated at the time of flower

induction.

The results were assessed by noting absence or presence of galls,

and their numbers and position on the spike,and analysed by different

techniques. For provision of data for use in taxonomic studies the 54.

weighted pair-group average linkage cluster analysis (Taxonomic

Distance Coefficient) was used.

The programme standardizes the data, if necessary, so that character

means are reduced to zero and variances to unity. A matrix of between

isolates (or between varieties) taxonomic distance coefficient is then

calculated and on this an average linkage cluster analysis is performed.

Isolates (or varieties) with the smallest distance between them are

first fused to form the nuclei of a cluster. Subsequent individuals

are added to existing clusters, which are then compared in terms of

the average distance between the members of the cluster and the new

individual. A full account of the programme with examples, is given by

David (1973).

3. Results

The frequency- of infections among the five plants of the 36 varieties of host tested (Table 3) showed that some hosts were heavily attacked while in others no gall formation at all was recorded.

The total number of 37 varieties were from 12 species of Tri'ticwn,

Aegilops and Hordelymus. The Iraqi isolate infected 12 varieties in seven species, the V.S.A. isolate infected 11 varieties in six species, the U.K. isolate infected 8 varieties in five species, 'the Indian isolate infected 5 varieties in three species and the Australian isolate infected only one variety in each of three species.

All the isolates infected at least one of the five varieties of the wild T. dicoccoides. The Iraqi isolate reproduced at low levels on 4 of the 5 varieties whereas other isolates reproduced only on one or two varieties, often in higher numbers (U.S.A. and U.K. isolates).

All the isolates except the U.K. isolate infected the first variety, 55.

Table 3. Number of galls produced from five plants of each variety of host range inoculated by soil or direct introduction with J2 of different isolates.

Host range Isolate Natural CPBI Species habitat No. No. Iraq Aust. USA U.K. India Total Triticum boeoticum Wild 11 1 eagiZopoides 12 2 13 3 3 3 15 4 18 5 1 1 T. b. thaoudar Wild 2 6 1 6 7 3 7 4 8 5 5 6 9 T. monococcum Cultivated 6 10 7 11 9 12 10 13 18 14 T. dicoccoides Wild 9 15 3 3 30 1 37 10 16 9 9 IB 76 17 2 2 IB :110 18 11 29 40 IB 113 19 2 1 3 T. dicoccum Wild 024 20 4 4 E26 21 2 2 E27 22 E28 23 E29 24 3 7 52 62 T. durum Cultivated Indian 25 runner Kubanka 26 5 48 1 54 Stewart 27 7 7 Aegilops squarrosa Wild A 28 2 s 3 4 9 B 29 2 2 4 C 30 1 3 4 G 31 8 2 10 L 32 5 7 12 Aegilops cylindrica Wild A 33 9 9 Triticwn poZonicum Cultivated 34 4 4 T. araraticwn Cultivated 35 _ 2 2 T. timopheevi Cultivated 36 One Hordelymus Wild 37 spike europaeus

Total 42 12 114 '56 66 290

CPBI = Cambridge Plant Breeding Institute. 56. while the Indian isolate alone infected the second variety. The wild

T. boeoticum aegiZopoides was infected by the U.K. and Iraqi isolates in low numbers. However, two of the five varieties were not infected at all. The wild T. boeoticum thaoudar was infected by the Iraqi and

U.S.A. isolates at low levels but two of the four varieties were not infected at all.

The cultivated T. dicoccum was not infected by the Australian isolate and prolific gall production occurred on only one variety infected by the Indian isolate.

The cultivated T. durum was not infected by the Australian isolate and also by the Indian isolate, whereas the Iraqi isolate infected two varieties and the U.S.A. isolate reproduced well on T. durum (variety

Kubanka).

The Aegilops squarrosa was infected by four isolates but none was a large infection. The U.S.A. isolate infected four varieties whereas the Australian isolate infected none.

The Aegilops cyZindrica was only infected by the Australian isolate, while the T. polonicum and T, araraticum were infected by the Iraqi and

U.S.A. isolates, respectively.

The T. monococcum of, which .five varieties were tested, and T. timopheevi were not infected by any isolate. Triticum dicoccoides variety No. 9 and variety No. IB 110 produced about 30 galls with the

U.S.A. and U.K. isolates respectively, while T. āicoccum variety No.

E29 and T. durum (variety Kubanka) produced about 50 galls with the

Indian and U.S.A. isolates, respectivēly.

However, the AegiZops cylindrica was the only host which produced many galls for Australian isolate (Table 3). The Hordelymus europaeus proved to be a good host for the Australian isolate. Results were not 57.

obtained for other isolates at the time of writing.

The position of the galls in the spikes varied according to the

isolate and host (Table 4), when the hosts were inoculated naturally

and artificially. Some isolates infected many spikelets in the same

floret in the same variety as for example in T. dicoccum variety No.

E29, where the Iraqi, U.K. and Indian isolates all infected spikelet

No. 4 floret No. 2. Some A. tritici isolates infected most spikelets

of one host, producing as many galls in the same spikelet as shown in

T. dicoccoides with the U.S.A. isolate.

Galls were formed generally on the spikelets between 2 to 8, but

most galls were produced between spikelets 2 to 5 (Table 5)..

Some isolates were infected in only one floret in one host as

the Iraqi isolates infected T. boeoticum aegiZopoides variety No. 18

in spikelet No. 1 floret No. 2, while the U.S.A. isolate infected the

whole of the spike as in T. dicoccoides Variety No. 9.

The Iraqi and U.K. isolates only shared infection of T. dicoccoides variety No. IB 110 and T. dicoccum variety No. E29 in about the same spikelets and florets (Table 4).

Four species appeared to be particularly suitable as hosts for

A. tritici. Of these the mostintense attack was that of the U.S.A. isolate on T. durum whereas the U.K. and U.S.A. isolates most heavily attacked T. dicoccoides and Aegilops squarrosa• The Indian isolate most heavily attacked T. dicoccum so caution must be exercised in interpreting this information.

The average linkage cluster analysis failed to cluster species of host on the basis of A. tritici parasitism (Figures 17 and 18). Table 4. An analysis of gall distribution by the position in spikelets and florets of host range infected by

A. tritici which produced galls, showing the actual position of the gall in the spike numbering from bottom to the top.

Triticum Triticum Species boeoticum boeoticum aegiZopoides thaoudar Triticum dicoccoides

CPBI No. 13 18 2 4 9 10 IB 76

• Spikelet 2 5 10 3 4 5 8 3 4 1 2 3` 4 5 ' 6 7 8 2 3 4 5 6 2 3 Floret 1 2 1 2 1 1 1 2 1 2 1. 2 2 Isolate 3 2 2 1 2 1 2 2 2 4 1 2 2 1 2 3 4 1 2 3 1 2 3 1 2 3 4 1 2 3 3 Iraqi 1 1 1 1 1 1 1

Australian 1 1 1

U. S. A. 2 1 2 1 2 1 1 1 1 1 2 1 1 3 3 2 3 1 3 1 3 1 1 1 2

U. K. 2 1

Indian 1 1 1 2 1 1 1 1 1

Total 2 1 1 2 1 2 1 1 2 1 1 1 1 1. 1 1 1 2 2 1 3 3 2 3 1 1 1 3 1 3 1 2 1 2 1 1 2 1 1 1 1 1 1 1

CPBI= Cambridge Plant Breeding Institute.

Continued.. Table 4. (Continued)

Species Triticum dicoccoides . Triticum dicoccum

CPBI No. IB 110 IB 113 E24 E26 E29

Spikelet 1 2 3 4 5 6 7 3 4 5 6 7 8 9 3 1 2 3 4 5 6 Floret sola 1 . 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 3 1 2 3 3 3 3 3 23 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3

Iraqi 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 kustralian

U. S. A. 1 1 1 1 1

U.K. 1 2 3 2 2 4 3 1 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Indian 1 1 1 1 3 2 2 2 3 3 2 2 3-3 3 2 3 3 2 2 2 2 2 2

Total 2 3 4 2 3 4 3 2 2 3 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 2 2 2 3 3 2 2 3 5 3 2 4 4 4 2 3 3 4 2

Continued..

U, • Table 4. (Continued)

Triticum durum Triticum Triticum Species polonicum araraticum

CPBI No. Kubanka Stewart

SPikelet 1 2 3 4 5 6 7 8 9 10 11 12 4 5 6 2 3 4 2 4 loret [so?ate 1 2 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 4 1 2 3 4 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 3 2 3 2 3 3 2 3 2 12

Iraqi 1 1 1 1 1 2 1 1 2 1 1 1 1 1

Australian

U.S. A. 2 1 2 2 1 2 2 1 2 2 1 2 2 1 2 2 1 . 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

I.T. K. 1

Indian

T otal 2 1 3 2 1 2 2 2 2 2 2 2 2 3 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 2 1 1 1 1 1 1 1

rn Continued... 0 Table 4. (Continued)

A c ylind Species AegiZops squarrosa yliriea

CPBI No. A B C Cl L A

Spikelet 2 3 4 2 3 4 2 4 3 4 5 6 . 3 4 6 7 8 2 4 5 Floret 2 1 2 1 1 2 1 1 2 3 2 3 1 2 Isolate 1 2 3 2 3 2 3 1 3 3 2 2 3 3 2 1 2 3 1 3

Iraqi 1 1 1

Australian 1 1 2 1 1 2 1 a U.S.A. 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1

U. K. 1 1 1 1 1 1 1 1 1 1 4

Indian 1 - 1 1 1

Total 1 1 1 2 1 2 1 1 1 1 1 2 1 1 3 1 1 1 2 1 1 2 2 2 1 1 4 1 1 2 1 1 2 1 62.

Table 5. Total number of galls formed in each spikelet of flowering head of host range.

Spikelet 1 2 3 4 5 6 7 8 9 10 11 12 13 Total Species

Triticum boeoticum 2 1 1 4 aegilopoides T. b. thaoudar 6 4 1 1 12

T. dicoccoides 3 15 18 20 15 9 8 3 91

T. dicoccum 4 9 12 13 .14 13 1 1 1 68 T. durum 3 6 6 8 9 10 4 3 3 3 3 2 1 61

T. potonicum 1 2 1 4

T. araraticum 1 1 2 AegiZops squarrosa 8 9 10 3 3 2 4 39

Aegilops cyZindrica 4 2 3 9

Total 10 46 53 59 46 35 15 12 4 4 3 2 1 290

3. 0643 2. 4359 . 4933 • . 5508 -0.0776 Triticum boeoticurn aegilopoides 13 Triticum dicoccum E 26 Aegilops squarrosa C Triticum boeoticum aegilopoides 18 Triticum .dicoccoides IB76 Triticum dicoccoides IB 113 Aegilops squarrosa A Triticurn boeoticum thaoudar 2 Aegilops squarrosa G Triticurn boeoticum thaoudar 4 Triticum dicoccum E 24 Aegilops squarrosa B Triticum araraticum Triticum dicoccoides 10 Aegilops squarrosa L Triticum durum Stewart Triticum polonicum Triticum dicoccoides 9 Triticum durum Kubanka Aegilops cylindrica A Triticum dicoccum E 29 Triticum dicoccoides 1B 110 3. 0643 2.4359 1.4933 0. 5508 -0.0776

Figure 17. Dendrogram to show relationships between ancestors of wheat varieties (22 varieties, data standardized)

using weighted pair—group method of average linkage cluster analysis and taxonomic distance coefficients. 1. 6634 1.511 b 1. 2838 1.0559 0.9800

IRAQI AUSTRALIAN INDIAN U. K. U. S. A.

1. 6634 1. 5116 1. 2838 1. 0559 0.9800 (Data standardized, 22 wheat varieties infected).

11.8605 10. 0125 7. 2405 4. 4685 2. 6205 I IRAQI AUSTRALIAN I U.K. INDIAN I 1 U.S.A. 11. 8605 10. 0125 7. 2405 4. 4685 2. 6205

(Data not standardized, 36 wheat varieties).

_ Figure 18. Dendrogram to show relationships between isolates of A. tritici, using weighted pair-group method

of average linkage cluster analysis and taxonomic distance coefficients. 65.

Similarly the relationships between isolates of A. tritici suggested by their.. host preferences appeared to be at variance with the results from the morphometric analysis in the previous section. 66.

C. Discussion

Despite the fact that J2, adult males and females of'all isolates

of A. tritici were grown under similar conditions (except the J2 from

galls grown in the country of origin), there were differences noted

when morphological measurements of different stages of A. tritici found

in literature from various parts of the world were compared (Bauer,

1823; Marcinowski, 1909; Byars, 1920; Goodey, 1932; Filipjev et al. 1941;

Thorne, 1961; Swarup at al., 1971; Paramonov, 1972). These differences

might be due to different methods used, the specimen ages (early stage

or late stage), the environmental effects and varieties of wheat cul-

tivated in the area.

Workers have not always agreed about the best methods of preparing nematodes for microscopical examination. This experiment was designed

to minimize the variations between the isolates of A. tritici, and all the five isolates were treated identically. This supports the findings of Stone (1971) on a standard method of processing Heterodera juveniles for identifying pathotypes.

Multivariate analysis was revealed as a useful means for separating all the geographical isolates of A. tritici. The analysis showed that when all characters are considered simultaneously in a multivariate analysis, certain isolates were always separated while others were not.

The Australian and U.S.A. isolates were always more closely related to each other than they were to the Iraqi and U.K. isolates which also formed a separate pair (Figure 1, 3, 4, 5, 6, 10, 11). The U.K. and

U.S.A. isolates were always separated as shown in Figures 6, 8, 10 and 11.

The pronounced linear relationship which is suggested by some of the data is difficult to account for, and does not seem to be capable of interpretation in a biological sense. It is known that principal component 67.

analysis sometimes produces a cluster pattern resembling an arc of

a circle (the"horse-shoe effect"), and it is possible that the

apparently linear scatter of points in Figures 2, 3, 4, and 5 is

connected with this statistical artefact. Lack of experience in the

interpretation of biometric data for nematode isolates of the kind

discussed here prevents further useful discussion.

The numerical methods chosen were not the only available ones,

and it is possible that alternative methods would have revealed some

features not brought out by the present techniques. Principal com-

ponent analysis is, however, a very well known and widely adopted method

of multivariate analysis, which makes very few assumptions about the

data, and is in some ways more flexible as a clustering technique than

the many hierarchical clustering methods that could have been used.

It also has the advantage of depicting better the relationship between

larger groups (perhaps at the expense of within-group structure),

whereas hierarchical methods have the converse property.

In the present case the within-group structure is of little or

no interest, and attention needs to be centred on differences between

the groups (i.e., isolates). The use of discriminant analysis (either

in the form of canonical variate analysis or the simple linear dis-

criminant function between two groups) again emphasises the interest

in distinguishing between groups, and also provides a method of describing

them in a way that reduces the overlap between them.

However, there were very significant differences in the measurements of J2 between isolates (Figures 6, 7, 8, and 14, j), The three-

dimensional model of canonical 'variates axes showed that all isolates

were almost entirely included within the variation shown by the Iraqi

and Indian isolates (Figure 7), The greatest differences occurred

between the U.K., Australian and U.S.A. isolates, whereas there was 68. much variation within and overlap between the Iraqi and Indian isolates.

This could be due to the fact that wheat was not native to U.S.A. and

Australia (New World), and the U.K. wheat was derived from the Old

World continents (Peterson, 1965). The Iraqi and Indian isolates of

A. tritici probably occupy a region near the centre of origin of wheat.

Therefore, the Iraqi isolate covered most the variation of the U.K. isolate as shown in Figures 6, 7 and 8, and the similarity between them, may reflect a common origin with the European wheat in the Med- iterranean region.

The other isolates of A. tritici came from various parts of the world. The results suggested that the Australian isolate was most similar to Iraqi and Indian isolates, again indicating possible origins.

The U.S.A. isolate was most related to the Indian isolate.

The J2 of A.tritiai-'were the most easily assessed -stage _because they are a relatively uniform developmental stage tolerant of many environmental conditions, which might have an effect directly or in- directly, on the morphology of the isolate. The influence of environment and host on the probable usefulness of these techniques was demonstrated by the results of the conformity of J2 measurements from galls grown in the country of origin with those produced in the U.K. under identical conditions. This showed that similar separation existed between the

U.S.A. and. U.K. isolates, except in the Australian isolate which overlapped with all other isolates when galls were produced in the U.K.

The galls which came from the hotter regions like India had many J2 and had thinner gall tissue surrounding them compared to galls from cold regions which had fewer J2 and thicker walls (Table 2 ). The temperatures at tropical regionmight be favourable to nematode as well as the host at certain times. The eggs hatch and the juveniles feed inside the galls, before the high temperature and dry season start 69.

affecting them, while in cool regions the temperature and drying

have less effect on the host and the nematodes., The adults may be

less affected by environmental conditions because they are covered by

plant tissue and they live for a short time. The effect of the host

on the adults is greater than environmental conditions,and the type

of host in the region could play an important part in the life-cycle

of the nematodes.

