Ecology and Biology of Nematodes Associated with Grass

ECOLOGY AND BIOLOGY OF NEMATODES ASSOCIATED WITH GRASS

by John Bridge B.Sc.(Hull), M.Sc.(McGi11)

A THESIS PRESENTED FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

in the Faculty of Science, University of London

Imperial College Field Station, January, 1971 Ashurst Lodge, Sunninghill, Ascot 2

ABSTRACT Nineteen genera of plant parasitic nematodes were found in grassland pasture soils, of these only Tylenchorhynchus and Tylenchus were present in all the fields sampled, and Helicoty- lenchus, Paratylenchus and Pratylenchus occurred in all but one of the fields. The other genera most frequently found were Aphelenchoides, Aphelenchus, Longidorus and Pratylenchoides. The vertical distribution of nematodes in grassland soil was studied. Certain plant parasitic genera had distinctly different soil depth preferences which did not vary at different sampling times throughout the year. Highest populations of

Tylenchorhynchus species occurred in the top 5 cm of soil, those of alenchus species in the upper 15 cm, Paratylenchus microdorus between 15 and 25 cm and Longidorus species below 30 cm. Pratylenchus and Helicotylenchus were irregularly distributed in the soil profile. The morphological characteristics of the ten species of Tylenchorhynchus found in grassland pasture soils are described and figured. Observations on the behaviour of seven alpnchorhynchus species on grass seedling roots showed that the method of feeding varied considerably between species. Some species were browsing ectoparasites others fed in aggregations on root tips, and two species exhibited both a sedentary ectoparasitic and a migratory semi- endoparasitic feeding behaviour. 3

Relationships between nematodes and growth of perennial ryegrass was studied. No significant reduction in top or root grOwth occurred in soil experiments when populations were equivalent to, or greater than, field populations. On agar plates it was observed that the feeding of Tylenchorhynchus maximus on root tips of seedlings caused damage which resulted in the reduction or cessation of main root growth.

Emergence from the egg of Tylenchorhynchus maximus and T. icarus larvae was observed. ACKNOWLEDGEMENTS

I am deeply indebted to my supervisor, Dr N.G.M. Hague, for his constant encouragement, advice and helpful criticisms throughout this work. To Miss F. Teare go my sincere thanks for her very valuable and ever willing assistance. I gratefully acknowledge the help and cooperation afforded me at all times by members of the Grassland Research Institute, Hurley, with especial thanks to Mr T.E. Williams, Dr J. Heard and

Mr E.A. Garwood. Officers of the National Agricultural Advisory Service at Shardlow, Reading and Cambridge assisted me in the survey of grassland pastures, to these and to the farmers who allowed me to sample from their fields I wish to offer my thanks.

I also wish to thank Mr J. Smith for technical assistance and Miss J. Williams for her help and capable typing of this thesis. Permission to work in the Department of Professor T.R.E.

Southwood is duly acknowledged.

Finally, thanks are due to the Ministry of Overseas

Development for allowing me to complete this work while in their

employ. 5 TABLE OF CONTENTS Page Title page •• •• .. • • .. •• •• •• 1

Abstract • • .• • • •• •• • •• •• •• •• 2

Acknowledgements .. 01 • • • • .. OO .. .. 4

Table of Contents • • •• • • •• • • •• .. •• 5

SECTION I General Introduction .. .. •• •• •• •. 7 SECTION II Distribution of nematodes associated with grass 12 Materials and Methods Sampling techniques ...... O. .. 12 Extraction procedures 00 • • SO 00 OS 1 4

A. Distribution of nematodes in grassland pastures •• 17

Results •• •• •• •• • • •• S. •• 17 Discussion .. • • • • •• •• •• .. 19 B. Vertical distribution of nematodes in grassland soils . 3o

Results •• •S. •• •5 •• •• •• •• 31 Discussion • • • • •S. • • •. •• •• 4o SECTION III Tylenchorhynchus species found in grassland pasture soils 44 Descriptions of Tylenchorhynchus species 45 SECTION IV The feeding behaviour of nematodes on grass roots •• • 61

Introduction • • •• •• • • • • • • •• 61 •• . . • • Materials and Methods • • • • • • • • 63 A. Feeding behaviour of Tylenchorhynchus species -.. 64

R esults ...... 00 40 04 O0

T. brew dens..O...... 0. 041 06 T. no thus ...... Oil 00 041 00 69 T. dubius IP* •• • • 0,6 40 • • • • 69 T. maximsu ...... OS 0, 00 O0 70 T. lamelliferus .. SO • • 0. OS OM 75 T. icarus and T. macrurus .. .. OS .. 77

Discussion 04, 06 00 00 • • 81

Page

B. Feeding behaviour of other nematodes ...... 84

Tylenchus filiformis ...... 84 Aphelenchoides saprophilus .. .. i. • • 86 Paratylenchus microdorus and P. projectus .. 86 Helicotylenchu,6 varicaudatus .. .. • • .. 88 Pratylenchus crenatus ...... 88

Discussion . . 0* 00 00 00 *40 OS 89 SECTION V Relationships of migratory nematodes (mainly Tylenchorhynchus and Paratylenchus) to growth of perennial ryegrass ...... 00 93

Introduction .. • • 00 00 ...... 93 a) Glasshouse experiments with Paratylenchus . .... - microdorus and a mixture of plant nematodes .. 4 Materials and Methods ...... 94 Results es •• •• •• •• •• •• •• 96 b) Controlled temperature experiments with two species of Tylenchorhynchus and Paratylenchus microdorus 97 Materials and Methods 00 00 00 00 00 97 Results • • 98 c) Root growth experiments with Tylenchorhynchus maximus and T. icarus on agar plates .. •• 99

Materials and Methods ...... 00 00 .. 99 Results 00 00 00 0* 00 04 •IF 100

Discussion .. 00 00 OS SO 00 00 •• 104 SECTION VI Some aspects of the biology of Tylenchorhynchus maximus and T. Icarus .. .. 00 0* .. 107

Fecundity and hatching of T. maximus .. .. • • 0 * 107

Emergence from the egg 040 ...... 108

SECTION VII General Discussion ...... 41 .. .. 112

References •• •• •• •• •• •• •• •• 120

Appendix Tables • .. •• •• •• •• 134 7

SECTION I. GENERAL INTRODUCTION Grass pasture plays a very important role in the agriculture of Britain. It was estimated in 1966 (Williams) that 70% of British agricultural output was livestock and livestock products. Grassland is the cheapest source of ruminant feed (Breese, 1968) and is therefore of great consequence to this industry. In 1969 permanent grassland comprised /ft.:, of the total acreage of crops and grass (excluding rough grazing) in England and :Tales (Anon., 1970). Perennial ryegrass (Lolium perenne) is the major sown grass species in long leys. It was estimated in 1959 (Baker, 1962) that ryegrass and ryegrass mixtures made up 79.25":, of different seed mixtures sown. Other grasses of importance are timothy (Phleum arvense), meadow fescue (Festuca pratensis) and cocksfoot (Dactjlis glomerata). Until recently grass has not been grown under intensive management as have arable crops, and consequently yields from pasture have not been as critically important. However, the loss of agricultural land to development schemes and the competition with the more economic arable crops has led to research being directed towards improving yields of grassland. Total acreage to permanent grassland has declined by nearly 600,000 acres since 1963 (Anon., 1970). Endemic diseases of grasses have to be considered in this improvement of yield. Grasses, as with other plants, are subject to attack by 8 many different pests and diseases: only a few of them have been recorded as of major significance, the others are considered to be less important. The major pests and diseases on grasses include aphids, leatherjackets, slugs, mildew (Erysiphe graminis), rusts (Puccinia spp.), choke (Epichloe typhina) and Ryegrass mosaic virus (Gair and Roberts, 1969). Nematode problems have been encountered mainly with the internal root feeding nematodes producing galls on grasses, Anguina spp., Ditylenchus spp. and Heloidogyne nassi (Goodey,

1959; Lewis and bley, 1966; Norton and Sass, 1966; Southey, 1969), although they are not considered of economic importance in this country. liacfadyen (1957) estimated that there were 1.8 to 120 million nematodes beneath one square metre of grassland soil. Although only a proportion are plant parasitic it does suggest that nematodes may be of considerable importance when damage to grasses is being assessed. However, the large number of ubiquitous, unobtrusive root feeding nematodes occurring in grass pasture soil have generally been ignored as causing economic losses in the field. This applies especially to the external root feeders.