When wheat plants (Maris Dove) were inoculated with each isolate,

most isolates infected the host and formed galls except the Indian one,

which might be due to the above facts.

The adult males showed less significant differences between the

Iraqi and U.K. isolates because the Iraqi isolate overlapped more than

others, while the U.K. was well separated from U.S.A. and Australia.

This separation was also true for adult females._

The eggs showed very wide variation. The large eggs may have

been formed when the females had more nutrient however, and because

of this I consider that they are a poor morphom etric character .

The number of adult males and females inside the galls showed

less significant differences between the isolates. This depended

on the number of J2 present at the time of floral primordia initiation.

Nine new species were established in the host range of A. tritici,

as shown in Table 3, which consisted of the cultivated and wild wheats.

The hostswithin Einkōrn, Emmer and Dinkel groups have more or less the

same morphology, but differences in the physiology exist (Peterson, 1965). 70.

These differences may affect the degree of infestation of

A. tritici, possibly due to the stimulation of J2 by the host response.

The results of the study on host range indicated that A. tritici

of each isolate could invade a number of different hosts depending on

availability of the hostsin the region where these isolates were es-

tablished. Isolate of A. tritici showed morphometric differences, which

could be due to differences in infection, as shown in Table 3, and also might be due to the physiological differences in activity. Some species were infected by all isolates, e.g. T. dicoccoides, while some species were infected with only some isolates.

Triticum dicoccoides, T. dicoccum and T. durum are in the same host group, hybrids between them are fully interfertile and they have similar morphology (Peterson, 1965). They were invaded by J2 of all isolates except the Australian isolate. This could be because the

Australian isolate has been more isolated than all the others and also perhaps because these hosts are less related to wheat varieties grown in

Australia which form its usual host range.

As Triticum durum is widely cultivated in U.S.A., it is not surprising the U.S.A. isolate of A. tritici formed many more galls on this species than on all other isolates.

The Triticton dicoccoides is considered to be an ancestor of modern wheats and some of the characters of this species are therefore present in all other groups, Anguina tritici might be specifically stimulated to invade the host, depending on the concentration of these characteristics, which are present in any one host.

A high frequency of gall production 'meant that the host had a veryY highhi g responseP to stimulation bby J2, allowing them to invade the tissue. In contrast, the presence of one gall in the host meant 71.

that the host had very little response to nematode, or that only a

few J2 could potentially be present there. Further studies will be

needed to discover the nature of the factors affecting the J2 and the

host. Furthermore, the stimulation of different hosts might be de-

pendent on the number of J2 present and feeding at a certain time when

floral structures are formed. This may have an effect on the place

of the galls on the spike. Also the spike formed in each variety at a

different time, which depended on the physiological and environmental

condition of the host. This too may have an effect on the nematodes

in a position to infect the developing head.

Mumford (1961) recorded that T. monococewn as a host for A. tritici

in U.S.A. whereas galls were not formed on testing T. monococcwn varieties

in the present study. This might be due to the variety used by Mumford which may be susceptible and the tested varieties were more resistant

to A. tritici.

The Aegitops squarrosa was an ancestor of the common wheat and still

grows within the region of the origin of wheat (Sakamoto, 1973). It

is often found as a weed in wheat fields, and natural hybrids between wheat and Ae.squarrosa are sometimes found (Peterson, 1965). Thus some isolates of A. tritici could infect Ae. squarrosa because of the sim-

ilarity in physiology with wheat. But there is a need to test many more varieties of host and isolates before a decision on this can be reached,

This study of morphology and host range has called into question

the role of environmental and host factors in determining the host

status of different plant species.

In Section five factors influencing infectivity are discussed 72. after consideration of the histopathological responses and an analysis of the invasion process in Section two. 73.

SECTION IV

HISTOPATHOLOGY AND THE INFECTION PROCESS.

1. Introduction

Plant parasitic nematodes cause damage to plants either by mechanical breakdown of cell or as a result of substances secreted during feeding. Kirkpatrick, Van Gundy and Mai (1964) classed plant parasitic nematodes as either ectoparasites, semi-endoparasites or endoparasites. All these various groups can be further divided into

(a) migratory nematodes, which feed for a short time at one site and then move to another, and (b) sedentary nematodes which remain at a selected feeding site for a long period or permanently.

From the work of Marcinowski (1909), Filipjev at al. (1941) and more recently Midha at ai. (1971) (see literature review page 11 ), it is not yet entirely clear to which group A. tritici should be assigned.

The relationship between the nematode and its host has been the subject of interesting studies on the histopathological effects. Sev- eral workers reported that A. tritici infected various parts of the host, but it is not always clear which parts were involved in gall formations. There is very little information available about the way in which the J2 infects the host's tissues.

This study investigates the histopathological effect of A, tritici on different susceptible hosts in conjunction with observations on the invasion process. 74.

2. Materials and Methods

Various stages of the infected wheat and wheat ancestor from the previous experiments were kept for histopathological studies on sectioned material. Infected and healthy plants were fixed for 6 hours in F.A.A. Fixative, and dehydrated by passing through alcohol series, 4 hours in each dilution of 50%, 70%, 95% and two changes in absolute alcohol. Then they were transferred to 100% butanol for

8 hours (two changes) and for embedding were transferred to 50 : 50 butanol/xylene (4 hours) and then to pure xylene (2 changes, 4 hours each). They were then transferred to 50 : 50 xylenelparaplast (M.P.

56-57°C) on a hot plate and left for 4 hours, and transferred to 100% (R) paraplast on a hot plate (2 changes, 4 hours each). A paraplast block was prepared in a paper boat, 12p sections were cut using a Reichert(R)

Rotary Microtome. The sections were placed on glass slides smeared with glycerine albumen as an adhesive, floated on a drop of water and flattened by keeping the slides on hot plates at about 50°C for 12 hours or overnight. The slides were passed through 2 changes of xylene

(5 minutes) and the sections rehydrated by passing through descending alcohol series (absolute alcohol.2 minutes; 95%, 70%, 50% and 30% alcohol, 2 minutes each),and stained with haematoxylin (Ehrlich) acid

(7 minutes); washed in running tap water (10 minutes), dehydrated again in ascending alcohol series (50%, 70%, 95% alcohol and absolute alcohol, 2 changes, 3 minutes each). The slides were then transferred to Xylene (2 changes, 4 minutes each), and mounted with Canada Balsam beneath a cover slip,

The results were assessed by examination of the sections. 75.

Results

Although nematodes could be observed around the developing spike for some time (many weeks) prior to infection (Figures 19 and 20; see next section for details), the first signs of invasion were seen after floral induction in the growing point. Immediately before invasion the primordia structures were stimulated to increase in size rapidly and were then invaded, in a matter of 2 - 1 day (Figures 21 and

22).

One or more second stage juveniles invaded the tissue (Figures

23, 24, 25, 26, 27 and 28). They formed a tunnel-like hole apparently between the cells, through which they entered the host tissues (Figures

29, 30, 31, 32 and 33) and began development.

The young infected spike was smaller and wider than the healthy one (Figures 34 and 35).

The infected tissues were bigger, deformed and deeper green in colour than healthy ones (Figures 36, 37, 38, 40 and 42). The second stage juveniles invaded the primordia of either one stamen only

(usually on the outer anther, Figures 38 and 39), the gynoecium (ovary) only (Figures 40 and 41), or both, to form the galls (Figures 42, 43, 44 and 45). These were invaded unequally in the florets of both Triticum and Aegilops species.

The uninfected parts in the same floret showed a decrease in growth either in the anthers, ovary or in both (Figures 21, 22, 38 and 42).

Those florets further up a spikelet than an infected floret were weaker and produced no grain, but those below were unaffected.

Alternatively, when all the florets in the spikelet were infected, galls were sometimes found in the whole spikelet (Figures 46 and 47); Figure 19. Cross section through the floret showing the second stage

juveniles around the floral tissue before infection takes

place (400X)

n = second stage juveniles.

Figure 20. Longitudinal section through the floret showing the J2

around the floral tissue before infection takes place (400x)

n = second stage juveniles. 76. Figure 21. Infected ovary showing increase in size, an entry hole

at the top, and a reduction in growth of other parts (400X)

h = invasion hole

o = Infected ovary primordium

a = anther primordia

Figure 22. Longitudinal section through the spikelet showing increase

in size, the infected anther and all the other parts normal (120X).

a = swelling indicating an infected anther (outer anther).

1 = lemma

o = ovary

p = palea

g = glume

r = rachilla 77. Figure 23. One second stage juvenile during invasion of the anther (400X)

a = anther

n = second stage juvenile

Figure 24. Spikelet showing three second stage juveniles invading

the ovary (400X)

n = three J2 half way into the ovary

g = glume

Figure 25. Longitudinal section through the floret showing one J2

during invasion, with an increase in size of the anther (400X)

n = second stage juvenile

a = anther

o = ovary

Figure 26. Longitudinal section through the floret showing two J2

invading the anther (400X)

n = second stage juveniles

a = anther

• -• •

• I • !rfi • ... • • So • •.. . .11 •••I.., . :• 1(.....' .•••. • • : •, f •4 • • VI 4. ,.,.•

V . •j •` ..1 I. 1"4:;(r •l" • Figure 27. Transverse section through the ovary showing three J2

invading the ovary-with others inside the ovary (400X)

n = second stage juveniles

ni = nematodes inside the ovary (different stages)

o = infected ovary

p = palea

1 = lemma

Figure 28. Transverse section through the ovary during invasion showing

J2 inside the ovary and one in half way (400X)

n = second stage juvenile in half way inside the ovary. 80.

• .. • s • r • • •

` •

• e • •

~• ~'y , • • • y • ••~ •• `;•♦ 1 ~• . • Figure 29. Longitudinal section through the floret showing the entry

hole in the anther (400X)

h = entry hole

a = anther

g = glume

I = lemma

p = palea

Figure 30. Cross section through the anther showing the entry hole.

The cells around the hole are intact (1600X).

h = entry hole. , _ A * 0

lf • • V . . •

♦ • • • * • • j

AO II: Ile 6i

Pi •a• ••• ••

• •• t

• Figure 31. Longitudinal section through entry hole showing intact cells

around the hole, J2 inside the anther. (400X). h = tunnel-like hole n = nematodes inside the anther (different stages).

a = infected anther

o = uninfected ovary

p = palea

I = lemma

g = glume

Figure 32. Cross section through the middle of the hole, showing

intact cells around the hole (400X)

h = entry hole

Figure 33. Longitudinal section through the hole illustrated in

Figure 31 showing the intact cells around the hole and the

shadow of the background of the hole appears intact as

well (1600X).

th = entry hole - like tunnel.

n = nematodes inside the anther (different stages). 83 . Figure 34. Healthy spike showing normal distribution of the

spikelets (120X)

Figure 35. Infected spike showing increase in width and decrease in

length (160X). 84. Figure 36. Healthy anthers and ovary at an early stage of development

(160X).

a = anther

o = ovary ps = primordium of the stigma

Figure 37. Healthy well developed anthers and ovary as anthesis

is approached (120X).

a = anther

o = ovary

fs = feathery stigma ul 00 Figure 38. Galling formed in the outer anther only (120X)

is = infected anther (outer anther)

a = uninfected anther

uno = uninfected ovary

fs = feathery stigma

ra = rachilla

Figure 39. Cross section through spikelet showing the gall in the

outer anther only all the other parts uninfected (120X)

is = infected anther

n = nematodes inside the anther (different stages)

a = uninfected anther

o = uninfected ovary

g = glume

p = palea

1 = lemma

ra = rachilla ra

-'\ •I pi iktr 14r Figure 40. Gall produced in the ovary all the other parts uninfected

(120X)

io= infected ovary

a = uninfected anther

ra = rachilla

Figure 41. Cross section through the spikelet showing the nematodes

inside the ovary, and the ovary bigger than the other parts

(120X)

io = infected ovary

n = nematodes inside the ovary (different stages)

o = uninfected ovary

g = glume

p = palea 1 = lemma

a = anther

ra = rachilla

Figure 42. Galls formed in the ovary and outer anther showing great

increase in size compared with the uninfected anther (120X).

ag = anther gall (outer anther)

og = ovary gall

a = uninfected anther

Figure 43. Galls produced in the ovary and outer anther showing increase

in size and the nematodes inside them. (120X).

ag = anther gall (outer anther)

og = ovary gall

'n = nematodes inside the ovary and outer anther (different stages)

ra = rachilla Q Figure 44. Longitudinal section through the floret showing joint

anther and ovary gall (160X)

n = nematodes inside the galls (different stages)

1 = lemma

p = palea

g = glume

r = rachilla

Figure 45. Longitudinal section through the floret showing anther gall

and ovary gall joint in the base (160X).

a = anther gall

o = ovary gall 89.

s.(het.* 2., • II t • ■

•

• i r: '~i•~1 r •

. ., .Y

i N'Ik lk '''' I ' . -....f,...-.... ,... 44

• L "•I• • ..n • ..

• 4

• Figure 46. Cross section through spikelet showing both florets in

the spikelet infected, gall in each form in outer anther

and ovary (120X)

n = nematodes inside the galls (different stages)

p = palea

1 = lemma

Figure 47. Cross section through spikelet showing the ovaries only infected (120X). io = infected ovary

a = anther

1 = lemma

p = palea

ra = rachilla

g = glume g0, 91. this is clearly observed in rye (Figure 48). This did not affect

the other spikelets, for each spikelet was independent of the other in the matter of gall formation.

Only rarely was an anther gall and seed found in the same

floret (Figure 49). Figure 48. Rye, showing the outer anther and the ovary. All spikelets

contained galls (120K)

og = ovary gall

ag = anther gall

a = uninfected anther (lateral anther)

ra = rachilla

Figure 49. Anther gall attached to a healthy seed in the same

floret (120X)

ag = outer anther gall

s = healthy seed. zog

ra 93.

4. Discussion will be Timing of invasion/related to development of flowering parts at the growing point. Prior to this time ectoparasitic, after stimulation of tissues. the J2 becomes endoparasitic before it begins development,and therefore the J2 should properly be designated as an endoparasite.

Observation from histopathological studies revealed that the degree of host response might be dependent on the number of J2 of A. tritici presences at any floral site. This is in agreement with the observation of Midha et al. (1974),who showed that the size of the developing galls varied greatly and was directly proportional to the number of adults present in the galls.

Investigators differ as to the origin and nature of the tissue constituting the flower gall. Filipjev et al. (1941); Midha et al.

(1974) showed that staminate tissue is mostly involved in the formation of the gall, while the dead ovary was generally seen attached to the gall later. Haberlandt (1877); Al-Sabie (1977) considered the galls to be of ovarian origin. Marcinowski (1909) thought that compound galls are formed by the fusion of single galls, depending upon the degree of maturity of the flower when infected, and galls can be formed from either an undifferentiated flower bud, staminate tissue, carpellate

tissue, tissue from between stamens or between stamens and carpels.

However, Thorne (1961) thought that juveniles entered the embryo seed

to form gall. The present results showed clearly that the primordia tissue of either outer anther, ovary or both were only involved to form the

gall in the varieties used in this study (Figures 38, 39, 40, 41, 42

and 43).

The J2 could only invade these tissues, and entered between 94.

apparently undamaged cells (Figures 31, 32 and 33). The cross-and longitudinal-sections showed clearly the entry hole and no disruptions in the cells where one or more juveniles entered the tissue (Figures

25, 26, 27, 28, 31, 32 and 33).

Barnard (1954) observed that, in the normal growth of wheat, the two lateral stamen primordia usually arise first, followed by those of the carpel (ovary) and anterior stamen primordia (outer anther).

No galls were formed on the lateral stamens (inner anthers) which arose first.

The J2 were observed feeding ectoparasitically at the growing point by Midha et al. (1971), and an effect on the host physiology due to changes in enzyme activity in different parts of the host were noted by Horovitz et al. (1969).

Interactions between the plant and the feeding J2 are only possible after the initiation of the ovary primordium and it may be suggested that the J2 responds to this by producing its own chemical which in turn induces swelling of host tissue. Nematodes may then be able to enter the responding tissue, easily separating cells to create the entry tube. Other juveniles often appear to join an initial juvenile in penetration, but Figures 31, 32 and 33 show no evidence of cell damage around the entry hole.

Galls formed at either ovary, outer anther or both. One gall formed when the J2 invaded either ovary or outer anther primordia

alone, while two galls formed when J2 invaded both ovary and outer anther

(Figures 38, 40 and 42).

When the J2 entered the tissue, they fed inside the infected

tissue and the host supplied these tissues with more nutrient. Thus 95. infection is due to changes in the structure and physiology of infected tissue. The uninfected parts of the floret were weaker, perhaps because they lacked nutrients. The uninfected florets above an infected floret in the same spikelet were also weaker and produced no grain because most of the nutrient had gone to the infected floret acting as a nutrient sink, while the uninfected florets below an infected floret usually grew normally. When an anther gall and seed formed in the same floret the nutrients must have been shared between them.

The results can be related with the resumption of nematode growth and development as was detailed by Al-Sabie (1977). The gonad primordia of the J2 rapidly begins to elongate and moulting to J3 and J4 takes place within 4 - 6 days and mature adults are present after 6 days.

This confirms the already well-known synchronization of host and parasite development in this species. 96.

SECTION V

FACTORS AFFECTING THE INFECTION PROCESS

1. Introduction

Many observations have been made on Anguina tritici infections

(see Literature Review and previous section). But a proper understanding of the host status of a plant may be affected by the various factors operating at the time nematode infection is imminent.

Experiments described in this section investigated the effect of different host ages, temperatures, numbers of inoculated J2 and the time of year at which inoculations were carried out. These included observations on an unclassified species of Anguina sp . from

Holcus mollis for comparison with A. tritici,(li.,:, isolate ).

2. Inoculation Before the Double Ridge Stage a. _ Materials and Methods

Wheat c.v. Maris Dove at the vegetative stage and shortly before double ridge initiation stage (Code number 32, Tattman and Makepeace,

1979) was inoculated artificially with J2 and kept at a temperature range of either 10 - 15°C or 16 - 20°C in a greenhouse.

The results were assessed to show the temperature effect on J2 and the plant. b. Results

Significant gall formation only occurred when J2 infected the host (penetrated swollen host tissues) at temperatures between 10 and

15°C. When the infested plants (plants with nematodes at or near potential sites of entry and gall formation) were kept at high temperature

(16 - 20°C), they showed no sign of an ability of the J2 to infect the 97.

host and no galls formed. The spikes of each plant grew rapidly and

developed faster and emerged from booting stage within one month.

When these plants were dissected, most of the J2 were found alive at the base of the head (next to the flag leaf node).

3. Inoculation During and After Flower Induction. a. Materials and Methods

Wheat c.v. Maris Dove was inoculated artificially with J2 during and after flower induction (determined by dissection of the growing point) and kept at 15+2°C in a greenhouse.

The results were assessed to show the effect of inoculum time on the host. b. Results

When the host was inoculated during flower induction at temperatures between 10 - 15°0, many J2 infected the host and galls were formed, but only after at least 24 days had elapsed from the time of inoculation. This period is termed the "latent period" and is referred to subsequently as such (see below and Figure 52). However, no infection was noticed when the J2 were inoculated after floral induction. They were again found in the base of the flowering head next to the node of the stem bearing the flag leaf.

4. Inoculation Throughout the Year Using Successive Generations. a. Materials and Methods.

Wheat c.v. Maris Dove was inocualted artificially with J2 during flower induction when the brown galls were formed (and before desiccation).

The J2 were extracted from the galls then concentrated by settling and removing excess water, These J2 were injected (inoculated) into another new wheat plant timed to be at the stage of flower induction, and so on for 98.

each new generation for one year.

The results were assessed to find how many generations per year.

b. Results

Five generations of A. tritici were obtained in the course of

a year (Table 6). The duration of each generation varied, depending

on the season. The shortest generation time took 60 days under field

conditions in summer, while the longest generation time was 87 days under field and greenhouse conditions, until brown galls were formed in autumn. The galls in winter were smaller in size (first and fifth generation), than at other seasons (Figure 50). The number of

J2 inside the winter galls were. 1249 (mean of 10 galls), while more than 3000 J2 were formed in galls from other seasons.

5. The Density of Artificial Inoculum.- a. Materials and Methods Second stage juveniles from 90 galls of each of 4 isolates (U.K., U.S.A., Iraqi and Australian). were allowed to revive. Each suspension was concentrated to give about 2500 J2 per 0.01 ml. Eight density levels were used (10,000, 5,000, 1,000, 500, 100, 50, 10 and 5) J2 per plant. Doses of 10,000 and 5,000 nematodes were given as 0.04 ml and 0.02 ml aliquots of the stock suspension. This was then pro- gressively diluted to give the lower doses.

Only one variety of wheat (c.v. Maris Dove) was used. Forty— eight plants (3 plant per pot) were inoculated with each dose. Half the plants (8 pots) were kept in field conditions, and the other half were kept at 15°C in a constant temperature room with 16/8 hours light/ dark cycle, supplied by white fluorescent tubes.

The results were assessed by counting the number and the position 99.

21 Seeds I • • %

• ,• Galls

M • Winter e d411' Galls + I 0 +

Figure 50. Comparison between the healthy seeds and different

galls. 100.

Table 6. The generation times of Anguina tritici throughout the year.

Generation Time Duration From To Days

First 25 November 1977 — 15 February 1978 83 Gh Second 16 February 1978 — 27 April 1978 71 Gh Third 28 April 1978 — 29 June 1978 63 Gh -)F Fourth 30 June 1978 — 28 August 1978 60 F Fifth 29 August 1978 — 23 November 1978 87 F 4 Gh

G = Greenhouse

F = Field. 101.

of green galls on the spikes at flower emergence. b. Results

Results were only obtained from field experiments, as all the plants which were kept at 15°C in a constant temperature room died earlier, due to a heavy attack by powdery mildew.

The number and the position of the galls varied among the density levels and isolates, for the plants; which were kept under field conditions.

At the density level giving highest infection for each nematode isolate, the galls were well distributed among the spikelets, while in the other densities, the galls were accumulated more or less in the middle of the spikes. Many galls were produced on spikelet number 7, with all isolates (Table 7).

The infected plants produced many galls at density levels between

100 to 1,000• second stage juveniles per flowering head depending on the isolate, while the Iraqi isolate produced as many galls as all the other isolates together. Few galls were produced at a density of 10,000

J2 (Figure 51). There were similar numbers of galls produced with

Iraqi and U.K. isolates at densities 50 and 5,000 J2,'while the similarity in the number of galls produced with U.S.A. and Australian isolates was at densities of 5, 10 and 100 J2 per head.

The galls were dissected before desiccation in the infected plant at density levels 5 to 50 and in some galls only one adult

(either male or female) was found, but a female in such a gall produced no eggs.

In order to compare the numbers of galls formed as a result of various inoculum densities in the different"isolates a Kolmogorov- 102 Table 7. Number and Position of the galls formed on 24 wheat c.v. Maris Dove plants for each isolate under each-density inoculum.

Isolate , UK USA ens 10 50 100 500 1000 5000 , 10000 Total 5 10 50 ,100 500 1000 '5000. 10000 Total Spikelet flore 1ō ulum 5 - ...... 1 1 1 1 2 1 1 - 4 1 1 1 3 2 2 1 1 1 1 ' 1 2 3 1 3 1 5 ' 4 1 1 2 N 3 1 1 • 1 1 2 3 2 10 N 2 2 1 1 1 2 7 1 2 2 7 3 1 2 3 2 8 2 2 4 1 1 4 1 1 1 3 2 3 2 12 2 1 1 2 4 6 14 1 2 2 3 2 10 3 5 3 2 7 17 2 2 4 1 1 . 2 5 1 1 1 1 4 3' 2 10 2 2 4 3 3 2 14 1 1 1 2 2 4 3 1 15 3 1 4 4 3 7 19 1 4 1 1 1 8 4 2 3 1 4 10 5 1 1 6 1 1 1 2 2 4 3 2 11 2 1 3 2 2 2 10 3 3 3 1 10 3 1 3 2 4 4 1 15 1 6 1 2 2 12 4 1 2 2 4 1 10 1 1 5 1 1 1 1 7 1 1 1 2 1 1 1 4 3 1 11 2 2 2 3 5 1 13 1 1 4 3 1 10 3 4 5 7 4 20 6 1 3 1 11 4 2 3 3 2 10 1 3 1 5 5 1 1 1 3 6 1 I 2 3 5 2 - 1 2 2 3 8 1 3 3 1 8 3 6 2 6 14 2 1 2 1 6 4 2 1 4 1 8 6 6 5 1 2 3 9 1 1 1 1 1 1 1 4 2 2 1 2 1 6 1 1 1 1 1 5 3 4 3 4 11 4 2 2 4 8 5• 5 10 2 3 2 5 1 1 3 4 2 6 3 3 . 4 2 2' 4 3 3 11 3 3 1 4 . 4 4 4 1 1 1 1 5 1 1 12 1 1 1 1 1 2 1 ,• 1 1 1 3 1 1 4 4 4 1 1 13 1 1 1 2 1 1 3 1 1 4 1 1 14 1 1 1 3 1 1 4 1 1 Total 8 7 11 75 42 52 77 5 277 3 4 6 21 67 41 47 29 218

Continued 103 Table 7. (Continued).

I Isolate IRAQ AUSTRALIA Spikelet 5 10 50 100; 500 1000 5000 10000 Total 5 ,10 150 100 500 1000 5000 10000 Total flore[ aity ~1uin 1 1 2 2 4 2 2 2 1 1 2 2 1 1 1 4 2 1 9 1 1 3 5 2 3 2 5 1 2 3 3 2 1 3 3 1 4 6 5 3 2 20 3 3 4 1 11 2 2 6 4 2 2 16 2 3 5 3 2 2 4 1 1 4 7 6 3 2 '23 2 2 4 1 9 2 • 4 5 5 3 2 19 2 4 1 7 3 4 2 1 7 2 2 5 1 1 4 8 6 4 3 26 2' 4 1 1 8 2 2 4 8 6 4 4 28 1 2 2 1 4 1 11 3 1 6 6 2 15 3 3 4 4 1 5 2 1 3 6 1 1 5 8 6 6 3 29 1 5 2 8 2 3 8 5 5 1 22 1 1 4 2 8 3 • 5 5 2 12 1 3 1 5 4 1 2 3 I 4 1 5 .

7 1 1 2 1N 8 7 7 2 32 1 1 2 4 2! 10 2 1 8 8 • 4 23 1 4 2. 7 I -1

3 1 6 5 3 16 1 2 2 4 1 1 11 4 4 1 1 6 2 2 4 8 1 2' 8 5 4 19 2 5 1 8 2 2 3 8 5 5 1 24 3 5 2 10 3 5 4 1 10 1 3 1 5 4 5 1 6 2 2 4 9 1 1 8 2 3 2 16 • 5 5 2 1 6 1 5 1 14 3 1: 4 3 5 2 7 1 1 4 4 1 5 1 1 2 10 1 6 3 1' 10 2 2_ 4 2 1 1 1 6 2 1 1 13 2 1 1 2 6 3 4 3 7 1 1 4 1 1 11 1 1 4 2 1 8 1 1 2 2 5 3 1 9 1 1 2 3 4 2 6 1 1 4 1 - 1 12 1 1 4 1 2 8 2 1 3 2 4 1 5 - 1 1 3 4 1' 5 1 1 4 1 1 13 1 4 1 5 1 1 2 4 4 1 1 3 1 1 1 1 2 14 1 3 3 1 1 2 4 4 1 1- 3 1 1 15 1 2 2 1 1 2 2 2 1 1 1 16 1 1 1 I 1 1 2 1 1 1 1 17 1 1 1 I I • 1 1 Total 2 3 13 47 233 119 80 29 526 2 6 17 23 141 112 21 4 199

. i

104. Figure 51, Number of galls formed on 24 plants for each isolate

under each density inoculum.

80 0 0 60 0 U.K. 40 0

20 0 H 0 0 0 1 _, 0 5 10 50 100 500 1000 5000 10000 Density of inoculum z 80 0 w 60 a U.S.A. 40 0 0 u 20 0

0 10 50 100 500 1000 5000 10000 Density of inoculum x 120 0

AUSTRALIA 80

0 40

20 O 0 O 0

G ,.a 0 5 10 50 100 500 1000 5000 10000 Density of inoculum a 240 0

0 0

10 50 100 500 1000 5000 10000 Density of inoculum 105.

Smirnov test was employed (Appendix 2). This provides a test of whether the distribution for any isolate at various inoculum densities differs significantly from the corresponding distribution for any other isolate. As 4 isolates were involved, there are 6 possible comparisons, all but one of which proved the differences to be statistically significant at probability levels of P < 0.001.

The remaining comparison (between the U.S.A. and Iraqi isolates) showed a significant difference at P < 0.01.

6. Transfer of Inoculum From One Plant to Another During the Period of Inoculation (Investigation of the latent period). a. Materials and Methods

A first set of 90 wheat plant c.v. Maris Dove were inoculated artificially with J2 during flower induction. Ten days after inoculation and during the period before invasion, 60 infested plants of this first set were dissected to extract the J2, while 30 remained as controls. The extracted J were concentrated and inoculated 2 into a second set of 50 uninfested plants one week younger than those in the first set from which the J2 had been removed. Starting at twelve days after inoculation, two plants of the re-inoculated second set were dissected every two days until infection was recorded. Controls of the first set were similarly dissected until infection was recorded. b. Results

Infection of the remaining wheat plant of the first set (controls) was first recorded 24 days after inoculation. Eleven of the 18 remaining controls were found to be infected at this time.

There-inoculated J2 developing on the second set of wheat plants to which they had been transferred after 10 days after exposure to plants of the first set, produced an infection only after 22 days on plants of the second set. A total of 31 plants of the 40 second set plants 106. remaining at 22 days post re-inoculation were infected.

These results are illustrated diagramatically in Figure 52.

7. Inoculation of Wheat with Anguina sp .from Creeping Soft

Grass and Inoculation of Creeping Soft Grass with A. tritici. a. Material and Methods

Two methods were used to infest the wheat and creeping soft grass (Holcus mollis L). The creeping soft grass is found growing naturally around Ashurst Lodge, but some uninfested grass was also donated by Kew Gardens, London.

1. Indirect inoculation

Seeds or seedlings of both plant species were inoculated by mixing the J2 and galls either A. tritici or Anguina sp . with the wet soil (John Innes compost No. 1), in bottom sealed clay pots.

2. Direct inoculation

At flower induction, plants of both species were'inoculated artificially with J2 of either Anguina tritici or Anguina sp . In both methods, there were controls in which the plants were inoculated with their own specific nematodes. The plants were kept either in a greenhouse or in the field.

The result was assessed to find the ability of J2 to invade the new host. b. Results

When wheat c.v. Maris Dove was inoculated with Anguina sp:. from HoZcus mollis (creeping soft grass), and creeping soft grass was inoculated with Anguina tritici, the results in both cases of reciprocal inoculations were negative as no galls were formed on either "host".

However, the juveniles in natural and artificial infested plants were still alive and accumulated in the bases of the flag leaf in both plants 107.

Figure 52. Diagram showing the inoculation of J2 during the

latent period

*------LATENT PERIOD FIRST SET

HOST 1 - 55 days old galling, initiated of further nematode .90 plants 30 plants development

J2 extractedt12 plants dissected no infection Inoculated 1000 J2 from 60 recorded plants . j

90 plants 18 plants (11 infected) day J2 inoculated 10; 24 351

SECOND SET

500 J2 Re—inoculated (from first set of 10 plants plants) HOST 2 - dissected 55 days old

50 plants 40 plants day J2 inoculated 01 I. f 22(31 infected)35 108.

when the spikes appeared.

8. Galls Produced on Naturally Infested Host Under Field Conditions

a. Materials and Methods.

This experiment investigated the number of galls produced on

naturally infested hosts under field conditions. Two hosts (wheat

c.v. Maris Dove and Rye c.v. Grazing) were used for U.K. isolate and

only one host (wheat c.v. Maris Dove) was used for Iraqi isolate

The seeds of each host were sown together with 50 galls of each isolate

tested in a randomized block design in the field during the Spring

of 1979.

After formation of the spike by each host, 9 different plants

from each host were selected at random and examined in order to

determine the number and position of the galls on the spike.

b. Results

The result in Table 8 showed that the U.K. isolate produced

more galls on wheat than the Iraqi isolate. The U.K. isolate infected

wheat better than rye because more galls were produced on the former

than latter. When the U.K. isolate infected wheat, more galls were

produced on spikelets 3 to 8, while the Iraqi isolate produced more

galls on spikelets 5 to 7. On rye, the U.K. isolate produced more

galls on spikelets 2 to 6, and the only florets in each spikelet

infected were the first and the second floret.