Ecological studies have established that certain plant parasitic nematode genera occur more commonly than others under grass. The presence of members of the Hoplolaiminae, Pratylenchus and Tylenchorhynchus in large numbers are considered to be typical of grassland habitat in South Africa (Vegte and Heyne, 1963). Tylenchorhynchus and HelicoVlenchus were common under Poa pratensis turf in Nebraska, .U.S.A. (Sumner, 1967). In North Dakota Tylenchorhynchus, Tylenchus, Pratylenchus, Paratylenchus and Xiphinema were among the most frequently occurring nematodes from forage grass (Pepper, 1963). Geraert (1967) in Belgium found that Tylenchorhynchus spp. were associated with pastures and he showed that T. dubius, T. lamelliferus, T. quadrifer and T. nanus exhibited a preference for meadows. Studies of the nematodes of grassland in Britain have been restricted to two localities, Moor House National Nature Reserve, Westmorland (Banage, 1962) and Broadbalk wilderness, Rothamstead Experimental Station (Yuen, 1966). Banage found that Tylenchorhynchus spp., Helicotylenchus spp., Tylenchus spp., Rotylenchus robustus, rratylenchus 2Eatensis and a species of Criconema were common in moorland soil under grass. In the ungrazed grassland of Broadbalk wilderness the dominant genera in order of abundance were Tylenchus, Paraty- lenchus, Helicotylenchus, Rotylenchus and Tylenchorhynchus. Other plant parasitic genera found associated with grass include Hypsoperine (Heald and Wells, 1967), Belonolaimus,

Hoplolaimus and Dolichodorus (Nutter and Christie, 1958), Scutellonema (Perry et al., 1959) and. Rotylenchulus (Loof and Oostenbrink, 1962). The association of nematodes with grasses is not necessarily an indication that they are causing damage to the grasses or even 1 0

using them as a source of food. Apart from the gall-forming

nematodes, genera that have been cited as causing damage to

grasses are Tylenchorhynchus (Havertz, 1957; Johnson, 1970;

Troll and Rohde, 1966), Longidorus (Thomas, 1969), Helicotylenchus

(Perry, 1958), Paratylenchus (Coursen and Jenkins, 1958),

Criconemoides (Johnson, 1970), Belonolaimus (Rhoades, 1962; ainchester and Burt, 1964; Johnson, 1970) and Pratylenchus

(Troll and Rohde, 1966).

Less research has been done on external root feeding

nematodes, with the exception of virus vectors, than on other

nematodes. Feeding by external root feeding nematodes produces

few easily recognisable symptoms and thus there is the difficulty

of relating their presence in the soil with damage to the host

plant.

Direct observations on the feeding behaviour of migratory

nematodes.on plant roots give a better understanding of the

relationship that exists between nematodes and their hosts.

Such observations have been made by a number of investigators

(Cohn, 1970; Fisher and Raski, 1967;Klinkenburg, 1963;. Ps- racer

et al., 1967; Rhoades and Linford, 1961; Sutherland, 1969) using in vitro culture techniques. They have established th-7.t the feeding behaviour of external root feeders varies between different

genera and species.

Aspects of the ecology and biology of nematodes associated with grass are here further investigated. The distribution of 11

nematode genera in grasslands and their vertical distribution

in the soil are studied. The study sets out to establish which

nematodes are conmonly found in grass pasture from farms in

England and how nematodes are distributed in the soil profile under grass. Determinations of damage to perennial ryegrass

by external root feeding nematodes, especially of the genus

Tylenchorhynchus are made and the taxonomy and feeding behaviour of species of Tylenchorhynchus are described in detail and com- parisons are made with observations on other genera. 12

SECTION II.

DISTRIBUTION OF NEMATODES ASSOCIATED 4ITH GRASS.

The distribution of plant parasitic nematodes in grassland

soils was determined by sampling a selection of farm pastures.

Many of the fields sampled were old permanent pastures that had

been down to grass for well over 20 years; the others were

grass leys that had been sown within the past 10 years. The

vertical distribution of nematodes under grass was investigated

to determine their seasonal fluctuation at different depths in

the soil profile and the, distribution of genera and species at

specific depths.

Materials and Methods

Sampling Techniques

Soil samples were taken from the selected fields'by means

of a 2 in diameter soil auger made from a cylinder of well-

boring steel with a lipped, tool steel cutting edgel) (Fig. 1).

It was driven into the ground with a 9 lb sledge-hammer - a

mild steel cap placed on the end of the cylinder prevented

damage to the head of the auger. Marks were painted on the auger

to indicate the depth at which samples were to be taken. With-

drawal from the ground was aided by a steel bar fitting into holes

drilled in the head of the cylinder. Soil was removed by inverting the auger and allowing the core of soil to slip out onto a metal tray. Little or no soil compression occurred using

1) From a design by E.A. Garwood, Grasslands Research Institute, Hurley. 13

Fig. 1. Two inch diam. soil auger. (-fter a design by

iz!.A. Garwood, Grasslands Research Institute, Hurley) 14

this method and the soil core could be cut neatly and accurately

into separate portions when sampling at different depths.

Samples were taken down to 40 cm in the selected grassland

pastures and soil from 0-20 and 20-40 cm depths was bulked

together. For the determination of seasonal vertical distribution

of nematodes two grass plots at Grasslands Research Institute,

Hurley, were chosen and 10 samples were taken at random on each

sampling date, down to a depth of 60 cm on one occasion and to ko cm at all other times. Soil cores were divided into 5 cm

portions and soil from each depth was bulked together.

Extraction Procedures

When possible, nematodes were extracted from the soil on the

day of sampling. Two, 200 ml soil subsamples from each field and

depth were used for extraction by two methods: the tray modific-

ation of the Baermann funnel method (;alitehead and Hemming, 1965)

for the majority of nematodes, and a sieving and sedimentation

method (Flegg, 1967) for mainly Xiphinema and Longidorus. The

nematode suspension from the tray method was allowed to settle

out in a tall, 1 litre beaker overnight. The supernatant was removed by a siphon tube (Fig. 2) set at a fixed depth with a

perspex block to give consistent results and to leave approximately

50 ml of water and nematodes in the beaker. The end of the siphon tube was bent upwards to prevent disturbance of nematodes lying in the base of the beaker.

Roots were extracted from the soil by a sieving and sedimentation technique. A weighed amount of soil was carefully broken 15

Fig. 2. Sinhon apparatus for ra.movin excess water from

nematode susrension. 16

up and soaked for 1 hour in a beaker after which time the

contents were stirred, allowed to settle for 20 secs and then

poured through a bank of sieves (2 mm, 355 p, 250 p, 105 p).

This process was repeated twice more and then the contents of

the top 3 sieves were washed into a beaker. The contents of the

105 p sieve were washed into a separate beaker which was filled

with water, stirred and allowed to settle for 30 secs before

pouring back through the 105 u sieve. Sieve contents were then added to the other washings. Roots and water were then poured

onto a weighed piece of 90 p nylon mesh and allowed to drain

on blotting paper before weighing.