9. Discussion

Inoculated juveniles are capable of invading the host tissue

only when the temperature is below 15°C. This is supported by the

observations under natural conditions of Filipjev et al. (1941); Kort

(1964), who showed that cool weather was important to J2 for invading ,ikelet 1 2 3 4 5 6 7 8 9 10 11 12 Total

)ret 1 2 1 2 3 1 2 3 1 2 3 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 1 2 3 1 2 3 1 2. 3 1 2 3 A- ris Dove 2 1 4 3 1 5 5 1 5 5 2 6 7 3 1 7 6 2 1 7 6 2 1 6 6 3 4 3 1 2 1 1 2 1 1 1 1 1 117 C. isolate

Ir is Dove 1 1 1 1 1 1 1 1 1 2 2 1 2 2 1 1 2 1 23 3qi isolate

Z plant 3 4 6 4 7 6 8 6 6 4 7 2 2 2 3 1 3 1 2 77 Z. isolate

Table 8. Number of galls of A. tritici (U.K. and Iraqi isolates) in individual spikelets and florets of wheat and rye

heads grown under field conditions and infected naturally from galls in the soil. 110.

the tissue.

Friend et al.- (1963) pointed out that the rate of morphological development of floral primordia of wheat was more rapid at higher temperatures. This might be the reason preventing the J2 from invading the floral primordia. Thus most J2 remained at the base of the flag leaf node, while the spike rapidly developed beyond the stage suitable for nematode invasion.

The observation of invading J2 showed that they could only invade 0 the tissue after .a period of 24 days from inoculation at 10 - 15 C, termed the latent period. The temperature decreased the growth rate of developing primordia. This would give more chance for J2 to accumulate on the young spike, and also might result in a physiological change in the nematode and plant during the period of 24 days at low temperature.

Horovitz et al. (1969) found that infestation of wheat with A. tritici caused marked changes in enzyme activity and this change was related to the development of the nematode. . The lack of invasion by J2 observed when the plant was inoculated after the period of flower induction might be due to the fact that outer anther and ovary primordia had already developed beyond the stage suitable for invasion.

The use of artificial inoculation in the present study proved a successful method of getting many generations per year, whereas under natural conditions there could only be one, or possible two, annual generations of A. tritici (Table 6).

The variation in the temperature and intensity, quality and duration of light might be the reason for longer generation time and smaller galls in winter (Figure 50). This may be due to physiological conditions in the plant during winter time, while all favourable growth factors are present in summer. It was observed that when juveniles were transferred from plant

to plant during the latent period slightly less time was taken to

invade the second set of host plants. This may be due to the different

growth rates between the plants, and the fact that the floral primordial

becomes available to the J2 when environmental conditions are variable from day to day.

The period of the growth for wheat plants varies from one region

to another as summarized in Figure 53. The Anguina tritici life cycle

is synchronized with the host in the regions of wheat growing in the

world. The duration of life cycle of the nematode and the host in

the semi-tropical of Mediterranean region is shorter than in the

temperate region due to the differences in the effects on the host of

the environmental conditions. The period of floral induction also varied, requiring a longer time to form in temperate regions. This period is very important for A. tritici to invade the host tissue.

Artificial inoculation of wheat plants showed that the infective stage needed an association with the flower primordia of at least 24 days (the latent period) for galling to be initiated. Development of A. tritici was faster in the semi-tropical region while its life cycle in temperate regions was slower because of the effect of low temperature on the growth of the host and nematodes.

The observation on final numbers of galls produced in different densities of juvenile inoculum per wheat head under field conditions confirmed that few galls formed at densities of 5,000 and 10,000 J2 per head. This resulted. in infection of the spikes with bacteria and fungus and consequently killed some wheat heads. Also the high density levels of J2 affected and upset growth of the head due to presence and competition in feeding there of J2. Moreover, some J2 112.

WIN I LP SPRING

n pee in.;

1, • .1 '!... !

Ihe gall

AUTUMN SUMMER

Figure 53. Diagramatic representation of the natural life cycle of

Anguina tritici showing invasion period, on different

wheat host growing in different regions. 113. were dead because they were present in the very small space around the head.

The maximum number of galls were reached at inoculation densities between 100 and 1,000 of J2 per head. This is dependent on the actual feeding rate of J2 and their presence in the right place in the spike.

However, when few nematodes inoculated, fewer galls were produced.

Perhaps more than one nematode may be needed to stimulate floral primordia to give the stimulus leading to structural change in the host.

The Iraqi isolate produced as many galls as all other isolates put

together; perhaps the J2 of this isolate can give a higher stimulus being native to the region in which Tritiewn species and related genera evolved.

It was observed that the-U.K.. isolate produce more galls on natural infested wheat than the Iraqi isolate, suggesting that the U.K. isolate is better adapted to environmental conditions and host in U.K.

than the imported Iraqi isolate. The fact that the U.K. isolate infects wheat more than rye suggest the possibility of host specificity to A. tritici.

Bonnett (1936) observed that the spikelet initials showed the

greatest elongation in the middle of the spike, while the terminal

spikelets were'the last to differentiate. This agrees with the observation of the greatest number of galls being produced in the middle of

the spike. Juveniles could invade the middle spikelet first while the

remaining J2 invaded the terminal ones (Table 7-8).

The food supply apparently had no effect on the sex ratio because

in some galls only one adult of either sex was present. Probably the

sex was determined by genetic factors. This point could be studied further. 114.

The females in such a gall produced no eggs. Probably because the female was young and unfertilized. Triantaphyllou et al.(1966) observed that development of the eggs proceeded following fertilization.

It is clear that wheat is adversely affected by invasion of

A. tritici. However, attempts at cross infection between the Anguina sp . from Hoicus mollis and A. tritici from triticum failed, suggesting a measure of host specificity among grass-galling Anguina species. 115.

SECTION VI

SURVIVAL OF SECOND STAGE JUVENILES UNDER DIFFERENT CONDITIONS

1. Introduction

It is known that some of the J2 of A. tritici can survive in the gall in dry conditions for as many as 38 years (Thorne, 1961), but less is known of survival of active juveniles in soil. When galls are introduced into moist soil, either during harvesting or planting, water is imbibed, and surviving juveniles became active.

Experiments were designed, therefore, to examine the survival of active J2 of A. tritici in more detail, particularly in soil with and without the host. Experiments were also designed to evaluate temperature effects during storage and the survival in air saturated water.

2. Survival of J2 under field conditions in the absence of the host. a. Materials and Methods.

The apparatus to study the survival of J2 under field conditions consisted of a polyvinylchloride (pvc) tube of diameter 2cm and a total length of 35cm filled with field soil, as shown in the diagram (Figure

54).

The soil to fill the tube was sieved (metal sieve, 355 microns aperture) and sterilized by autoclaving. Two nylon meshes.HD10 (heavy duty 10 microns aperture, Simon Precision Textiles, Henry Simon Limited,

Stockport, Cheshire, England), were inserted at 25cm and 30cm to prevent migration of J2 from the tube into the surrounding soil. These meshes were inserted by sandwiching them between two pieces of the tube which were glued together using solvent cement (Hunter Plastics Industries

Limited, catalogue number SC 953), The bottom 5cm of the tube (containing 116.

Figure 54. Diagram showing the apparatus for survival of J2 in the soil

pvc_ tube

25cm

35cm

HD 10 nylon mesh 5cm

Adapter I- HD 10 nylon 5cm mesh 5mm

Cork

Filter paper 117.

the second mesh), could be taken apart to check the soil between these

two meshes. The joint between these tubes was made up with a third

tube just large enough to make a tight fit•as shown in the diagram

(adaptor). The bottom end of the tube was plugged by a cork with a 5mm

diameter hole. A filter paper was stuck to the end of this cork with 21 a moisture resistant glue (PritI . This tube was buried vertically

in the soil leaving lcm above the soil surface. Thus, this apparatus

enabled the soil in it to be maintained under similar conditions to

that in the surrounding soil.

Standardization of the galls.

The galls were standardized by sieving to select uniform size and

approximate weight. Two sieves of aperture size 2.8mm and 2.3mm were

used to select out large and small galls.

The experiment was designed to show the survival of J2 in field cōnditions after the growing season of the host is complete, and the galls are shed in the soil and have remained in the absence of the host. In natural conditions this period occurs in autumn and winter.

The experiment was conducted between November 1978 to July 1979. Five standard galls were tied in anylon gauze and buried 1 - 2cm deep in the soil within the tube. Twelve tubes were prepared this way and two tubes sampled every 75 days for 450 days.

The total numbers of living and dead J2 from the galls and soil, in the above described tubes, was counted. The galls were washed and dissected to release the juveniles. These were then stained in 0.1%

Phloxine B (Fenner, 1962) to pick out the dead ones which were stained pink, from the living ones which remained clear. The J2 from the soil were extracted by sugar centrifuge flotation (Caveness and

Jensen, 1955). These juveniles were also stained with Phloxine B.

Active juveniles were separated by using 84p sieve. 118.

The floral primordia of wheat c.v. Maris Dove were inoculated with the extracted J2 from the soil and the galls.

The total numbers of living and dead J2 inside the gall and the soil were extracted and counted. The presence or absence of.gall on the plants was also noted. b. Results

When the galls were recovered from the soil periodically, the number of J2 inside the gall dropped dramatically from February to the end of

June. The number of J alive in the soil increased sharply from February 2 to mid-April (Figure 55). However, the total of the number of J2 inside the gall and those alive in the soil decreased and all the juveniles were dead by mid-September in the absence of the host. By the end of

June most J2 were found in the soil, while the few J2 then remaining inside the gall had disappeared by mid-September (Figure 55).

When the J2 were extracted from the soil and galls, they were inoculated artificially onto wheat plants c.v. Maris Dove at the floral induction stage. Few galls were present on the spikes in February, but many galls appeared on the spikes with inoculations carried out in

April and June.

3. Survival of J2 under field conditions in the presence of the host a. Materials and Methods.

This experiment was designed to show the effect of the host on the gall as would be caused when the galls are sown with the seeds in spring under natural conditions (May to November,-1979). The experiment is similar to the previous experiment except that a spring wheat seed was also sown at the same time when the galls were inserted in the tube.

Two tubes were sampled every two weeks until the death of the plant and then every 75 days for 174 days. In addition to counting the J2 in the 119.

Figure 55. Survival of J2 of A. tritici placed in the soil in winter

under field conditions but without the host.

5x104 KEY Total J2 inside the gall — Total J2 in soil. Total J2 alive in soil

104 ) le sca

ic 3 hm 10 it r a (log

iles en juv

e tag s

on 2 10 sec f o ber Num

10 r I I N DJ FMAMJ JASON TIME (MONTH) 120.

galls and the soil, the plants were also dissected and any juveniles found recorded. Any galls formed on the plants were counted and then dissected, to record the number of males and females. The results were expressed as in experiment No. 2. b. Results

When galls were recovered from the tube soil periodically, the

were extracted from the galls, soil and wheat plant. The number of

J2 inside the gall decreased sharply from June to the end of August due to dry conditions in July 1979. The number of J2 in the soil of the tube increased from June to mid-July and dropped again from mid-August.

By mid-November, all the J2 in the soil were dead, while the J2 were inactive inside the gall (Figure 56) and were not further studied.

Many J2 were found in the growing wheat planted in the tube during the period of floral induction under natural conditions. The greatest number of J2 were present in the plant and in the soil by mid-July. Few J2 infested the plant after gall formation.

The number of males and females 03, J4 and adults) varied from spikelet to spikelet (Table 9). At the end of July, the maximum number of males was 26 at spikelet number 5, while the greatest number of females was 48 in spikelet number 7, By mid-August, however, the host had more spikelets, the maximum number of males was 39 at spikelet 6, while many females were present at spikelet 5 and 6. Few nematodes were found above spikelet 8 and below spikelet 4.

In general, for both times, most nematodes were present. in Spikelets number 5 and 7 (Table 9).

The sex ratio (females:males) was bigger in the galls at the end of July than at mid-August, but varied,

121. Figure 56. Survival of J2 of A. tritici placed in the soil with the

host in spring under field conditions.

4 2x10

4 10 ) le sca ic • hm it

r 3 KEY a I 1 10 - Total J2 inside the / gall. (log — Total J outside the 1 2 all (soil) ' Total J in the host

iles 2 - but still remaining. l ectoparasitic /

Juven

e 1 I 1 tag •: 1 s d J 1 I i ~ 1 secon

f 2

o 1p. - I ber I i I Num t. _ I \ 1 1 I t I 1 i 7 i \....:.... 1 [ i 1 ND JFMAMJ J ASOND

TIME (MONTH) 122.

Table 9. Number of Males and Females in two different plants and times.

Spikelet Time 4 5 6 7 8 Top Total Sex (>8)

End of Male 13 26 15 18 17 6 95 July

55 days Female 41 39 36 48. 19 10 193

Mid-August Male 10 12 39 10 7 2 80

84 days Female 5 49 47 32 9 1 143

Total 69 126 137 108 52 19 511

Females Sex ratio Males

Sex ratio (end of July) = 193 - 2.03

143 - 1.79 Sex ratio (mid-August) = 80

Sex ratio (overall) = 1.92 123.

4. Survival of J2 in different media with temperature and their

subsequent infectivity. a. Materials and Methods.

The survival of J2 from A. tritici (U.K. isolate) and Anguina sp.:. from creeping soft grass (Hoicus mollis L.) was studied. The juveniles from 20 galls were allowed to revive and active juveniles were separated.

In order to obtain consistent aliquats of juveniles from the stock suspension, an apparatus was constructed to keep the juveniles uniformly suspended. This consisted of a funnel containing 200 ml of distilled water-through the, bottom of which air was bubbled through an air stone ( connected to an aquarium pump (Rena "super"). One ml of suspension was removed by wide mouth pippette, placed in a plastic petridish (5cm diameter) and the juveniles counted. This was repeated 45 times and the mean calculated. The results showed very low variability (standard error = 0.746).

1 ml of nematode suspension was placed on:-

1. 10 ml of water agar (1.5%) at 33°C before it solidified.

2. 10 ml of water agar (1.5%) at 33°C before it solidified with antibiotics (Streptomycin sulphate (Sigma) at 1 mg/ml and Penicillin-G

(Sigma) at 1,000 units/ml, in autoclaved agar added before it set), and

3. 25 grams of moist soil.

20 petridishes of each different media were kept at 5°C, 15°C and

25°C (altogether 60 petridishes per treatment). After 75 days interval, juveniles were extracted from 4 petridishes from each treatment and temperature. For complete extraction, Whitehead's tray method followed by sugar centrifugal flotation was used.

The results were assessed by counting the active nematodes, and 124.

by inoculating the floral primordia to measure their infectivity

(viability) to form galls after each interval of 75 days for 375 days. b. Results

The J2 of Anguina tritici survived better after 75 days at 5°C and 15°C in water agar and soil than in water agar containing antibiotic

(Table 10). After 150 days, the highest percentage of J2 survived at 5°C for all media, while 25°C gave lowest survival.

The J2 could not survive more than 225 days, in soil at 15°C and

25°Cpbut at 25°C,in either water agar or water agar with antibiotic, they could not survive more than 300 days. The percentages of J2 surviving decreased sharply after 150 days.

All the treatments showed similar curves except at 25°C in soil

(Figure 57), where it showed a rapid decrease in the surviving number of J2.

The J2 of Anguina sp . (from creeping soft grass) showed different survival ability, depending on the treatments. They survived better than

Anguina tritici at 5°C and 15°C which were the most favourable temperatures for all treatments (Table 11). The J2 of Anguina sp. survived longer than 375 days at 15 C for all treatments, while the J2 in water agar with antibiotic showed the highest percentage survival at 375 days (Figure

58). All the J2 were dead after 300 days at 25°C in either water agar or water agar with antibiotic except the J2 in soil which were dead after

225 days (Table 11), the shortest survival period for this Anguina sp.

(Figure 58).

Differences between treatments in both species had a similar significance at probability level P= 0,001 in either time, media, temperatures or in the interactions time x media, time x temperatures, media x temperatures or times x media x temperatures. (Appendix 3). 125.

Table 10. Percentage of live J2 of A. tritici recovered at various times following incubation under different conditions.

Treatment Temperature Time (days) oC 75 150 225 330 375

5 88.29 70.57 1.00 0.97 0.47

Water 15 92.04 62.89 1.27 0.60 0.34 agar

25 72.27 62.30 0.03 0 0

5 71.85 61.43 1.75 1.42 0.98

Water 15 73.79 43.45 1.41 0.70 0.54 agar with 25 75.22 36.71 0.03 0 0 antibiotic -- 5 87.91 61.33 0.73 0.63 0.17

Soil 15 81.25 50.27 0 0 0

25 59.36 5.06 0 0 0

Mean initial numbers used in each treatment 750 (see Figure 57). 800 KEY 700 - L75C° water agar Hā z 15C° water agar 625C° water agar 600 - 15C° water agar + Antibiotic w >E i(15C° water agar + Antibiotic c5u 500- Q - 25C° water agar + Antibiotic 0 soil z 15C0 soil Li 400 - w 71 25C° soil

~- 300 -

= 200 -

• 100- O 7 "

150 200 250 300 350 400 TIME (ORY5 )

(U.K. isolate) on different media and at different temperatures. Figure 57. Survival of J2 of A. tritici 127.

Table 11. Percentage of live J2 of Anguind sp . from Hoicus mol Us surviving on different media at various temperatures with

time.

Treatment Temperature Time (days) o C 75 150 225 300 375

5 92.84 61.68 37.67 6.09 1.31 Water agar 15 86.42 71.18 71.10 30.20 18.69

25 83.83 52.04 7.51 1.02 0

5 79.98 64.71 50.93 17.65 4.88

Water agar 15 88.37 70.70 63.05 55.98 37.85 with Antft, otic 25 60.91 33.89 1.47 0.21 0

5 87.11 57.90 6.78 4.53 0.37

Soil 15 51.51 29.31 11.70 10.27 9.45

25 34.44 1.75 0.45 0 0

Mean initial numbers used in each treatment 620 (see Figure 58). KEY D L75C° water agar 015C° water agar H 60U '25C° water agar 4 I SC° water agar + Antibiotic Kl5C° water agar + Antibiotic 500 Lu .025C° water agar + Antibiotic

(r) R 50.5C° soil 0 400 2:25C° soil z U W tn 300

> 200 cn

100 - 0

50 100 150 200 250 300 350 400 TIME (DRYS)

on different media and at various temperatures. Figure 58. Survival of J2 of Angitina sp.. from Holeus mo Uis 129.

The extracted J2 of either A. tritici or Anguina sp . from all treatments could form galls after 225 days when the wheat c.v. Maris

Dove at the floral induction stage was inoculated artificially.

Most cultures were contaminated with fungi, unidentified mites and bacterial colonies.

5. Survival of second stage juveniles in air saturated water a. Materials and Methods.

A known number of J2 from each different isolate were kept agitated in 100 ml of distilled water in a conical flask. The agitation was provided by a continuous supply of air from a Rena 'super" aquarium pumP .

The results were assessed each week, by counting the live J2, using biological stain (Phloxin B 1%), which stained the dead nematodes pink. b. Results

The percentage of J2 showed a significant difference in response to air saturated water with time for each isolate. Most of the J2 were dead between 28 and 35 days (Figure 59) depending on the isolates, except that those from the U.S.A. survived until 42 days (Appendix 4).

The U.S.A. isolate survived best throughout, while the Indian isolate survived worst (Figure 59). The Australian isolate showed an average. 50% survival for three weeks continuously, being somewhat similar to the U.S.A. pattern. The U.K. and Iraqi isolates rapidly lost 'viability as did the Anguina sp . from creeping soft grass. 100 -

KEY 80 O UK 7 70 ID IRAQ } p 0 AUSTRALIA GO USA INDIA N X )< 50 O d UK ( grass). ti o 40

z 30 Ise 20

10-

45 40 10 15 30 35 TIME 20FtYS 1 25

Figure 59. Survival of J2 of different isolates of Anguina tritici and Anguina sp . from Holcus mollis in water

bubbled with air. 131.

6. Discussion

Observations on the effects of increasing the number of juveniles in the soil, provide evidence of relationships between the soil moisture and A. tritici. Some conclusions about the nature of this relationship can be drawn from these experiments.

After the seed has been harvested, the galls fall to the soil.

Fielding (1951) showed that the J2 remained in a desiccated stage inside the gall while the soil was dried. However, the rainy season affected the soil moisture as well as the gall. The galls which had imbibed water and the juveniles revived, more or less active, depending on the soil temperature.

The rate of emergence of juveniles is dependent on the decomposition of the galls in the soil. The rate of decomposition of the galls depends on the soil ecology (Marcinowski, 1909; Leukel,--1957)..--This may explain why the juveniles take such a long time to emerge from the gall in winter (Figure 55). While the galls were decomposing, the number of J2 increased in the soil as J2 were still active. As the soil temperature increased to mid-April, most of the J2 werefound in the soil. The J2 remained active and many were found outside the galls in moist soil until the end of June. Dry season conditions after July 1979 caused a rapid death of all juveniles then in the soil. 'Many J2 died in the soil and inside the gall due to fungus attack and lost their ability for survival.

The number of J2 increased faster in the soil when the seeds and the galls were sown together in spring (Figure 56), probably due to faster gall decomposition which may affect the emergence of the J2 from the galls.

The growth of wheat in spring and summer of 1979.was very good 132:

compared to spring/summer 1980. The J2 normally infest the plant at tillering stage (Midha et aZ., 1972; Al-Sabie, 1977). The number of J2 increased in the host gradually, while many J2 were in the soil.

The number of J2 reached the maximum in the host before floral induction in the wheat, but after the spike formation, there were very few J2 in the host (ectoparasitic). This may be due to the inability of some juveniles to invade the floral primordia soon enough.

The observation showing large numbers of J2 in the soil emphasises that relatively few J2 were able to infest the host. Perhaps because they had reduced host finding and penetrating abilities.

In the second experiment, all the J2 were in the tube as well as the soil, but few J2 infested the host and most of the others were still in the soil. Midha et aZ. (1972) found that no galls were formed until 1000 juveniles per _1000_g soil were present. However, they showed that the marginal juvenile level in the soil for initiation of plant, infection appeared to be 10,000 juveniles or 2 galls per 1000 g soil. The only reason for this might be that many J2 lost their ability to infest the host. The effect of the dry 1979 season was to kill the active

J2 in the galls as well as in the soil.

From the observation on the effect of constant temperatures and

different media on the survival of the J2 (Tables 10 and 11), it appeared

that few J2 could infect the floral primordia after they have been active for 225 days. The J2 in the soil were unable to infest the host, but

when they were placed on the floral primordia they could invade, causing

gall formation as shown in two different experiments.

When the J2 were inoculated artificially after they had been

active for 300 days, no infection occurred. This might be due to the

low numbers inoculated and also due to the J2 losing their viability 133.

during the long active period.

Most adult males and females of A, tritici were found in spikelets

5 to 7 (Table 9), which confirmed previous observation. The number of galls produced depended on the number of inoculated J2 as was shown in the previous section. The J2 of the U.K. isolate inoculated in

February gave fewer galls than in June. This fact may be due to the actual number of J2 present and the unseasonal time of invasion which was used.

The survival of active J2 was affected by different factors such as temperatures and media. Anguina tritici and Anguina sp . could not survive well at 25°C. This might be because the food reserves inside the body are used up quickly. The 5°C and 15°C were the best temperatures for both species, perhaps being in the normal range of soil temperatures.

Mites and fungus growth were found in all treatments. These might be the direct cause of the decrease in the number of the J2, while the media had an indirect effect on the J2. Many organisms were present in the soil treatment and the effect of these organisms is likely to be dependent on the temperature. The water agar and water agar with antibiotic limited the fungus growth, but more mites were present.

The percentage of J2 of A. tritici surviving was less than the

Anguina sp . from Holcus 7noliis, possibly because A. tritici could not infest the host after a certain period as mentioned above and survival in an active state for a long time is probably without survival value in the life cycle of this nematode, In contrast, Anguina sp has adapted to survival for a long period because its host is a perennial grass. The active J2 of Anguina sp . could infect the rhizome immediately after they emerged from the gall. The creeping soft grass is adapted 134.

to growth in temperate regions (Hubbard,1978),•-and this may explain why.Anguina sp . survived better than A. tritici.

Nematode movement in the soil is very slow and little energy is spent on movement (Lee and Atkinson, 1976). In contrast, survival of

J2 in air saturated water was affected quickly. Thus the continuous movement by bubbling air through the suspension might have affected

the J2 by either causing physiological changes in the nematodes during

enforced activity or causing the food reserves to be exhausted faster

than would normally occur.

As isolates differed in their ability to survive continuous air

bubbling it may be supposed that this reflected differences in their metabolism.

Some nematodes can survive anaerobiosis e.g. Meloidogyne naasi

(Ogunfowora, 1978), ApheZenchus avenae (Cooper and Van Gundy, 1970),

but the literature contains no reference to this ability in J2 of

A. tritici. This topic is further investigated in the next section

dealing with respiration in A. tritici juveniles. 135.

SECTION VII

THE OXYGEN CONSUMPTION OF A. TRITICI OF J2- OF DIFFERENT ISOLATES

1. Introduction

A preliminary and comparative study of the respiration of J2 from single and mixed galls of different isolates was made as one possible approach to the understanding of the physiological basis of variation within the species. The effect of temperature on oxygen consumption was also investigated.

Toxic chemicals are commonly used to investigate the metabolic chemistry of organisms and polarographic assay provides a simple method for studying oxygen consumption under the influence of the toxic substances.

In this respect, the third experiment was concerned with the effect of aqueous cyanide concentrations on different J2 of A. tritici isolates.

Cyanide was used to reveal the type of respiration and therefore the versatility of its response to unfavourable (potentially anaerobic) conditions.

2. Oxygen consumption of J2 from single and mixed galls of the U.K.

and Iraqi isolates. a. Materials and Methods

Oxygen consumption apparatus

The rate of 02 uptake was measured by a polarographic method, using a Rank Oxygen electrode and all assays were done at constant 20°C, unless otherwise stated. The incubation chamber was rinsed five times after each treatment and washed out by a miniature nozzle connected to an aspirator; the inner wall of the chamber was kept clean.

The oxygen electrode consists of platinum wire sealed in perspex over which is overlaid a thin Teflon membrane. The platinum wire corresponds to the cathode. A silver rim placed in the periphery of a 136.

shallow depression around the slightly elevated platinum electrode represents the anode. The latter is kept immersed in saturated KC1; to prevent the Ag electrode from drying out. A circular band of thin R) single ply 1{leenex tissue is placed on the Ag rim which is thus kept well moist by a layer of saturated KC1.

When a D.C. potential is applied (0.6v) across the two electrodes, which are in direct; contact through the Teflon membrane with an oxy- genated solution, 02 diffuses through the thin porous Teflon membrane and on arrival at the negative Pt electrodes undergoes an electrolytic change at its surface:

02 + 2e + 2H+ H2 02

H2 02 + 2e + 2H -+ 2H20

Thus the oxygen concentration of the solution is directly proportional to the weak but significant electric current thus generated which when moderately amplified can be measured on a sensitive galvanometer.

A single pen recorder was used in conjunction with Rank, Oxygen. electrodes. The potentiometer range of which was from 0 - 1.0 WV, The running rate of the paper on which a pen trace was recorded was 1.25cm/nin.

Further details can be obtained from the Rank Oxygen operating Instruction

Manual and from Eastbrook and Pullman, 1967, Methods in Enzymology Vol. 10 pages 42 - 45.

Working

After setting up the electrodes in the manner described; 1 ml distilled water was dispensed into the incubation chamber and stirred by means of a magnetic stirrer acting on a miniature glass coated "Flea" which was placed within the incubation chamber. The double jacketed incubation chamber was maintained at 20°C by constantly recirculated 137.

water thermostatically controlled. The air saturated state i.e. when the gaseous 02 has equilibrated with dissolved 02 in the 1 ml of water in the incubation chamber, a linear trace was obtained. At this point the trace was adjusted to 0.9 mv. Thus a base line was obtained corresponding to the quantity of 02 which dissolved in 1 ml of distilled water. The horizontal linear trace indicated that the 1 ml of distilled water was saturated with air at 20°C and 760 mm Hg': atmospheric pressure, for which the 02 concentration was calculated from the International critical tables. The base line was checked between runs.

The second stage juveniles from a single gall and from 20 mixed galls were allowed to revive at 20°C for one hour before use. Active juveniles were washed several times with distilled water in order to wash off all traces of debris and Corynebacterium tritici (Hutchinson).

A suspension of worms was made, which was used in all estimations of

02 uptake. Three replicates and four separate measurements were used for each isolate. Twenty and sixteen measurements were used for the U.K. and Iraqi isolates respectively with mixed galls.

One millilitre of J2 suspension was added to the well cleaned incubation chamber and sealed with the perspex plug. In sealing the chamber, no air bubbles were allowed to be trapped beneath the plug as bubbles in the chamber solution give distorted results.

The recording was concluded when a trace with a steady slope was obtained (usually 15 — 20 minutes). In order to obtain the dry weight of the nematodes, the nematodes were carefully pipetted out in suspension followed by three rinses and dispensed into preweighed small foil crucibles

4cm '.x 2.5 cm diameter (made out by impressing tin foil into small glass crucible) which were oven dried at 45°C in a desiccator containing phosphorus pentoxide (P202) for three days, the difference in weight 138.

taken as the dry weight in milligrams.

The results were assessed to find if there were any differences

between the single and the mixed galls of each isolate.

b. Results

The rates of 02 consumption for nematodes from a single gall

showed that there were little if any differences between galls of each isolate (Figure 60). However, the nematodes of each isolate differed

from each other in the rates of 02 consumptions. The rates of 02 con-

sumption remained at about the same level for the nematodes from each

of the three single galls of the U.K. isolate, except for one which at 25

hours decreased the rate of 02 uptake.

The J2 of the Iraqi isolate started to consume 02 at a level much

lower than that of the U.K. isolate. The rates of 02 consumption in-

creased sharply until 25 hours after revival for the J2 for all the

three replicates of the Iraqi isolate. This level was maintained for

50 hours.

The F-test showed there were significant differences between

isolates with time. Also the t-test showed significant results between

replicates at time one and two, while the t-test was less significant

at time three and four (Appendix 5).

When nematodes from a number of galls were mixed, the rates of

02 consumption was greater for U.K. isolate after 20 hours, while the Iraqi isolate remained almost unchanged until 80 hours (Figure 61).

Only one measurement decreased after 72 hours and 122 for U.K. and Iraqi

isolates, respectively, where the U.K. isolate decrease was greater

than that of the Iraqi. isolate. Erratic readings were occasionally

attributable to recorder malfunction. The 02 uptake decreased for U.K.

isolate after 160 hours to the same level of 02 uptake as Iraqi isolate

after 126 hours.

N. MI /

T. N RY D MG / ON TI P M SU N O C EN G S OXY E MOL CRO I 0 1 M 0 10 15 20 25 30 35 40 45 5G TIME (HOURS)

Figure 60. Oxygen consumption at 20°C of three single galls of Anguina tritici isolate. • Z

IRAQ

20 40 60 80 100 . 120 140 160 180 200 TIME ( HOURS 1 .

o. Figure 61. Oxygen consumption at 20 C of mixed ga11 of Anguina tritici of two isolates. 141.

3. Oxygen consumption at different temperatures of J2 from mixed

galls of each isolate. a. Materials and Methods

The J2 from old galls of each isolate (U.K., Iraqi, U.S.A., Indian) and for each temperature at 5, 10, 15, 20, 25 or 30°C, were allowed to revive 24 hours before use. Active juveniles were separated by using

84p sieves overnight. The J2 were washed as previously (in experiment

2.a).

All assays were done in the Rank Oxygen electrode thermostatically regulated to constant temperature at 5, 10, 15, 20, 25 or 30°C for three replicates of each isolate as in the experiment 2.a.

The horizontal linear trace indicated that the 1 ml of distilled water was saturated with air at 5, 10, 15, 20, 25 or 30°C and 760 mm Hg atmospheric pressure, for which the 02 concentration was calculated from the International critical tables. Base line was checked between treatment. The results were assessed to find the oxygen consumption of the juveniles at different temperatures for each isolate, b. Results

The rates of 02 consumption differed from one isolate to another depending on the temperatures ('Figure 62). The U.S.A. isolate gave the highest 02 uptake at 5°C, while the Iraqi and -U.K. isolates gave the lowest 02 uptake. The 02 uptake decreased for U.S.A. and Indian isolates at 10°C, while the others increased (Figure 62). At 15°C, the U.S.A. isolate reached the maximum 02 uptake, while the Indian and

U.K. isolates increased their rates of 02 uptake sharply, but the Iraqi remained. unchanged. The U.S.A. dropped sharply at 20°C and gradually to 30°C. The U.K. had a small decrease at 20°C but increased again to reach the maximum 02 consumption at 25°C. The Indian isolate continued to increase gradually in 02 uptake until 25°C and dropped at 30°C. 142.

Figure 62. Oxygen consumption of second stage juveniles of Anguina tritici

of different isolates at different temperatures.

4.0

3.5

3.0

2.5

4-1 3

b 2.0 ov

N ° 1.5 0

O 3-' 1.0 u 1

0.5

0.0 0 5 10 15 20 25 30 Temperature °C 143.

The Iraqi isolate reached the maximum 02 uptake at 20°C and decreased at 25°C and 30°C.

. The 02 uptakesfor Iraqi and U.K. isolates were the lowest at 5°C to 15°C while the U.S.A. isolate was almost the highest at these temperatures (Figure 62 and Appendix 6).