Nematodes were killed at 52°C and fixed in 2°/, formalin

plus calcium carbonate. Part of the nematode suspension, equivalent to 10% of the final volume, was used after fixation for the counting and generic determination of nematodes extracted by the tray method. The total numbers of Xiphinema and Longidorus were counted. Identification to species level was made after processing to glycerine (Baker, 1953). 17

A. DISTRIBUTION OF NEMATODES IN GRi,SSLAND PASTURES.

Surveys of two grassland habitats in Britain, Westmorland and Broadbalk Wilderness (Banage, 1962; Yuen, 1966) have suggested that certain plant parasitic genera, such as, Tylencho- rhynchus, Tylenchus, Ielicotylenchus and Rotylenchus, are commonly found associated with grass in this country although it is difficult to base conclusions on results from these two isolated localities. In the present study soil samples were collected in

August, 1969 from grassland pastures of 15 farms in the counties of Berkshire, Buckinghamshire, Cambridgeshire, Essex, Leicester- shire, Hampshire, and Hertfordshire. The majority of the pastures were used for the grazing of livestock. Results from a pasture at Grasslands Research Institute taken in June, 1969 are included here.

Results

The grassland pastures occurred on 9 different soil types varying from loam sand to silty clay loam (Table 4). Vegetation was varied, Perennial ryegrass (Lolium perenne), Agrostis spp. and Cocksfoot (Dactylis glomerata) being the dominant grasses present and Trifolium spp. the most frequently occurring non- graminaceous plants in the fields (Table 4). Two of the fields had been down to grass for approximately 100 years and other permanent pastures for 20 to 56 years. Many of the fields had not been ploughed within the.living memory of the farmers and it 18

was difficult to ascertain the exact age of the pastures. Five

leys are represented of 2 to 9 year stands.

A list of plant-parasitic nematodes occurring in the soils

is presented in Table 1 together with the frequency of occurrence

and population means. Populations and number of species in the

fields are given in Tables 2 and 3. Nineteen genera were found

altogether and of these only Tylenchorhynchus and Tylenchus were

present in all the fields sampled. Helicotylenchus, Paratylenchus

and Pratylenchus occurred in 15 out of the 16 fields. The other genera most frequently found were Aphelenchoides, Aphelenchus,

Longidorus and Pratylenchoides. The genera Paratylenchus,

Tylenchorhynchus, Tylenchus and Helicotylenchus occurred in the

highest numbers.

Ten species representing the genus Tylenchorhynchus were T. dubius were the most abundant; found, of these T. quadrifer, T. ica7u7-T.-Facrurusitnd T. dubius

and T. lamelliferus were the most commonly recorded.

Helicotylenchus, Longidorus and Pratylenchus each had 6

representative species; H. vulgaris, L. macrosoma, P; penetrans

and P. thornei were found in the most fields. Paratylenchus had

3 species present, P. microdorus occurring in 15 of the fields.

Two species of Pratylenchoides were present, P. laticauda and P. crenicauda although the former was only found in one field.

Trichodorus occurred in small numbers in 3 of the fields sampled.

Genera with only one species present were Hemicycliophora typica, 19

Nacroposthonia rustics, Rotylenchus raodeyl., Truhurus sculptus

and Xiphinema diversicaudatum. Largest total nematode populations were consistently found in the upper 20 cm of soil (Table 3). Large numbers of

Paratylenchus spp. in some of the fields accounted for high nematode populations in the 20-40 cm soil level. In 8 of the fields sampled Longidorus spp. were more abundant below 20 cm.

Discussion

The results indicate that the plant parasitic genera,

Tylenchorhynchus, Tylenchus, Helicotylenchus, Paratylenchus and

Pratylenchus, are commonly found in grassland soils. They support the findings of Banage (1962) and Yuen (1966) in England and investigations in other countries (Geraert, 1967; De Iaeseneer,

1963; Pepper, 1963; Sumner, 1967; Vegte and Heyne, 1963) although

Rotylenchus was less abundant than had been suggested.

The majority of species did not show preferences for particular soil types (Tables 1 and 4). The spread of Tylencho- rhynchus species in all soil types agrees with results from

Germany (De Maeseneer, 1963). However, Geraert (1967) in Belgium found that T. dubius and T. microdorus prefer sandy soils, and

T. lamelliferus prefers heavy soils. All three species occurred in both soil types although T. dubius was the only species to be found in very sandy conditions.

Longidorus spp. were distributed in most soil types in con- 20

trast to the findings of other workers which suggest that Longidorus

prefers coarser soils (Jones et al., 1969). Xiphinema diversi-

caudatum populations occurred in both sandy loam and silty clay

loam as opposed to the findings that they seem to favour medium

or heavy soils (Harrison and dinslow, 1961). Highest populations

of Trichodorus were found in very sandy soil and in the same locality Hemicycli2phora was also present.

The unique habitat afforded by a complete cover of grass with little or no disturbance of host or environment throughout the year would be expected to produce an equilibrium of the soil fauna populations in old permanent pastures. Lowest plant parasitic nematode populations were found on 4 of the 5 young leys and highest populations had built up in some, but not all, of the permanent pastures of 20 or more years standing. Grass- land pastures degenerate over the years and the original high yield grasses are replaced by other plants. A survey in 1959 (Baker, 1962) showed that only one-fifth of the permanent pasture of this country is of good quality containing appreciable quantities of perennial ryegrass, the other pasture is dominated by inferior grasses, such as, Agrostis. The mixture of vegetation was apparent in the permanent pastures sampled (Table 4) and this is likely to affect nematode populations. Occurrence of nematode species in the fields could not be related to the different grass hosts because of the many grasses and other plants present at each site. 21 Table 1. Plant parasitic nematodes recovered from the upper 20 cm of grass pasture soils.

No. of fields Farms found Population Nematodes found (see Table 4) means/200 ml (out of 16) soil + SE

Aphelenchoides spp. 14 1,2,4,5,6,7, 1+1 (9) 8,9,10,11,12, 13,14,15 Aphelenchus spp. 14 1,2,4,5,7,8,9, 32 (16) 10,11,12,13, 14,15,16

Criconemoides spp. 7 1,3,4,9,10,12, 15 (4) 14

C. informis 1 5 Ditylenchus spp. 4 1,3,13,16 5 (o)

Helicotylenchus 15 240 (32) H. californicus 1 12 65 H. canadiensis 1 14 125 H. digonicus 6 4,8,9,11,12,13 112 (54) H. pseudorobustus 3 5,6,14 115 (50) H. varicaudatus 10 1,2,3,4,9,10, 148 (37) 13,14,15,16 H. vulgaris 8 8,9,10,11,12, 115 (37) 13,15,16

Hemicycliophora typica 5 Heterodera larvae 7 3,8,10,12,14, 21 (10) 15,16

Longidorus 10 18 (9) L. caespiticola 3 8,15,16 L. elongatus 2 3,15 L. goodeyi 1 16 L. leptocephalus 1 1 L. macrosoma 5 3,4,10,11,13 L. profundorum 2 8,14 22

Table 1. cont.

Farms found Population No. of fields Nematodes found (see Table 4) means/200 ml (out of 16) soil + SE

Nacroposthonia rustica 5 2,6,8,15,16 13 (4) Meloidogyne larvae 3 1,4,7 5 (o) Paratylenchus 15 400 (110)

P. goodeyi 1 3 35 P. microdorus 15 1,2,3,4,6,7, 341 (104) 8,9,10,11,12, 13,14,15,16 P. projectus 5 3,7,12,14,15 170 (39) Pratylenchoides 11 65 (26)

P. crenicauda 11 2,3,4,6,7,9, 50 (21) 10,11,12,14,16 P. laticauda 1 9 140 Pratylenchus 15 117 (31)

P. crenatus 3 3,4,5 20 (5) P. flakkensis 1 15 15 P. minyus 3 1,4,11 63 (21) P. penetrans 5 1,8,13,14,16 67 (20) P. pratensis 4 2,6,7,16 126 (66) P. thornei 6 4,5,6,8,9,12 87 (82)

Rotylenchus goodeyi 3 5,7,14 90 (33)

Trichodorus 2 90 (35) T. christiei 1 1 T. primitivus 1 1 T. sp. 1 7 Trophurus sculptus 4 4,6,9,13 16 (4) Tylenchorhynchus 16 336 (92)

T. brevidens 5 5,6,9,15,16 84 (36) T. dubius 8 1,3,4,7,9,11, 124 (34) 15,16 T. icarus 3 3,4,7 207 (72) 23 Table 1. cont. No. of fields Farms found Population Nematodes found (see Table 4) means E55 ml (out of 16) soil + SE

T. lamelliferus 8 3,6,7,9,11, 58 (16) 12,14,15 T. macrurus 5 3,8,9,12,13; 166 (106) T. maximus 1 4 95 T. microdorus 2 4,9 T. nothus 7 2,5,9,10,12, 56 (48) 13,14 T. obscurus 2 9,16 53 (3) T. quadrifer 5 3,7,8,14,15 224 (58) Tylenchus spp. 16 1,2,3,4,5,6,7, 342 (43) 8,9,10,11,12, 13,14,15,16 Xiphinema diversicaudatum 5 2,13,14,15,16 4 (1) 24

Table 2. Populations of nematode genera and (in brackets) number of species from top 20 cm of soil in fields sampled. Means of two subsawples/200 ml of soil.

Nematodes Fields sampled 1 2 3 4 5 6 7 8

Aphelenchoides 5 55 - 3 105 50 70 - Aphelenchus 15 15 - - 20 - 20 20 Criconemoides 5(1) - 35(1) 5(1) - _ _ - Ditylenchus 5 - 5 - - - - - Helicotylenchus 25(1) 45(1) 315(1) 230(3) 75(1) 55(1) - 280(2) Hemicycliophora 5(1) - - _ _ - _ - Heterodera - - 10 - - - 5 Longidorus 1(1) - 48(2) 1(1) - - - 93(2) Macroposthonia - 5(1) - - - 35(1) - 20(1) Meloidogyne 10 - - - - - 90 - Paratylenchus 45(1) 100(1) 980(3)1530(1) - 90(1) 655(2) 130(1) Pratylenchoides - 50(1) 5(1) 5(1) - 5(1) 10(1) _ Pratylenchus 225(2) 320(2) 30(1) 55(2) 25(2) 160(2) 45(1) 20(2) Rotylenchus - - - - 25(1) - 110(1) _ Trichodorus 125(2) - - - - _ - Trophurus - - - 5(1) - 10(1) - - Tylenchorhynchus190(2) 30(1)1560(6) 525(5) 10(2) 30(2) 450(4) 310(2) Tylenchus 110(3)115(2)175(3) 370(3) 345(4) 700(4) 430(3) 230(3) Xiphinema - 2(1) ------

Total plant parasites 765 737 3163 2731 6o5 1135 1880 1103 Total nematodes 5945 3050 8360 7235 2355 5550 6135 4260 25 Table 2. cont. Fields sampled Nematodes 9 10 11 12 13 14 15 16

Aphelenchoides 15 35 65 10 40 5 30 _ Aphelenchus - 20 30 95 100 10 10 5 Criconemoides 20(1) 5(1) - 10(1) - 20(1) - _ Ditylenchus - - - - 5 - - 5 Helicotylenchus 270(3) 275(2) 345(2) 305(3) 150(4) 600(3) 260(2) 375(2) Hemicycliophora ------Heterodera - 5 - 5 - 25 5 20 Longidorus - 6(1) 7(1) - 1(1) 2(1) 15(2) 8(2) Macroposthonia ------15(1) 5(1) Meloidogyne ------Paratylenchus 610(1) 95(1) 90(1) 400(2) 50(1) 565(2) 520(2) 145(1) Pratylenchoides 170(2) 215(1) 30(1) 20(1) - 15(1) - 185(1) Pratylenchus 125(1) - 45(1) 435(1) 80(1) 90(1) 30(2) 65(2) Rotylenchus - - - - - 135(1) - - Trichodorus ------Trophurus 25(1) - - - 25(1) - - - Tylenchorhynchus 245(7) 185(1) 185(4) 95(3) 175(2) 520(3) 310(3) 490(3) Tylenchus 290(2) 335(2) 290(2) 470(3) 210(3) 445(3) 275(2) 685(4) Xiphinema - - - - - 5(1) 6(1) 3(1)

Total plant 1770 1176 1087 1847 836 2437 1476 1991 parasites Total 5205 5345 7725 6385 2215 8750 5450 9535 nematodes

Table 3. Nematode populations at 0-20 and 20-40 cm soil depths in the fields sampled.

Nos. of nematodes/200 ml soil. Means of two subsamples..

0-20 cm depth 20-40 cm depth

VI Ea Fields 0 4 4.) -4-) 0 (a 0 0 V) U) 0 0 0 Ca Ca (a g Z ;S44 Ci) CO i 0 0 $4 H 0 (1) 1 0 ki) H •—I 0 co Pi .4 .4 H 0 Pi H T.S c., 0 ••• .0 r 0 0 '0 H 1"--4 H 4 4 .3 -ri H V? A -4-) A' -1-) H H "PV) c".5t; 4C), 0 .. • a • :1,0 • c..1 r' I 0 0 c1 p., • 0 H 4 Si FA Pi PI Pi 4) 4.. 4-1 E: ,-4 .. 34 $4 Pi. ab Pi. -Pcj 471-1 -PO 'i' . -• $1 f'+ cl pi 0 al o "3. 0 0 P. $• Pi 0 Pi 0 Pi 0 0 E-i 0 E-1 0 Lu r { to i-1 Ell E-• ..l• E-1 Er 0 10 P.: ca 14 02 E-I A 4 i 1 190 45 1 765 5945 15 110 4 482 2007 ! 2 30 100737 3050 - 190 - 537 962 3 1560 980 4.6 3163 836o 5 1920 48 2283 4043 4 530 1530 2 2731 7235 - 1405 14 1402 2357 1 5 10 - - 605 2355 - AM ••• 340 900 6 4o go - 1135 5550 - 85 - 325 57o 7 45o 655 - 1880 6135 120 270 - 550 2250 8 310 130 93 1103 4260 - 85 150 471 1285 9 270 610 - 1770 5205 - 65 - 291 9 10 185 95 6 1176 5345 5 530 43 3(6)33 11 185 go 7 1087 7725 - 155 17 333 903 12 g5 400 - 1847 6385 - 75 - 872 1705 13 200 50 1 836 2215 - - 6 123 238 14 52o 565 2 2437 8750 - 210 9 508 1108 15 310 52o 15 1476 5450 55 750 4o 1422 3082 16 4go 145 8 1991 9535 20 170 3 400 1020 27

Table 4. Grasslands sampled for nematodes.

-.•:1011•••••••••••-

Yrs down 1) to con- Predominant Address Other vegetation Soil tinuous vegetation type grass

1 Brickwall Farm 8 Lolium perenne, Agropyron repens, LS Sible Eeddingham Dactylis glomerate Festuca sp., Essex Agrostis sp. Capsella bursa Taraxacum officinale

2 Filldilch Farm 5 Lolium perenne Dactylis glomerate, FEL Swanmore Phleum sp., Holcus Hampshire lanatus, Trifolium sp.

3 More Place Farm 35+ Lolium perenne, Dactylis glomerate, L Much Hadham Agrostis sp. Festuca sp., Herts. Poa pratensis, Phleum sp., Trifolium sp., Ranunculus sp.

4 G.R.I. 7 Lolium perenne L Hurley, Berks.

5 Belney Farm 8-9 Lolium perenne, Trifolium sp., ZyL Southwick Rolcus M01118, Ranunculus sp., Hants. Phleum sp., Hordeum pretense, Festuca sp. Carex sp.

6 Valentines Farm 80-100 Lolium perenne, Ranunculus sp. ZL Barnet Poa annua, Herts. Holcus lanatus, Festuca sp. 28

Table 4. cont.