4. The effect of different concentrations of aqueous NaCN on the

Oxygen consumption of second stage juveniles of Anguina tritici. a. Materials and Methods

The J2 from 15 galls of each isolate were suspended in distilled water and divided to six equal portions.

Each portion of J2 was adjusted to give an average 10 mg wet weight per ml. and kept at 5, 10, 15, 20, 25 or 30°C for 24 hours.

1 ml of the J2 suspension was then allowed to equilibrate and sealed with a perspex plug. The test cyanide solution was dispensed into the incubation chamber via a capillary port in the perspex plug by using micro syringe. In sealing the chamber no air bubbles were allowed to be trapped beneath the plug. Measurements were made as in experiment

2.a, 3.a.

The cyanide (NaCN) was made as follows:

A stock solution of 0.5 M NaCN was kept during each treatment.

20p1 of this stock solution was added to 1 ml of test suspension which produced within the chamber a cyanide concentration of 10.E M; _3 41 of 0.5 ji NaCN was dispensed when a 10 M NaCN solution was required; -4 0.2p1 of 0.5.M NaCN when a 10 11 NaCN concentration was required in the chamber. For the purpose of accuracy after the J2 were dispensed into the chamber the recording for the controls were obtained for 10 - 15 minutes. The same suspension was then resaturated and 0.2p1 of 0,5 M 144.

NaCN was added. The different slope was noted which corresponds to -4 the rate of 02 uptake in 10 M NaCN. The same procedure was used with

, other concentrations for each isolate and temperature. The results

were assesed to find the 02 uptake of the J2 in different concentrations

of NaCN at different temperatures.

b. Results

The NaCN has a lethal effect on the J2 of all isolates; after

4 minutes the 02 consumption stopped for all isolates at all concentrations. -3 The J2 were dead within 4 minutes in 10-2 and 10 M NaCN, but survived -4 for 15 minutes at 10 M NaCN.

5. Discussion

In the present studies a temperature of 20°C was employed since

this is about the normal environmental condition under which wheat

grows (Peterson, 1965).

There were only small differences in the rate of 02 consumption

between the J2 from different single galls of the same isolate. These

differences in the U.K. isolate might be due to the fluctuations in some

readings obtained from the Random Block Design which were a result

of recorder failure. The stability reading of the Iraqi isolate showed

that there were no differences between the single galls of the same

isolate. Possibly, this might be because each isolate is exposed

to similar environmental and physiological influence.

The initially high rate of respiration of the U.K. isolate after

hydration of juveniles might indicate that either the U.K. isolate

revived faster or the metabolic rate of the J2 increased faster as their

tissues absorbed more water. The J2 from Iraqi isolate took a longer

time to reach the highest level of respiration (Figure 60). This may

be due to the effect of the way the galls and the juveniles are initially

desiccated. 145.

However, the rates of 02 consumption of the two isolates were less significantly different after 25 hours. Possibly most of the J2 of both isolates may recover their activity and resume their respiration normally.

The J2 of mixed galls showed different respiration from the single gall. When all the J2 of each isolate were placed in water they could not revive immediately. This might be because the Oxygen in the water was not enough for the respiration of all -the._J2. Those of the U.K.-isolate increased their respiration sharply after 20 hours

(Figure 61). This might be due to the activity of the J2, since as they moved near the surface of the water there might be a tendency for them to agitate the water surface to increase the 02 dissolving in the water. A fall in the respiration of those in the Iraqi isolate until

78 hours, might possibly be due to less metabolic activity. Both isolates might switch on their metabolism to another physiological activity thereby increasing their respiration after 78 hours (Figure 61).

These observations agree with those of Bhatt et al. (1970) who earlier on studied the respiration of A. tritici. All these changes in respiration within the species might be due to different physiological activity switched on by different sugar metabolism levels (Evans pers. comm.). The changes in the sugar level affected the respiration rate of oxygen uptake in the J2 during different times. The sugar levels might be varied between the isolates due to environmental and host interaction.

The abnormal measurements, like the ones at 72 and 125 hours, with

U.K. and Iraqi isolates respectively, might be because of recorder failure.

Such variable results on the 02 'uptake of A. tritici of different 146. isolateswere obtained, perhaps because of the effect of temperature on the respiration of each isolate. The results (Figure 62) agree with the findings-of-Bhatt et al. (1970) who reported that the rate of most biological processes, including respiration, increased with an increase in temperature, reaching a maximum, beyond which the rate of activity tended to decline. They also showed that the op-

timum temperature for respiration varied with species. In their estimation of oxygen uptake by ApheZenchus avenae,Cooper and Van Gundy

(1970) quoted figures of 5.6 -5.8p1 02/mg dry wt./hr. which differed from findings by Awan (1975) who noted figures of 4.89 - 4.64u1 02/mg dry wt./hr. These changes in the respiration within species of ApheZenchus avenae were due to physiological differences between them.

Awan (1975) used 10 mg of nematodes in suspension and got good responses. The same amounts of J2 of A. tritici were used in the -4 cyanide experiment, but the J2 of all isolates died in 10 'M NaCN within 15 minutes. This probably meant that the nematodes have a completely aerobic respiration and are unlikely to tolerate anaerobiosis. Moreover, the cyanide was inhibiting respiration even faster at high concentrations -2 of 10 , 10-3 M NaCN and hence the nematodes died within 4 minutes.

The results show that A. tritici isolates are very sensitive to cyanide because the cyanide inhibits cytochrome enzymes completely.

Bryant et al. (1967) obtained nearly 70% inhibition of oxygen uptake by Caenorhabditis briggsae treated with 10^4 M HCN. This might suggest that at least part of an operative Tricarboxylic acid (TCA) cycle occurs in the nematodes.

Results with single galls were generally consistent (Figure 60)

suggesting that even though shortage of galls of some isolates limited the

experimental material, serious errors are unlikely to have been caused. 147.

SECTION VIII

EFFECT OF OSMOTIC STRESS ON J2 OF DIFFERENT ISOLATES

1. Introduction

The ability of nematodes to osmoregulate can be of great importance in some soils, where it can prevent loss of essential solutes from the nematode which might cause its subsequent death. The J2 of Anguina tritici exist in the soil at the active stage, infesting the young plant as ectoparasites and remain there until the floral induction in the host, when they infect the floral primordia and become endoparasites. During this part of its life cycle, the osmoregulatory process could be expected to play an important part in J2 survival and also when they move from the gall to the soil and finally to the host plant.

A change in the volume of nematodes during osmotic stress is usually estimated from changes in body length (Stephenson, 1942; Lee, 1960;

Wright and Newall, 1976). If the length measurement method is to be used, then account must be taken of changes in diameter, depending on the methods used and the nematodes.

In the present investigation, the J2 of A. tritici of different isolates were measured in different concentrations of NaC1 at different times with and without the presence of a "balanced" salt solution.

2. Materials and Methods

The pilot experiment was done using different concentrations of

NaC1 in distilled water (0.05, 0.1, 0.15, 0.25 and 0.5 M NaCl). Due to variability and lack of clear trends a new incubation media was used instead of distilled water, an artificial tap water made by adding the following salts to distilled water (Greenaway, 1970):- 148.

NaCl 0.350 mM (20.455 mg/L),KC1 0.044 mM (3.28 mg/L), Ca(HCO3)2 1.000.mM

(146.347 mg/L) and Mg(HCO3)2 0.4 mM (58.539 mg/L).

Solutions of different concentration were prepared by adding sodium choride (salt) to the artificial tap water as follows:-

0.05 M (2.9221 g/L), 0.10 M (5.8443 g/L), 0.15 M (8.7664 g/L), 0.25 M

(14.6107 g/L) and 0.50 M (29.2214 g/L).

These solutions were stored and kept covered in airtight glass bottles with plastic lids to avoid evaporation. Active second stage juveniles, in artificial tap water, of each of five isolates (U.K., Iraqi,

U.S.A., Australian and Indian) were used.

Leucocyte migration plat's (SterilinR; with 12 rings were used to prepare hanging drops on 19 mm circular coverslips, sealed to the rings with petroleum jelly "vaseline" to produce an airtight seal. This prevented evaporation, so maintaining the concentration of the solution, and provided an oxygen supply.

Each drop (0.02 ml) contained one nematode and the liquid had various osmotic pressures (0.05, 0.1, 0.15, 0.25 and 0.5 M NaC1 and distilled water). The solution was delivered by 1 ml disposable syringe, a new syringe being used for each different solution to prevent contamination.

Twelve replicates of each solution were made; juveniles were measured as soon as they were put in the drop and then at 30 'minute intervals. for 4 hours and a final measurement after 24 hours, using a video tape system connected to the microscope (see Appendix 7) and tracing nematode outlines from single frames of tape.

3. Results

In a pilot experiment the U.K. and Indian isolates showed little consistency and no clear trends in osmoregulation (Figure 63). However,

▪

149.

Figure 63. The pilot experiment showing the irregular changes in body

length of J2 in distilled water supplemented with NaC1 only.

+4 U.K. +3 th

/ ,;111 •• • ,

leng +2 .

. '4( in A A \ye % •••••

+1 •••• ••• e . "" Z. \• • • // • •.••• .: •••. • • %„ . ••••••',. • hang 0

c °- -- e -1 \/' •N tag •N /. N. -2 Percen -3 0 30 60 90 120 150 180 210 Key Time(minutes) 0.05 M

0.15 M 0.25 h — 0.5 M India +3 ...... _ .—L5 ..•• •-• • re.'•••■ _ ...-• +2 \ V —.5-.-. o••••*'...... --•• .- — — 7/45\ . / \ +1 - . \ ....1= - .. \ . / \ 0 ), 0 • ------•-... \ .,.. . -----4 ...... • / .P4 -1 `,••••••4• .• \ •••.. • . ■ -2 N ■ . 0 ...... N .... • ■... %•....X...... ":., .... 'u .. ...1/4 -3 ■...... „.... • t ..'• a() • /V I •• t • . / i w 3.4 414 -5 Ni -6

0 30 60 94 120 150 1180 2-10. Time (minutes) 150.

when NaC1 was included in an artificial tap water,Anguina tritici

appeared to be capable of some osmoregulation of its body fluids, but

it did not show extensive adjustment to alterations in external ion

concentrations. It was able to osmoregulate in dilute media between

0.05 M NaC1 and 0.10 M NaCl (Figures 64, 65, 66, 67 and 68).

The second stage juveniles shortened their original body length between 2.7% for Indian isolate and 4% for Australian isolate, in the

first half an hour in 0.5 M NaCI. After three and a half hours the

J2 had lost between 4.7 and 7% of body length in the same concentration for Indian and Australian isolates, respectively. The J2 lost their

ability to osmoregulate after 24 hours in a hypertonic solution depending

on the isolates. The Iraqi, U.K., Australian and Indian decreased 6.6%,

7.6%, 10.4% and 17.1% in body length respectively (Figures 64, 65, 66,

67 and 68).

The U.S.A. and Australian isolates decreased body length by a similar amount, while the Iraqi and'U.K. isolates had a similar decrease. All the J,, were dead after two days when they were kept in the 0.5 M NaCl.

All the J2 decreased in body length after 24 hours in 0,1 M NaCl between -0.4% for U.S.A. isolate and -2.6% for Indian isolate.

The J2 of U.S.A. isolate increased in length in distilled water,

0.05 M or 0.1 ~I NaC.I, but they decreased in length in all other concentrations (Figure 66). The Australian isolate showed the same increase except at 0.1 M NaC1, in which the length fluctuated more than the U.S.A. isolate (Figure 67). The Iraqi isolate showed the greatest ability to osmoregulate; body length was-unchanged for at least 4 hours in 0,05 M and 0.15 M NaC.l (Figure 65).

151.

Figure 64. The change in length of second stage juveniles of

U.K. isolate of Anguina tritici in distilled water

and artificial tap water supplemented with various

concentrations of sodium chloride.

+5 U.K. isolate

+4 +3

•

-3 • -4 -5 Key DW -6 0.05 M 0.1 M P<0.001 -7 0.15—M 0.25 M -8 0.5 M

• 30 60 90 120 150 180 210 1440

(24 hrs) Time (minutes) 152.

Figure 65. The change in length of J2 of Iraqi isolate of A. tritici

in distilled water and artificial tap water supplemented

with various concentrations of sodium chloride.

+5 Iraqi isolate h t +4

leng +3 in e +2 hang +1 c e tag 0 ...... ery—'_'_._. ;_. _..~ n ?~ .. ~ • .....' • —1 Perce

• - 2

-3

-4 Key -5 DW • 0.05 M -6 0.1 M 0.001 0.15 M - 7 0.25 M 0.5 M 1 I 1 1 I /\ I 30 60 90 120 150 180 210 1440v

(24 hrs

Time (Minutes) 153.

Figure 66. The change in length of J2 of U.S.A. isolate of A. tritici

in distilled water and artificial tap water supplemented

with various concentrations of sodium chloride.

+3 +2

+1 h t

ng 0

le -1 in e • y ...... •. -2 \ .

hang ...... S. c e -3 tag n -4 rce U.S.A. isolate

Pe -5

-6 Key -7 DW 0.05 M -8 0.1 li. 0.15 M p < 0.001 -9 0.25 M 0.5 M -10

-11 l I 1 1 1 I 1 V I 0 30 60 90 120 150 180 210 v 1440

Time (Minutes) 154.

Figure 67. The change in length of J2 of Australian isolate of A. tritici in distilled water and artificial tap water supplemented with

various concentrations of sodium chloride

+1 h t 0 leng

in -1 e 2 hang c e -3 tag en Perc

Australian isolate

p < 0.001 Key DW 0.05 M 0.1 M- 0.15 M 0.25 M 0.5 M

1 1 1 I 1 I I /1 30 60 90 120 150 180 210 144

Time (Minutes)

155.

Figure 68. The change in length of J2 of Indian isolate of A. tritici

in distilled water and artificial tap water supplemented with

various concdntrations of sodium chloride.

+ 1 - 0

•

- 1 •

—2

_ • —3

—4

Indian isolate • -.. • —5 bow• —6 P < 0.001

• — 7 • —8 U —9

—10 Key

—11 DW 0.05 M —12 0.1 M 0.15 M 0.25 M —13 0.5 M —14

—15

—16

—17

l , I /\ 0 30 60 90 120 150 180 210 v 1440

Time (Minutes) 156.

After one hour, the body lengths for all isolates were decreased steadily in 0.25 M NaCl, except Indian (Figure 68). The J2 in 0.25 M

NaC.l were dead after 6 days while the other juveniles in either 0.15, 0.1

0.05 M NaCl or distilled water remained alive for more than 12 days.

The analysis of variance for this experiment is shown in the Appendix 8.

4. Discussion

Experiments on osmoregulation have in the past been difficult to make accurately. The problems of measuring moving specimens was most difficult. The use of a video system however, provided an answer to the problem. Once the picture of single frames of the nematode in one plane was stopped, measurement of the juveniles length was possible.

The diameter of the nematode has a considerable effect on the

Andrassy's formula V /V = L d22 d12 (L = Length, d = diameter, 1 2 2 /L1 V = volume). The maximum diameter was very difficult to measure accurately, since this appears as faint lines on the screen. However, all the J2 of all isolates gave constant diameter throughout.

The diameter of the juveniles was ignored and the length of the body was taken as the only valid measurement and this was found to differ considerably. The ability of soil inhabiting nematodes to osmoregulate and control their body volume is probably a necessary feature of their existence (Lee and Atkinson, 1976). Osmotic concentrations expressed originally as freezing point depression values have been converted to the molarity of NaC1 solution with an equivalent osmotic pressure, by the relationship -1.0°C = 293 mmol/Liter NaCl (Krogh, 1939),

Anguina tritici did appear to be a very poor osmoregulator since it required between 0.05 to 0.1'M NaCl to osmoregulate. This however, depends on the physiology of the isolate as this is thought to be about 157.

equal to the osmotic pressure exerted by soil water (Wallace, 1971).

The inability of A. tritici to return to its original body length at

higher salt concentrations than 0.1 M NaC.1 suggests that it is rarely

exposed to very hyperosmotic conditions.

All the isolates have more or less the same decrease in length except at 0.5 M NaC1 concentration during which the lengths differed

(Figures 64, 65, 66, 67 and 68).

The Iraqi and U.K. isolates showed a fairly similar reduction in body length after 24 hours. The U.S.A.. and Australian isolates also showed similar reduction in length, but higher than those in the Iraqi and

U.K. isolates, while the Indian isolate failed completely to maintain its body length at the same level as other isolates. Wright et al. (1976) stated that nematodes are capable of regulating their volume in hypo- tonic (low osmotic) media, but not those hypertonic to their tissues.