Yrs down 1) to con- Predominant Address Other vegetation Soil tinuous vegetation type grass

7 Pendley Farm 20 Lolium perenne, Phleum sp., Cirsium ZL Tring Poa sp. sp., Urtica dioica, Bucks. Trifolium sp., Achillea millefolium

8 Manor Farm 20+ Lolium perenne, Trifolium sp., ZL Scraptoft Dactylis glomerata Potentilla sp., Leics. Plantago sp., Taraxacum officinale, Bellis perennis

9 The Grange 50+ Lolium perenne, Phleum sp., Poa sp CL Knapwell Dactylis glomerata Lordeum pratense, Agrostis sp. Lolium sp., Festuca sp., Cynosurus cristatus, Galium sp.

10 Lockington Farm 25 Lolium perenne, Trifolium sp., SZyCL Lockington Agrostis sp. Plantago sp., Leics. Cirsium sp.

11 Firs Farm 100+ Lolium perenne, Dactylis glomerata ZyCL Caxton Agrostis sp. Poa annua, Trifolium sp.

12 Antershill Hall 10+ Lolium perenne Dactylis glomerata ZyCL Phleum sp., Holcus lanatus, Trifolium sp.

Table 4. cont.

Yrs down Predominant 1) Address to con- Other vegetation Soil tinuous vegetation type grass

13 Marsh Hill Farm 2 Lolium perenne Phleum sp., Poa ZyCL Aylesbury sp., Trifolium Bucks. sp., Cerastium sp., Achillea millefolium

14 Lamport Hall 30+ Dactylic glomerata Phleum sp., ZyCL Lamport Lolium perenne, Trifolium sp., Northants. Agrostis sp. Ranunculus sp., Cirsium sp., Prunella vulgaris

15 Overhall Farm 35 Lolium perenne, Dactylis glomerata ZyCL Gilston Agrostis sp. Phleum sp., Hordeum Harlow pratense, Poa sp., Essex Trifolitu sp., Achillea millefolium, Cirsium sp.

16 Childerley Hall 56+ Lolium keyenne, Dactylis glomerata OZL Nadingley Agrostis sp., Phleum sp., Cambs. Trifolium sp. Poa sp.

)Soil types: LS - Loam sand FSL - Fine sandy loam L - Loam ZyL - Silty loam ZL - Silt loam CL - Clay loam SZyCL - Sandy silty clay loam ZyCL Silty clay loam OZL - Organic silt loam 30

B. VERTICAL DISTRIBUTION OF NEMATODES IN GRASSLAND SOILS.

The vertical distribution of nematodes in grassland soils

has been studied by Yuen (1966) and 'dallace and Greet (1964) in

Britain. Yuen concentrated on Helicotylenchus vulgaris and

Rotylenchus pumilis, and Wallace and Greet restricted their study to Tylenchorhynchus icarus. De Maeseneer (1963) has investigated the distribution of total plant parasitic populations at depth in German meadow soils. The influence of depth on population levels of nematodes in other soils has been studied by a number of workers (Harrison and Winslow, 1961; Wallace, 1962; Hoff and

Mai, 1964; Tseng et al., 1968; Flegg, 1968).

During sampling of grass pastures there appeared to be a localisation of Tylenchorhynchus species at specific depths in the soil. Further samples were taken throughout the year in one pasture to determine if the distribution at different depths of

Tylenchorhynchus and other genera changed during the year due to seasonal and other varying environmental conditions. The investigation was concentrated on two field plots,

Highfield and Lime Kiln II, at Grasslands Research Institute,

Hurley. The first plot measuring 10 x 15 m, a 3 year pure stand of Lolium perenne var. 824 on Highfield, was sampled only once.

The second plot, 10 x 10 m, mainly a perennial ryegrass pasture on Lime Kiln seeded 6 years previously, was sampled 5 times throughout the year. Both fields had loam soil overlying sandy 31

clay loam with chalk at varying depths.

Results

The nematode species found in Lime Kiln soil are presented in Table 1 (farm 4). Species in Highfield differ by the notable absences of Tylenchorhynchus maximus, T. microdorus and Praty- lenchoides crenicauda. Longidorus macrosoma was replaced by L. profundorum. In both fields 3 species of Helicotylenchus were present (H. digonicus, H. varicaudatus, H. vulgaris) and Tylencho- rhynchus species common to both were T. dubius, T. icarus and T. brevidens. Paratylenchus microdorus was the only representative of this genus in the two fields. The results from the one sample at Highfield are presented in fig. 3 and Appendix Table 1 and those from Lime Kiln in figs. 4-8 and Appendix Table 2. Greatest numbers of free-living and plant parasitic nematodes were found in the top 20 cm of soil in both fields at all sampling times and populations generally decreased with depth below this

layer. Very few nematodes were found below 35 cm soil depth. Tylenchorhynchus

The distribution of Tylenchorhynchus was on the whole restricted to the upper 20 cm of soil. The dominant species in order of abundance were T. dubius, T. icarus and T. maximus: highest respective populations of 1910, 1260 and 570 per 200 ml soil were recorded in the 0-5 cm soil layer (Fig. 7). Maximum 32

numbers consistently occurred in the top 5 cm of soil samples taken from Lime Kiln throughout the year (Fig. 8). Only occasionally were Tylenchorhynchus spr. found below 20 cm. Both T. brevidens and T. microdorus were present in small numbers. Paratylenchus Paratylenchus microdorus was found at all depths sairipled, but highest numbers were found between 15-25 cm, maximum populations fluctuating from 2,880 to 39,850 per 200 ml soil at this depth in Lime Kiln (Figs. 4 and 7). Highest populations occurred in the winter months of January and December and concentration of nematodes at the 15-25 cm soil depth was constant at the different sampling times (Fig. 8). P. microdorus constituted the bulk of plant parasitic nematodes in the soil. Longidorus Longidorus profundorum in Highfield and L. macrosoma in Lime Kiln were only extracted from the deeper soil layers. Highest numbers occurred in the 30-40 cm soil layer and L. macrosoma was found to be present at 55-60 cm when samples were taken to this depth in March (Appendix Table 2 b). Low populations of both species were found, the highest for L. macrosoma being 46 per 200 ml soil at the 30-35 cm depth. Distribution of L. macrosoma was constant throughout the year at the lower soil depths differing from both that of Tylenchorhynchus and Paratylenchus (Fig. 8). 33

Other .plant feeding nematodes Highest populations of Tylenchus spp. were recovered from the top 15 cm of soil at all times and numbers decreased with depth down to 40 cm. Pratylenchus (P. minyus, P. thornei, P. crenatus) and Helicotylenchus were irregularly distributed in the soil profile with highest populations occurring in different soil depths at the various sampling times. Both genera were recovered from the 35-40 cm soil layer, and Pratylenchus occurred in soil from the 55-60 cm depth taken in March (Appendix Table 2 b). In Highfield populations of Pratylenchus increased with depth and maximum numbers were extracted from the 35-40 cm soil layer (Fig. 3). Pratylenchoides crenicauda and Meloidogyne occurred in small numbers generally below 20 cm soil depth.

Soil temperatures were seasonal at all depths, decreasing with depth during the warm months and increasing with depth during the winter. A maximum of 16.5°C was recorded at the 0-5 cm soil depth in June and a minimum temperature of 2.800 at

10-25 cm in March and 0-5 cm in December. Soil moisture was always highest in the 0-5 cm soil layer and decreased with depth at all sampling times (Figs. 3,4,5 and 7). Grass roots were concentrated in the upper 5 cm of soil in both fields (Figs. 3 and 7).

Soil temperature °C 5 10 15

Root content gm.(dry wt./100 gm. of soil) 0.5 1.0 1.5 No. of nematodes / 200m1. soil Moisture content Z dry wt. of soil 2000 4000 6000 MOO 10000 12000 14000 10 20 30

0-5

5-10 Paratylenchus • • Roots rcr°637"—'1/ 10-15

\ 15-20

Vx. 20-25 Total nematodes S. 25-30 • • 8 30-35 2210.68

340 -• r

No. of nematodes / 200 ml. of soil

200 400 600 600 1000 1200 50 100 150 200 250 300 350 400

Tylinchorhynchus 0-5 shibius

5-10

Tylenctiorhynchus 10-15 Icarus •".• I / E 15-20 O I / Helicatylenchus Tyleachus spp. S 20-25 • O 25-30 •

PrMyfinictim sm. 30-35 2210.60 / 35-40 •

Fig. 3 Vertical distribution of nematodes and soil temperature, root content and moisture content in Highfield grassland on 22.10.68. 35

Soil temperature °C 5 10 15 1 No. of nematodes / 200m1. of soil Moisture content 1 dry wt. of soil 10 20 30 2000 4000 6000 8000 10000

0 0-5 • \.s. Paratywochus 5-10- • Temp. —filo ii icr°d°rus Moisture N• 10-15 - E 15-20 1 0/ o. 20-25 Total nematodes s/ 25-30 7/

30-35 -• x 22.1.69 1. a 35-40

No. of nematodes / 200 ml of soil 200 400 600 800 200 400 600

T.maximus 0-5 • 0 x • stS Pratylenchus 5-10 ( 5P12- x . Tylenchus Tylenchorhynchus spp. Icarus 10-15 • T.dubius

15-20 •0

20-25

25-30 4 HeliCotyl.:; us Longidorus p macrosoma 30-35 • /

35-40 V

Fig. 4 Vertical distribution of nematodes, and temperature and moisture content in Lime Kiln grassland on 22.1.69.

Soil temperature °C 5 10 15 1 I . Na of nematodes /200 ml of soil Moisture content % dry wt. of soil 2000 4000 6000 8000 10000 10 20- 30 I I I

0-5 -4 \ P. microdorus 5-10 — • Total x cf nematodes 10-15 x o U 15-20 Moisture x / o. 13 20-25 X 0 <--"'"?ernp. 1 25-30 X 0

29.4.69 I 30-35 —•X x I 35-40 •X X 0

Na of nematodes / 200 ml of soil 100 200 300 400 500 100 200

0-5 x T. maximus Pratylenchus 11 fi spp. 5-10 Ox Tylenchus Tylenchorhynchus sPIL Icarus 10-15 0 /// T. dubius / Helicotylenchus :F▪ 15-20 • , SPP. o. I /N 13 20-25 •

25-30 11.# 1"I'gicismacrosoma 30-35

35-40

Fig. 5 Vertical distribution of nematodes, and temperature and moisture content in Lime Kiln grassland on 29.4.69. 37

Soil temperature °C 5 10 15 No. of nematodes / 200 ml of soil 2000 6000 10000 14000 I

0-5 - • xO \ P. microdorus • • 5-10 ra\ Total nematodes 10-15 • 0 E 15-20 • O

A' 20-25 ir• Temperature 25-30 • x

30-35

\ 35-40 • x

No. of nematodes / 200 ml of soil 600 800 200 400 600

T.maximus Pratylenchus app. 0-5 X

5-10 o t T. dublus Tylenchus = 10-15 Ty lencMrhynchus sP13. I/ Icarus .c 15-20 • /\t/ al CI 20-25 I

25-30 4,___,LmOdoms .104- mac rosoma 30-35 II/---He"cota?chus

35-40 • Ill

Fig. 6 Vertical distribution of nematodes, and temperature in Lime Kiln grassland on 25.6.69.

Soil temperature 'So

Root content gm(drywt0-7100grn / of soil) 0-1 0-3 0.5 No. of nematodes /200m1 of soil Moisture content z dry wt. of soil 10000 30000 50000 10 20 30

0-5 0 x

5-10 . I 7 Total . • R7oots nematodes \ I I 10-15 o• . P. mi crodorus ...f r__, 15-20 , x Moisture is 1 Temp. / 20-25 • 0) x

I a 25-30 1 0 I H 30-35 0 / 16.12-69 . • / \ / 35-40 - • o r

No. of nematodes /200 ml of soil 500 1000 1500 2000 100 200 300 400 500

T. maximus 0-5 PrMylmOm 5-10 o Tylenchus

E T. dubius 10-15 sPI). Tylenehorhynchus II/ Icarus ugg:= 61 15-20 Lf ✓ x i \ 20-25 ri x

25-30 • ////e

30-35 - Helicotylenchus WIN Itic/ 35-40 .ox •

Fig. 7 Vertical distribution of nematodes, and temperature, root content and moisture content in Lime Kiln grassland on 16.12.69.

Sampling dates JFMAMJJASOND 11111T I I I I I I I

0-5 0 0 0 0 o

5-10

E 10-15 U

-c 15-20 1B--IN III ii 0 20-25 e a

25-30

30-35 • •

35-40 • •

o o Tylenchorhynchus spp.

■ ■ Paratylenchus microdorus

• • Longidorus macrosoma

Fig. 8 Maximum concentrations of Tylenchorhynchus spp., Paratylenchus microdorus and Longidorus macrosoma at different depths from January to December, 1969 in Lime Kiln grassland. 40

Discussion

The results have indicated that many of the nematode species

under grass have distinctly different soil depth preferences

which do not vary at different sampling times. Tylenchorhynchus

spp., Paratylenchus microdorus, Longidorus macrosoma, L.

profundorum and Tylenchus spp. were consistently found at specific

soil depths, however, species of Helicotylenchus and Pratylenchus

were more variably distributed in the soil and maximum numbers

were concentrated at different depths throughout the year.

Why should nematodes in the same soil profile be distributed

at different depths? It has been suggested that the depth at

which plant parasitic nematdoes occur is influenced mainly by the

distribution of plant roots, but other factors, such as, temp-

erature, water, aeration and soil type, must be taken into consider-

ation because of their effect on reproduction and survival

(Wallace, 19,.5). Soil aeration has been shown to fluctuate in

the soil profile and this may influence the vertical distribution

of nematodes. Low oxygen levels have been reported to reduce

populations of plant parasitic nematodes (Stolzy et al., 1962;

Van Gundy and Stolzy, 1961; Wallace, 1956). Van Gundy et al.

(1962) found that the amount of oxygen available to nematodes.

decreased with soil depth and showed that certain nematode

species were more sensitive to reduced oxygen levels than others.

In my experiments soil temperature varied considerably at all 41

soil levels, increasing with depth during the cold winter months

and decreasing with depth during the summer. The soil acts in

an insulatory capacity and the surface layers heat up and cool

down more rapidly than the lower depths. There was no relation-

ship between temperature and the distribution of nematodes in the soil profile.

The vertical distribution of all species of Tylenchorhynchus

was related to root distribution; the greatest concentration of

grass roots occurred in the upper 5 cm of soil in both fields and

decreased markedly below this depth. Higher moisture levels in

the upper soil layers may partly account for the prevalence of

Tylenchorhynchus and Tylenchus in these layers. Wallace and Greet

(1964) showed that the largest populations of Tylenchorhynchus

icarus at about 5 cm depth in a plot of timothy and meadow fescue

corresponded to the greatest root concentration and numbers in the

top 5 cm decreased during dry periods. The restriction of

Tylenchorhynchus to the upper 20 cm of soil under grass confirms

the results by De Maeseneer (1963) who showed that ten species of

,Tylenchorhynchus were confined mainly to the top 20 cm of meadow

soil; the predominance of Tylenchorhynchus spp. in the 0-20 cm

soil layer in the survey of grassland pastures agrees with this

evidence. The complete vegetative cover afforded by grass pasture seems to be an important contributory factor in the vertical distribution of Tylenchorhynchus. Mukhopadhyay and 42

Prasad (1968) found that, in general, largest numbers of

Tylenchorhynchus were recovered from 10-20 cm under arable crops

and under fallow the 20-30 cm layer supported the maximum number

of nematodes.