These differences between the isolates of the same species are possibly due to the nature of the environmental conditionswhere they existed and these might affect the physiological balance of the nematodes as shown in the result of the present study.

The pilot experiment was a complete failure, due to the use of single salt solutions (Figure 63). The U.K. and Indian isolates showed non-regulation in NaCl. This might be due to an effect on cuticle permeability, which possibly caused an upset in the balance of the salts in the cuticle, giving rise to irregular changes in the body length. Newell (1979) obtained the same result as in the present experiment, namely that Enoplus communis and Enoplus brevis failed to regulate their volume when placed in 0.55 M NaCl, but in an osmotically equivalent medium (balanced salts), which also contained Ca and K ions, volume regulation occurred. 158.

There is a clear evidence from the pilot experiment and other workers

that the use of single salt solution does not give a valid impression

of regulatory ability under natural conditions.

The J2 have the ability to regulate their volume according to the

environment in which they live, which may be in the soil, on the plant

(between the tissues) or in the tissue. Further studies need to measure

the osmotic pressure in the galls in which all the stages of nematode

exist.

The body wall of nematodes is differentially permeable to water

and to various ions. The Lipid membrane content of the outer cortical

layer of the cuticle may probably constitute a major permeability

barrier (Bird, 1971).

Bhatt et ai. (1970) demonstrated that the ionic composition of

certain elements such as potassium did not stimulate respiration of

J2 of A. tritici.

L 159.

SECTION IX

GENERAL DISCUSSION

As a result of the work reported in this thesis it is possible to highlight a number of interesting aspects of the biology of Anguina tritici. Although many detailed studies of the survival of J2 in the dry gall have been made, little or no information is available on the survival of active second stage juveniles in various conditions.

Under field conditions, J2 are at first in a desiccated stage inside the galls on the host. At the end of the growing season, galls may be either harvested with the seeds or left to fall to the ground.

During the rainy season, the galls imbibe water and the J2 inside them revive.

My studies have shown that in the winter months when the temperature is low, J2 were released slowly from the gall and this subsequently led to relatively few J2 in the soil at this time. However, many J2 were observed in the soil during the spring, either resulting from their faster release from galls or from the accidental sowing of seeds and galls together.

The temperature, moisture and the thickness of the gall walls are very important for release of J2 from the gall. These observations agree with those of Marcinowski (1909); Leukel (1924); Swarup et al. (1971).

Further studies are needed to find out whether other factors which affect the softening and decomposition of galls are important for slower or faster release of J2 in the soil thereby influencing intensity of infestation. Jenkins and Taylor (1967) pointed out that the adaptive value of quiescence is of little consequence, once the galls enter the 160. soil, but it is of significance in the long distance dissemination of A. tritici; once active juveniles are in soil, they appear to lack a resistant stage.

The present study showed that the osmoregulatory ability of

A. tritici was affected by concentration of salts in soil. The J2 could probably survive longer in the soil with salt concentrations equivalent to 0.05 - 0.1 M NaCl as the J2 can regulate their body volume in this range. This finding agrees with the work of Bhatt et al- (1970). They tested the influence of osmotic pressure on oxygen consumption of

A. tritici. Also they showed that the J2 respired within the range of osmotic pressures from 0 - 2.0 14 NaC1 and that only sodium ions stimulated respiration.

The oxygen consumption of the J2 of all isolates studied increased with an increase in the temperature, reaching a maximum beyond which the rate of activity tended to decline, depending on the isolate. The J2 were very sensitive to cyanide and this suggests the presence of the classical aerobic form of respiration alone as there was no residual oxygen consumption and no continued activity by the J2 as might have been expected if alternative respiratory pathways were available.

Further work needs to be done on the influence of soil environment on physiology and respiration of A. tritici.

Infestation of the host is determined by the number of J2 able to penetrate between the tissues of the host. It was observed that there were many J2 in soil and few between the host tissues, and this agrees with the work of Midha et al. (1972). 14arcinowski (1909) stated that the J seem to lack the capacity for selecting the proper host; they attack 2 the first plant they meet. Al-Sabie (1977) tested the attraction of

J2 by seedlings and found only random movements of J2 in all treatments; 161. no accumulation was observed at any part of the wheat plant. The plant is usually infested during the tillering stage (Midha et ca., 1972;

Al-Sabie, 1977). Further studies are needed on infestation of the host.

After penetration, Marcinowski (1909) and others showed that the

J2 accumulate on the growing point causing different effects on the host such as more tillering, stunting, twisting and distortion of leaves.

However, Horovitz et ai•(1969) found remarkable changes in enzymes in the host during infestation and infection of the tissue. The J2 remain on the developing growing point, feeding ectoparasitically according to Midha et al. (1971), but Al-Sabie (1977) showed that it is not necessary for J2 to feed ectoparasitically for a long time during the host's vegetative stage. Juveniles accumulated on the wheat head exposed to nematodes-on an agar surface, suggesting that attraction was involved.

Further studies are needed to determine whether ectoparasitic feeding by the J2 on the host's reproductive tissues, during-infestation, -is- necessary to stimulate development and to supplement the stored food reserves, providing more energy for invasion of the flowering head.

Artificial inoculation of wheat at floral induction stage showed that some J2 from galls or from soil could invade the flower primordia after a latent period of 24 days when the temperature was below 15°C; some active J2 were still able to infect even after 225 days of storage.

This latent period could be very important for what it reveals about the necessity for changes in the J2 and in the host. The existence of a latent period suggests that induction of swelling in the host might be a controlling factor in the timing of J2 infection. It also might be due to some physiological interactions between nematodes and host associated with this period, but this requires further sophisticated experiments. The success of artificial inoculation has had the practical consequence of allowing protection for the J2 from many hazards in the soil and in the host before reaching the wheat head at the susceptible 162. double ridge stage, which makes the study easier, and allows more generation per year whenever the wheat floral primordia is inoculated.

The histopathological study revealed that the infective J2 make their entry to the host between the cell spaces of swollen tissue and that one or more J2 could enter the tissue from the same hole, although it is not known how long it takes for the J2 to make the entry. These observations disagree with Gupta et aZ. (1968) because they stated that the galls formed from joining the two sides of the growing point of the floral parts, thus enclosing. a variable number of J2 originally present at the growing point.

It is very clear that J2 could invade floral primordia of either ovary, outer anther or both. These observations agree in principle with the observations of other workers (Marcinowski, 1909;'Midha et al., 1974).

Rapid development of the whole of the body of J2 during and after invasion was observed by Al—Sabie (1977). The specific factors from the host which influence this development are not known.

For the first time artificial inoculation allows galls to be produced throughout the year from successive generations of J2 or from any other inoculation during the year. However, the galls in the cool winter season are smaller and contain fewer J2 inside the gall than in the warm seasons, This might be due to the physiological changes in the host during different seasons, which affect the stimulation and reproduction of A. tritici,

There is a question as yet without an answer about J2. The J2 of a new generation are produced from the egg and -J1 in a new gall, Why are the J2 not able to develop further in the same gall in which they were produced? If these 32 are inoculated to another floral 163.

primordia, they are able to invade and develop normally.

The number of adult females usually equals or slightly exceeds

the number of adult males in the same gall. In some galls only one nematode was found and this could be either male or female. It seems

that food does not affect the sex ratio inside the gall. Anguina tritici is amphimictic and the males are required to fertilize the

eggs of the females (Triantaphyllou et al., 1966). Apparently, the

sex ratio is controlled by genetic factors and modified by success

in penetrating under the influence of food or environmental conditions.

Further studies are needed to find out the factors affecting the sex ratio within the galls.

The survival of other stages (J3, J4, adult males and females)

is not known, and whether they could infect another host if placed in

the right place on a developing floral primordium at the right time.

Considerable differences• between the isolates of A. tritici were

seen in the body lengths, oesophagi and stylets in all stages.

The morphometric analysis can be interpreted to suggest that the

J2 of the Iraqi and Indian isolates have similar properties and en-

compassed much of the morphological variation found in the U.K., U.S.A.

and Australian isolates. The adult males and females showed that the

Iraqi and U.K. isolates were well separated from the U.S.A. and Australian

isolates, while overlap was present within these pairs.

Differences in morphology are in general agreement with observations

of other workers (Bauer, 1823, Byars, 1920, Limber, 1938). These

differences between the isolates might be more influenced by environmental

conditions than was shown by this study. In order to investigate this,

each of the isolates should be infected separately on the host under 164. identical conditions for several years. It would be desirable to conduct similar tests with these isolates in the geographical locations of the world from where they were obtained.

In overall morphological characteristics, the U.K. isolate is longer than other isolates and the Australian isolate is the smallest. The

Indian isolate failed to osmoregulate after 24 hours in 0.5 M NaC1 whereas other isolates osmoregulated within the same range and the Iraqi and

U.K. isolates are more efficient in this respect than others. The Iraqi,

U.S.A. and U.K. isolates attack many hosts in descending order, whereas the

Indian and Australian isolates attack few hosts. The U.S.A. isolates produced more galls than others.

It seems impossible yet to generalize on the usefulness of host range data either as a taxonomic tool or to provide evidence on the origin of Anguina trttici. All isolates infected the spring wheat variety c.v. Maris Dove except the Indian isolate. The host range test showed that A. tritici could infect nine new host species which are ancestors of wheat (Table 3). Among the nine new hosts, Trz`ticwn boeoticum aegiiopoides, T. b. thaoudcr, Aegilops squarrosa, Ae•cylindrica and

Hordelymus europaeus, have a wild habitat.

The modern wheat (Triticum aestivwn) shows some morphological and physiological characteristics of these ancestors of wheat, which is the clearest evidence that A. triti'ci infected these wild wheat hosts before the modern wheat. Triticwn dtcoccoides was infected by all isolates and gave many-more galls than all other hosts. Peterson (1965) stated that Tr2''t2ewn dicoccoides was the central host which carried the genomes of all other ancestral species. It is necessary to find out whether similarity exists in physiological activity (such as enzymes or hormone production) between all wheat ancestors and relate this to 165.

the ability of these hosts to resist infection by A. tritici.

Anguina tritici was the first plant parasitic nematode and has been studied for a very long time. However, it is still a major problem wherever wheat is grown in most countries (such as Middle East,

India, Europe...etc.), even after using modern mechanical seed cleaning which eliminates the galls of the planted seed. These infestations might now more clearly be attributed to the growing of wild wheat naturally in the same field with cultivated wheat as weeds.

Thus the best approach to practical control of A. tritici may be by eradication of the wild wheat hosts which this study showed to be an often prolific source of infection. Eradication of infected cultivated wheat hosts and flooding the field with water after harvesting are other potentially useful cultural control measures.

Varieties tested suggest that some are resistant. Moreover, others have considered more extensive tests in searching for sources of resistance and have been unsuccessful and in view of the wide host range, sources of resistance may be difficult to find and incorporate into cultivated varieties.

However, further work might investigate how quickly poor hosts become readily attacked by locally adapted isolates of Anguina tritici. 166.

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APPENDICES 180.

Appendix 1. The means and standard deviations of 20 individuals of

each isolate. Body length of J2 (mm), from galls grown in the country of origin (Old J2).

Isolate U.K. Australian U.S.A. Iraqi Indian

Mean 0.916 0.850 0.860 0.884 0.870 Standard 0.032 0.065 0.037 deviation 0.038 0.042

Analysis of variance

Source of variation DF SS MS F Individual 19 0.04224 0.00222 1.144 NS Isolate 4 0.05230 0.01307 6.737 *** Residual 76 0.14719 0.00194 Total 99 0.24173

Body length of J2 (mm),from galls produced in the U.K. (New J2)

Isolate U.K. Australian U.S.A. Iraqi

Mean 0.914 0.874 0,850 0.868 Standard 0.034 0.04 0.032 0.032 deviation

Analysis of variance

Source of variation DF SS MS F

Individual 19 0.02054 0.00108 0.871 NS Isolate 3 0.04393 0.01464 11.806 *** Residual 57 0.07040 0.00124 Total 79 0.13487

*** P < 0.001

I8l,

Appendix 1. (Continued)

Oesophagus length of J2 (U), rom Old J2

Isolate U.K. Australian U.S.A. Iraqi Indian Mean 221.039 204.284 205.584 214.935 202.856 Standard deviation 5.519 5.607 9.582 9.188 8.592

Analysis of variance

Source of variation DF SS MS F

Individual 19 2296.8 120.9 2.529 ** Isolate • 4 4981.8 1245.5 26.057 *** Residual 76 3630.5 47.8 Total 99 10909.1

Oesophagus length of J2 (u), from New J2

Isolate U.K. Australian U.S.A. Iraqi

Mean 217.667 210.907 212.467 216.753 Standard deviation 5.114 6.825 6.226 8.818

Analysis of variance

Source of variation DF SS MS F

Individual 19 729.8 38.4 0.763 NS Isolate 3 642.6 214.2 4.258 ** Residual 57 2866.2 50.3 Total 79 4238.6

*** P < 0.001 ** P < 0.01 182.

Appendix 1. (Continued

Stylet length of J2 (p), from Old J2

Isolate U.K. Australian U.S.A. Iraqi Indian

Mean 7.400 7.075 5.970 7.205 6.880 Standard deviation 0.611 0.664 0.653 1.548 0.743

Analysis of variance

Source of variation DF SS MS F Individual 19 20.550 1.082 1.393 NS Isolate 4 24.775 6.194 7.972 *** Residual 76 59.049 0.777 Total 99 104.374

Stylet length of J2 (ii'), from New J2

Isolate U.K. Australian U.S.A. Iraqi

Mean 7.530 7.075 6.165 7.595 Standard deviation 0.534 0.664 0.715 1.138

Analysis of variance

Source of variation DF SS MS F

Individual 19 9.527 0.501 0.741 NS Isolate 3 26.089 8.696 12.864 *** Residual 57 38.553 0.676 Total 79 74.140

*** P < 0.001 183.

Appendix 1. (Continued)

Gonad length of J2 (u), from Old J2

Isolate U.K. Australian U.S.A. Iraqi Indian

Mean 22.339 25.585 36.431 31.495 34.955 Standard deviation 2.443 3.069 6.574 6.073 7.000

Analysis of variance

Source of variation DF SS MS F

Individual 19 747.5 39.3 1.494 NS Isolate 4 2924.0 731.0 27.795 *** Residual 76 1997.8 26.3 Total 99 . 5669.3

Gonad length of J2 (+),from New J2

Isolate U.K.. Australian U.S.A. Iraqi

Mean. 24.027 27.142 33.946 29.353 Standard deviation 2.040 2.453 4.249 3.682

Analysis of variance

Source of variation.. DF SS 'MS. F

Individual 19 221.4 11.7 1.17 NS Isolate 3 1043.7 347.9 34.79 *** Residual.. 57 572,7 10.0 Total 79 1837.8

*** P 4 0.001 184.

Appendix 1. (Continued)

Body length of adult males (mm), from galls produced in the U.K.

Isolate U.K. Australian U.S.A. Iraqi Mean 2.49 1.99 2.11 2.35 Standard deviation 0.13 0.24 0.18 0.17

Analysis of variance

Source of variation DF SS NS F

Individual 19 0.4858 0.0256 0.717 NS Isolate 3 3.1055 1.0352 28.997 *** Residual 57 2.0371 0.0357 Total 79 5.6284

Oesophagus length of adult males (U), from galls produced in the U.K.

Isolate U.K. Australian U.S.A. Iraqi

Mean 226.28 206.42 228.14 211.43 Standard deviation 18.01 16.63 22.96 13.78

Analysis of variance

Source of variation DF SS 'MS F

Individual 19 8608` 453 1.573 NS Isolate 3 6987 2329 8.087 *** Residual 57 16403 288 Total 79 31998

*** P 4 0.001 185.

Appendix 1. (Continued)

Stylet length of adult males (),from galls produced in the U.K.

Isolate U.K. Australian U.S.A. Iraqi

Mean 10.86 10.36 10.86 9.79 Standard deviation 0.98 1.13 0.86 1.07

Analysis of variance

Source of variation DF SS MS F

Individual 19 16.46 0.87 0.813 NS Isolate 3 15.64 5.21 4.869 ** Residual 57 61.04 1.07 Total 79 93.15

Greatest body width of adult males (),from galls produced in the U.K.

Isolate U.K. Australian U.S.A. Iraqi

Mean 103.00 80.58 83.86 99.86 Standard deviation 13.13 6.62 10.58 14.20

Analysis of variance

Source of variation DF SS MS F

Individual 19 2208 116 0.841 NS Isolate 3 7590 2530 18.333 *** Residual 57 7851 138 Total 79 17649

*** P < 0.001 ** P < 0.01 •186

Appendix 1. (Continued)

Tail length of adult males (ii),from galls produced in the U.K.