The distribution of Longidorus macrosoma and L. profundorum

remained constant at the lower soil depths where there was the

smallest quantity of grass roots and where aeration is considered

to be low. Flegg (1968)-similarly found an increase in numbers

of L. macrosoma with depth under a hawthron hedge and L.

profundorum around pear roots did not parallel root distribution.

A possible explanation for the vertical distribution of these

nematodes may be that Longidorus favours the different soil

texture and structure found at the lower depths although no

preference for different soil types was indicated by the distrib-

ution of Longidorus in the grassland survey.

The highest populations of Paratylenchus microdorus in Lime

Kiln and Iiighfield were constantly found between 10 and 25 cm

contrasting markedly with the distribution patterns for both

Tylenchorhynchus and Longidorus. The high total nematode popul-

ations at the lower depths and at 20-40 cm in the other fields were mainly due to presence of large numbers of P. microdorus.

There appeared to be no relationship between nematode numbers

and root distribution. 43

Helicotylenchus and Pratylenchus were found at all depths in the soil and populations varied considerably during the year at the different depths. Yuen (1966) found that there was no optimum depth at which H. vulgaris occurred under grass.

Pratylenchus spp. are endoparasitic in grass roots and Helicoty- lenchus spp. are also possibly so (Cohen, personal commun.) and the migration of nematodes between roots and the soil may partly explain the variability of their depth distribution.

Highest overall nematode populations were extracted from soil in December and lowest populations in March. Similar population changes at the different sampling times were apparent for many of the nematode species; however, interpretation of such results as being directly attributable to seasonal variations are difficult when length of soil storage time, room temperature during extraction, the general activity of the nematodes at different soil temperatures and their reaction to temperature changes have to be taken into consideration.

The results emphasise the importance of understanding the distribution patterns of nematodes at different depths around plant roots when general survey work is undertaken. 44. SECTION III.

TYLENCHORHYNCHUS SPECIES FOUND IN GRASSLAND PASTURE SOILS The genus Tylenchorhynchus was first thoroughly reviewed by Allen (1955) when it constituted 34 species, 22 of which were described as new in his paper. A key published by Loof (1959) included another 8 species that had been proposed since 1955, and, in a compendium of the genus by Tarjan (1964), a further 23 species were added to the rapidly growing list. Guiran (1967) included 70 valid species in a key of Tylenchorhynchus and between 1967 and the beginning of 1970 twenty seven additional species were described. There is now the incredible number of 150 species in

this group and a possible 30 new species to be described (Sher, personal communication) making their identification exceedingly difficult. The genus contains species that differ widely in their morphological characteristics and even as early as 1955 Allen had suggested that separation of the diverse forms into two or more genera was possible. Thorne and Malek (1968) erected two closely related genera, Nagelus and Geocenamus, and Siddiqi (1970) proposed a new genus, Merlinius, for those species of Tylencho- rhynchus that differed mainly by having 6 incisures in the lateral field and shape of the male spicule. Siddigits proposals are not widely accepted as there still remain striking morphological differences between species in his two genera and his suggestions only serve to increase the complexity of the group. Until the group is reviewed in the light of new evidence and all the morphological characters are taken into consideration it seems wiser to accept the various species under one genus. 45

Descriptions of Tylenchorhynchus species

Tylenchorhynchus obscurus Allen, 1955 (Fig. 9, A-B)

Measurements: 394? L = 0.807-0.829 mm; a = 28-31; b = 5.2-7.2; c = 14-17; V = 53-58%; stylet = 26-28.5 p. • 3(Sd L = 0.800-0.845 mm; a = 29-33.5; b = 4.9-6.1; c = 12-14; T = 42.3-55.2%; stylet = 26-28 u; spicule = 28-32.5 p; gubernaculum = 10-11.5 p. Female: Body tapering at both ends, in a closed C-shape when relaxed by gentle heat. Lip region rounded, set off from body by a slight depression, 7 annules. Weak head skeleton. Stylet slender, 26-28.5 p long, with small rounded basal knobs. Pyriform basal oesophageal bulb. Tail 2.2-3.0 times anal body width, tapering with rounded, annulated terminus. Six incisures in lateral field. Phasmids opening at middle or posterior to middle of tail. Male: Similar to female. Bursa envelops tail, Spicule 28-32.5 p in length with notched distal portion.

These specimens of Tylenchorhynchus obscurus were marginally different from those described by Allen (1955) in body and stylet size, and having a more rounded lip region, but no other morgh- °logical differences were apparent.. T. obscurus can be recognised 46 by the long slender stylet, tapering tail and 6 incisures in the lateral field.

Tylenchorhynchus maximus Allen, 1955 (Fig. 9, D, E)

Measurements: L = 1.007-1.304 mm; a = 39-47; b = 6.6-7.7; c = 17-22; V = 49.2-53.7%; stylet = 20-24 ii. Female: Cylindrical body, often in a spiral shape when relaxed by gentle heat. Lip region bluntly rounded, set off from body by a slight depression, 7 annules. Weak head skeleton. Stylet slender, 20-24 p long, with rounded basal knobs. Four incisures in lateral field. Tail cylindrical, with bluntly rounded, annulated terminus. Tail 2.7-3.8 times anal body width. Post anal intestinal sac present, extending two-thirds of tail length. Cuticle coarsely annulated. Phasmids opening at middle to posterior middle of tail. No males.

Tylenchorhynchus maximus can be recognised by its large size and slender proportions, cylindrical tail with annulated terminus, coarse annulation of the cuticle and presence of a post-anal intestinal sac. T. maximus is similar in some respects to T. dubius from which it differs by its larger size, longer stylet, almost continuous lip region and the absence of males. 47

50µ

Fig. 9 Tylenchorhynchus obscurus: A, anterior region of female; B, female tail; C, male tail. T. maximus: D, anterior region of female; E, female tail. 48

Tylenchorhynchus brevidens Allen, 1955 (Fig. 10, 11-C)

Measurements: 10vi? L = 0.526-0.676 mm; a = 21.5-27; b = 4.5-5.1; c = 12-14.5; V = 53-58.5%; stylet = 14-15.5 u. 1 L = 0.632 mm; a = 35; b = 4.6; c = 11.9; T = 62.1%; stylet = 15 u; spicule 22.5 u; gubernaculum = 7.5 u. Female: Body tapering at both ends. Lip region flattened almost continuous with body contour, having 5-6 annules. leak head skeleton. Stylet 14-15.5 i long, with small rounded basal knobs. Six incimres in lateral field. Tapering tail with terminus flattened and smooth, 2.4 to 3.3 anal body widths long. Phasmids opening posterior to middle of tail. Male: Similar to female. Bursa envelops tail. Spicule 22.5 u long with notched distal portion.

Tylenchorhynchus brevidens can be distinguished by shape of the head, the tapering tail with a smooth flattened terminus, and the small stylet. It differs from T. microdorus in having an almost continuous blunt lip region, generally longer stylet and a more flattened tail terminus, and from T. nothus by length of stylet and absence of annulations on the tail terminus. 49

Tylenchorhynchus microdorus Geraert, 1966 (Fig. 10, D-F)

Measurements: 5isli L = 0.545-0.636 mm; a = 24.5-29; b = 4.4-5.2; c = 13-14.5; V = 55.4-57.6%; stylet 11-14 12. 2SS L = 0.561-0.586 mm; a = 24.5-26.5; b = 5.0-6.0; c = 10.5-12.5; T = 53.4-64%; stylet 11-12.5 p; spicule = 20-23 u; gubernaculum 7-7.5 u Female: Body tapering at both ends. Lip region low and rounded, set off by a slight constriction, 5-6 annules. Indistinct head skeleton. Stylet short, 11-14 u, with small backward sloping basal knobs. Six incisures in the lateral field. Tail conical with rounded terminus without annulations, 2.5 to 2.9 anal body widths long. Phasmids opening posterior to middle of tail. Cuticle finely annulated. Male: Similar to female. Bursa moderately developed enveloping tail.