Isolate U.K. Australian U.S.A Iraqi

Mean 86.76 74.44 86.14 84.14 Standard deviation 4.40 4.78 5.74 7.61

Analysis of variance

Source of variation DF SS MS F

Individual 19 888.3 46.8 1.631 NS Isolate 3 1970.0 656.7 22.881 *** Residual 57 1638.6 28.7 Total 79 4496.9

Spicule length'of adult males'(u), from galls produced in the U.K.

Isolate U.K. Australian U.S.A. Iraqi

Mean 31.86 32.57 35.00 29.86 Standard deviation 4.66 1.43 3.33 3.99

Analysis of variance

Source of variation DF SS MS F

Individual 19 240.4 12.7 1.000 NS Isolate 3 270.6 90.2 7.102 *** Residual 57 723.4 12.7 Total 79 1234.4

*** P < 0.001 •187.

Appendix 1. (Continued

Body length of adult-females -(mm), from-galls produced in the-U.K.

Isolate U.K. Australian U.S.A. Iraqi

Mean 4.42 3.01 2.83 4.24 Standard devia- 0.37 0.44 0.40 0.42 tion

Analysis of variance

Source of variation DF SS MS F

Individual 19 2.460 0.129 0.725 NS Isolate 3 40.636 13.545 76.096.*** Residual 57 10.132 0.178 Total 79 53.229

Oesophagus length of adult females (u), from galls produced in the U.K.

Isolate U.K. Australian U.S.A.. Iraqi

Mean 223.84 204.27 204.36 235.30 Standard devia- 14.30 11.59 58.21 12.19 tion

Analysis of variance

Source of variation. DF SS 'MS.. '7

Individual 19 10932 575 1.132 NS Isolate 3 - 10441 3480 6,850 *** Residual 57 28965 508 Total 79 50338

***P 4c0.001 188.

Appendix 1 (Continued)

Stylet length of adult females (p), from galls produced in the U.K.

Isolate U.K. Australian U.S.A. Iraqi

Mean 11.79 10.40 11.36 9.57 Standard deviation 0.64 0.79 1.18 0.94

Analysis of variance

Source of variation DF SS MS •F

Individual 19 12.680 0.667 0.761 NS Isolate 3 59.101 19.700 22.489 *** Residual 57 49.950 0.876 Total 79 121.731

Greatest body width of adult females (ii),from galls produced in the U.K.

Isolate U.K. Australian U.S.A. Iraqi

Mean 214.13 130.82. 160.57 198.57 Standard deviation 25.30 16.01 31.77 29.31

Analysis of variance

Source of variation DF SS • 'MS 7

Individual 19 12405 653 0.940 NS Isolate 3 85494 28498 41.004 *** Residual 57 39605 695 Total 79 137504

*** P < 0.001 189.

Appendix 1 (Continued)

Tail length of adult females (p),from galls produced in the U.K.

Isolate U.K. Australian U.S.A. Iraqi Mean 86.29 92.01 89.57 89.90 Standard 3.66 0.89 7.83 6.93 deviation

Analysis of variance

Source of variation DF SS MS •F

Individual 19 420.2 22.1 0.654 NS Isolate 3 335.8 111.9 3.311 * Residual 57 1925.0 33.8 Total 79 2680.9

Length from vulva to end of the tail of adult females (min), from galls produced in the U.K.

Isolate U.K. Australian U.S.A. Iraqi

Mean 0.398 0.361 0.345 0.413 Standard 0.048 0.047 0.04 0.052 deviation

Analysis of variance

Source of variation DF SS 14S F

Individual 19 0.05915 0.00311 1.646 NS Isolate 3 0.06012 0.02004 10.603 *** Residual 57 0.10791 0.00189 Total 79 .0.22718

*** P < 0.001 * P < 0.05 190.

Appendix 1 (Continued)

Eggs length (p), from galls produced in the U.K.

Isolate U.K. Australian U.S.A. Iraqi

Mean 90.58 96.31 92.58 102.41 Standard deviation 4.23 9.57 8.91 14.12

Analysis of variance

Source of variation DF SS MS F

Individual 19 2483.3 130.7 1.525 NS Isolate 3 1622.1 540.7 6.309 *** Residual 57 4886.4 85.7 Total 79 8991.8

Eggs width (U),from galls produced in the U.K.

Isolate U.K. Australian U.S.A. Iraqi

Mean 44.13 34.74 39.24 35.77 Standard deviation 2.82 0 3.55 2.37

Analysis of variance

Source of variation DF SS MS F

Individual •19 170.74 8.99 1.572 NS Isolate 3 1077.02 359.01 62.764 *** Residual 57 326.19 5.72 Total 79 1573.95

*** P < 0.001 191.

Appendix 1 (Continued)

Adults inside the galls produced in the U.K.

. Isolate U.K. Australian U.S.A. Iraqi Sex Female Male Female Male Female Male Female Male Replicate 1 6 4 21 15 19 18 3 2 2 2 2 17 6 29 27 4 3 3 6 5 4 4 13 17 5 5 4 19 16 3 1 1 1 5 3 5 7 5 8 6 8 7 3 3 6 6 3 11 6 3 2 3 2 7 3 1 2 2 5 3 2 2

Mean 7 5.143 9.429 5.714 11.143 10.714 3.571 2.857 Standard 5.598 5.014 7.323 4.572 10.007 10.012 1.134 1.069 deviation

Analysis of variance

Source of variation DF SS ES F

Isolate 3 433.1964 144.3988 3.4606 * Sex 1 39.4464 39.4464 0.935 NS Isolate-Sex 3 23.3393 7.7798 0.186 NS Residual 48 2002.8571 41.7262 Total 55 2498.8393

*Pic 0.05 192.

Appendix 2. Kolmogorov-Smirnov test of Chi- squared (x2) for

distribution of the random galls between two isolates

(UK/USA).

5 10 50 100 500 1000 5000 10000

UK 8 15 26 101 143 195/ 272/ 277 /277 /277 277 /277 ~77 277 277 277

USA 3 7 13 34 101/ 142 189 218 1218 /2 8 /218 /218 218 /218 /218 /218

UK 0.029 0.054 0.094 0.365 0.516 0.704 0.982 1.0

USA 0.014 0.032 0.060 0.156 0.463 0.651 0.867 1.0

1 0.015 0.022 0.034 0.209 0.053 0.053 0.114 0

Max,

D = 0.209

= 4 D2 n1n2 nl + n2

277 x 218 = 4 (0.209)2 21.315 X2 277 + 218

X2 = 21.315 for df = 2 is P < 0.001

Reference to this text

SIEGEL, S. (1956). Nonparametric statistics for the behavioural

sciences. McGraw-Hill Book Company, New York, Toronto and

London, 312 pp. Appendix 3. Survival of J2 in different media with temperature Anquina tritici Analysis of variance

Source of variance DF SS MS F

Time 5 195614407.9 3912281.59 4597.5339 ***

Media 2 84590.0833 42295.0417 49.70319 ***

Temperature 2 130219.1944 65109.5972 76.51381 *** Time-Media 10 210707.5278 21070.7528 24.76138 ***

Time-temperature 10 253165.75 25316.575 29.75088 ***

Media-temperature 4 56261.2222 14065.3056 16.5289 ***

Time-Media-temperature 20 187939.00 9396.95 11.04286 ***

Error 162 137854.25 850.9522

Total 215 20622E+08

*** P 4 0.001 Appendix 3. (Continued)

Anguina spp.

Analysis of variance

Source of variance . DE. SS MS

Time 5 8843426.8148 1768685,363 970,1287 *** Media 2 583733.0648 291866.5324 160.0896 **

Temperature 2 810482.0093 405241.0046 222.2758 ***

Time-Media 10 332721.1574 33272.1157 18.2499 *

Time-Temperature 10 489546.3796 48954.638 26.8518 *

Media-Temperature 4 214483.7963 53620.9491 29.4112 *

Time-Media-Temperature 20 291414.8148 14570.7407 7.9921 *

Error 162 295349.5 1823.1451

Total 215 11861E+08

*** P < 0.001

** P < 0.01

* P < 0.05 195.

Appendix 4. Survival of J2 in air saturated water of different isolates

U.K.

Time (day) Replicate 0 7 14 21

1 63 41 23 3

2 59 52 19 2

3 65 56 21 1 4 69 44 23 0

5 78 43 18 0

x 66.8 47.2 20.8 1.2

S 7.275 6.458 2.28 1.304

Analysis of variance

Source of variation DF SS MS F

Time 3 12500.800 4166.9333 165.355 ***

Error 16 403.200 25.200

Total 19 12904.00 '

*** P 4 0.001

x = Mean

S = Standard deviation 196.

Appendix 4. (Continued)

Iraqi

Replicate Time (day) 0 7 14 21 28

1 668 609 398 86 5

2 689 557 383 85 7

3 681 546 424 82 4 4 676 556 419 82 7 5 691 563 419 85 8

x 681 566.2 408.6 84 6.2

S 9.46 24.69 17.47 1.87 1.64

Analysis of variance

Source of 'variation DF SS MS F

Time 4 1742912.96 435728.24 2154.085 ***

Error 20 4045.6 202.28

Total 24 1746958.56

*** P ( 0.001 197.

Appendix 4. (Continued)

Australian

Replicate Time (day) 0 7 14 21 28 35

1 355 199 190 133 9 1

2 371 194 180 174 30 1

3 346 185 183 155 26 0

4 352 192 191 156 36 0 5 357 191 187 151 28 0

x 356.2 192.2 186.2 153.8 27.8 0.4 S 9.26 5.07 4.66 14.62 6.18 0.55

Analysis of variance

Source of variation DF SS MS F

Time 5 414456.1667 82891.2333 1290.805***

Error 24 1541.20 64.2167

Total 29 415997.3667

*** P 4 0.001 198.

Appendix 4. (Continued)

U.S.A.

Time (day) Replicate 0 7 14 21 28 35

1 292 284 258 239 53 11

2 275 261 263 204 32 4 3 281 271 254 216 45 15

4 293 269 275 199 36 10 5 279 263 259 211 49 6

x 284 269.6 261.8 213.8 43 9.2 S 8.06 9.04 8.04 15.51 8.8 4.32

Analysis of variance

Source of variation DF SS MS F

Time 5 381479.7667 76295.9533 512.397 ***

Error 24 3573.600 148.9

Total 29 385053.3667

*** P < 0.001 199.

Appendix 4. (Continued)

Indian

Replicate Time (day) 0 7 14 21

1 705 161 44 13 2 724 149 40 10

3 719 156 43 14

4 739 163 45 9 5 711 158 44 11

x 719.6 157.4 43.2 11.4

S 13.07 5.41 1.92 2.07

Analysis of variance

Source of variation DF SS MS F

Time 3 1638127.4 546042.4667 10495.771 ***

Error 16 832.4 52.025

Total 19 1638959.8

*** P 4 0.001 200.

Appendix 4. (Continued)

Anguina spp (from creeping soft grass) U.K.

Time (day) Replicate 0 7 14 21 28 35

1 730 617 115 30 14 5

2 785 702 84 58 12 2 3 725 546 68 62 15 4

4 719 626 58 41 7 3 5 769 651 83 53 11 0

x 745.6 628.4 81.6 48.8 11.8 2.8

S 29.475 56.686 21.594 13.142 3.114 1.924

Analysis of variance

Source of variation DF SS MS F

Time 5 2877164.1667 575432.8333 72.911***

Error 24 18938.00 789.0833

Total 29 2896102.1667

*** P < 0.001 201.

Appendix Micromoles 02 consumption/mg dry wt./min. of two isolates

Time Isolate 1 2 3 4

2.908 2.906 2.804 2.883 UK 2.974 2.938 2.865 2.895 3.043 2.971 2.343 2.878

x 2.975 2.938 2.671 2.867

1.18 1.46 3.08 3.22 Iraq 1.17 1.41 3.1 3.22 1.15 1.43 3.12 3.24

x 1.167 1.433 3.100 3.227

t P < 0.001 P < 0.001 P < 0.01 P < 0.02

t = xl x2 S~1 17-1 Pooled standard deviation = 0.105

Analysis of variance

Source of variation DF .SS MS F

Isolate 1 2.4244 2,4244 218.41*** Time 3 4.3842 1.4614 131.66*** Isolate /Time 3 6.3295 2.1098 190.07*** Error 16 0.1771 0.0111

Total 23 13.3152

*** P < 0.001 202.

Appendix 6. Oxygen concumption of J2 of Anguina tritici of different isolates at different temperatures. micromoles 02 consumption/mg dry wt./min.

Isolate Temperature U.K. Iraqi Indian U.S.A.

0.405 0.321 1.283 1.732 5°C 0.315 0.356 1.253 1.639 0.381 0.383 1.251 1.732 Mean 0.367 0.353 1.26 1.7 S 0.0467 0.0310 0.0179 0.0535

0.602 1.065 1.000 1.888 10°C 0.494 0.797 1.002 1.36 0.952 0.609 1.018 1.484 Mean 0.683 0.824 1.01 1.58 S 0.240 0.229 0.0099 0.276

1.272 0.698 2.047 2.715 15°C 1.503 0.577 1.692 2.416 2.792 0.684 3.04 4.069 Mean 1.86 0.653 2.26 3.07 S 0.819 0.0661 0.699 0.881

1.847 2.594 1.741 2.024 20°C 0.586 2.714 2.919 1.109 0.724 2.727 2.895 2.990 Mean 0.785 2.68 1.55 2.04 S 0.236 0.0736 1.17 0,141 203.

Appendix 6. .(Continued)

Isolate U.K. Iraqi Indian U.S.A. Temperature

3.956 2.412 3.495 1.898 25°C 3.559 2.388 3.908 1.454 3.640 2.165 2.189 1.97 Mean 3.72 2.32 3.20 1.77 S 0.210 0.136 0.898 0.280

5.038 1.583 2.664 1.595. 30°C 1.732 1.478 2.228 1.379 3.335 1.782 3.113 1.896 Mean 3.37 1.61 2.67 2.62 S 1.65 0.155 0.443 0.260

Analysis of variance

DF SS MS F Isolate 35.398 1.799 5.452 ** Temperature-. 5 31.720 6.344 19.224 ***

ISOlate/Temperature 15 27.409 1.827 5.536 ***

Error 48 15.840 0.330

Total 71 80.367

*** P < 0.001 **. P < 0.01 204.

Appendix 7.

The video tape/microscope system consisted of:-

1. Microscope - wild (R) with 10 x eye pieces and 4 x object.

2. Camera - CC TV Hitachi (R)

(R) 3. Video recorder - National time laps VTR NV 8030

4. Tape-Sony (R) High density V-62

(R) 5. Television - Hitach 205.

Appendix 8.

The effect of distilled water and artificial tap water supplemented

with 0.05, 0.1, 0.15, 0.25 and 0.5 M NaCl,on the length of J2 of A. tritici at different time for each isolate.

Analysis of variance U.K.

Source of variation DF SS MS F

Concentration. 5 0.036107 0.007221 43.76 ***

Time 8 0.003063 0.000383 2.32 *

Error 40 0.006607 0.000165 Total 53 0.045777

*** P < 0.001

* P < 0.05

Analysis of variance Iraqi Isolate

Source of variation DF SS MS F

Concentration 5 0.0464512 0.0092902 103.686***

Time 8 0.0016244 0.0002030 2.266 *

Error 40 0.0035843 0.0000896 Total 53 0.0516599

*** P < 0.001

* P < 0.05 206.

Appendix 8. (Continued)

Analysis of variance U.S.A.

Source of variation DF SS MS F

Concentration 5 0.014377 0.002875 68.359***

Time 8 0.002666 0.000333 2.6 * Error 40 0.005135 0.000128

Total 53 0.022178

*** P < 0.001 * P < 0.025

Analysis of variance Australian

Source of variation DF SS MS F

Concentration 5 0.013594 0.002719 15.537***

Time 8 0.002894 0.000362 2.069 NS

Error 40 0.006989 0.000175 Total 53 0.023476

*** P < 0.001

Analysis of variance Indian

Source of variation DF SS MS F

Concentration 5 0.021927 0.004385 12.78***

Time 8 0.010976 0.001372 4.00**

Error 40 0.013914 0.000348 Total 53 0.046816

*** P < 0.001 * P < 0.01 207.

Appendix 8. (Continued)

Analysis of variance for all isolates, concentration and time.

Source of variation DF SS MS F

Isolate 4 1.820 0.455 315.635 ***

Concentration 5 1.064 0.213 147.671 ***

Time 8 0.199 0.025 17.245 *** Isolate + concentration 20 0.542 0.027 18.796 *** Isolate + time 32 0.067 0.002 1.442 *

Concentration + time 40 0.353 0.009 6.118 ***

Isolate + concentration + time 160 0.142 0.001 0.618 NS

Residual 2970 4.282 0.001

Total 3239 8.469

*** P < 0.001

* P < 0.05