Spicule with notched distal portion 20 to 23 u long.

Tylenchorhynchus microdorus can be recognised by the small stylet with backward projecting basal knobs, tapering tail with no annulations on terminus, and head offset by a slight constriction, Differs from the closely related T. brevidens by shape of head, indistinct head skeleton and rounded tail tip, and from T. nothus

A D

sop i

Fig. 10 Tylenchorhynchus brevidens: A, head; B, female tail; C, male tail. T. microdorus: D, head; E, female tail; F, male tail. 51

by size of stylet, head shape and lack of annulations on tail terminus.

Tylenchorhynchus dubius (Butschli, 1873) Filipjev, 1936 (Fig. 11, A-C)

Measurements: 1029 L = 0.658-0.753 mm; a = 23.5-35; b = 5.1-5.9; c = 13.5-17; V = 54.7-57.4%; stylet = 17.5-19 g 5cUL = 0.606-0.699 mm; a = 30.34; b = 5.2-6.3; c = 13-17; T = 56.5-57.3%; stylet = 16.5-19 u; spicule = 21-24.5 g; gubernaculum = 9-11.5 g Female: Cylindrical body gently tapering at anterior and posterior ends. Lip region rounded, set off from body by a marked constriction, 6-7 annules. Slight head skeleton. Stylet 17.5-19 u long, with rounded, backward sloping basal knobs. Cylindrical tail with a broadly rounded annulated terminus, 2.5-3..5 anal body widths in length. Four incisures in the lateral field. Phasmids opening anterior to middle of tail. Post anal intestinal sac present extending one-third to one-half length of tail. Cuticle finely annulated. Male: Similar to female. Bursa enveloping tail. Spicule with distal portion pointed, 21-24.5 p in length. 52

Tylenchorhynchus dubius can be recognised by the lip region

set off by a marked constriction, presence of a post anal

intestinal sac and four incisiires in the lateral field. It is similar to T. maximus from which it differs by the set off lip region and shorter body length.

Tylenchorhynchus nothus Allen, 1955 (Fig. 11, D-F)

Measurements: an L = 0.584-0.746 mm; a = 25-28; b = 4.2-5.6; c = 11-13.5; V = 54.4-58%; stylet = 15-18 p.

d' L = 0.550; a = 27.5; b = 5.0; c = 10.5; T = 51%; stylet = 18 11; spicule = 21 p; gubernaculum = 8.5 .i. Female: Body tapering at both ends. Lip region rounded continuous with body contour, 5 to 6 annules. Indistinct head skeleton. Stylet slender, 15 to 18 A long, with small, rounded basal knobs. Six incisures in the lateral field. Tail elongate-conoid, 2.6-3.6 times anal body width, with annulated terminus. Phasmids opening at middle of tail. Cuticle finely annulated. Male: Similar to female. Bursa envelops tail. Spicules 21 A in length with notched distal portion.

Tylenchorhynchus nothus is a difficult species to identify but

A D

Fig. 11 Tylenchorhynchus dubius: A, head; B, female tail; C, male tail. T. nothus: D, head; E, female tail; F, male tail. 54

can be distinguished by the tapering of the body at both ends, rounded lip region continuous with body contour and the elongate- conoid tail with annulated terminus. It differs from T. brevidena and T. microdorus by shape ofthe lip region and presence of annulations on the tail terminus.

Tylenchorhynchus lamelliferus (de Man, 1880) Filipjev, 1936 (Fig. 12, A-C)

Measurements: 10n L = 0.806-1.045 mm; a = 28.5-32.5; b = 5.5-6.9; c = 15-21; V = 51.3-56.2%; stylet = 21-27 11 53S L = 0.757-0.933; a = 26-31; b = 5.4-6.8; c = 19-23; T = 56.8- 63.6%; stylet = 21.5-24.5 p; spicule = 32.5-38 p; gubernaculum = 14-17 u Female: Body tapering at both ends, lying in an open C position when relaxed by gentle heat. Lip region continuous with body contour, bluntly rounded, having 5 to 6 annules. Faint head skeleton. Stylet 21 to 27 p long, delicate with small rounded basal knobs. Four incisalres in lateral field. Cuticle finely annulated with prominent longitudinal ridges, 15 to 17 near middle of body. Tail clavate with rounded, bulbous distal end; annulations extend round terminus of tail. Phasmids opening just anterior to middle of tail.

Male: Similar to female. Bursa curved for three-quarters of its length 55

and then straight, enveloping tail. Spicule 32.5-38 p in length with distal portion pointed.

Tylenchorhynchus lamelliferus is a unique species characterised by shape of the female tail, bhe shape of the male bursa, the prominent longitudinal striations, and the long, delicate stylet.

Tylenchorhynchus quadrifer Andrassy, 1954

Measurements: 10w L = 0.624-0.720 mm; a = 24-29.5; b = 4.9-6.0; c = 14.5-24.5; V = 53.2-57.4; stylet = 16.5-18 p 5 EL = 0.638-0.699 mm; a = 25.5-30.5; b = 4.9-5.5; c = 14-20; T = 49.7-64.6%; stylet = 16.5-18 p, spicule = 23-25 p; gubernaculum = 7.5-9 s Female: Cylindrical body gently tapering at both ends. Lip region rounded, set off from body contour by a slight constriction, 5-6 annules. moderate head skeleton. Stylet 16.5 to 18 p. long with rounded basal knobs. Six incisures in lateral field. Cuticle has fine longitudinal striations forming a superficial network. Tail cylindrical with flattened terminus that is not annulated, 1.6 to 2.6 anal body widths long. Phasmids opening at middle of tail. Male: Similar to female. Bursa envelops tail. Lateral field is areolated on tail. Spicule 23-25 p in length with notched distal 56

Sap

Fig. 12 Tylenchorhynchus lamelliferus: A, head; B, female tail; C, male tail. T. quadrifer: D, head; E, female tail; F, male tail. 57 end.

Tylenchorhynchus euadrifer is characterised by the fine longitudinal striations forming a superficial network, conspicuous smooth tail terminus, and six incisures in the lateral field. Guiran (1967) synonymised T. ornatus Allen, 1955 with T. quadrifer.

Tylenchorhynchus icarus Wallace and Greet, 1964 (Fig. 13, A-0

Measurements: 10 L = 1.299-1.795 mm; a = 30-41; b = 6.5-7.9; c = 19.5-24.5; V = 52-57.7%; stylet = 34.5-58.5 )1 5 crorL = 1.191-1.622 mm; a = 32-40.5; b = 5.8-6.9; c = 15.5-18.5; T = 44-68.2%; stylet = 34-36 p; spicule = 38-41 p; gubernaculum = 12-13 p Female: Body cylindrical, tapering at anterior end$ in an open C position when relaxed by gentle heat. Lip region bluntly rounded, continuous with body contour, 6 to 8 annules. Head skeleton strongly developed. Stylet large and robust, 34.5 to 38.5 n in length, with well developed, rounded basal knobs. Six incisures in the lateral field. Tail cylindrical, broadly rounded, 2.0 to 2.6 times anal body width in length. Annules present on tail terminus narrower than other tail annules. Cuticle coaraely annulated. Phasmids conspicuous, opening at middle of tail. 58

Male: Similar to female. Bursa envelops tail. Spicules 38 to 41 u long with notched distal end.

Tylenchorhynchus icarus can be distinguished by its large size, robust stylet, well developed head skeleton, and broadly rounded, cylindrical tail.

Tylenchorhynchus macrurus (Goodey, 1932) Filipjev, 1936 (Fig. 13, D-F)

Measurements: 10 ?? L = 0.766-1.011 mm; a = 22.5-33; b = 4.9-5.7; c = 14-16.5; V = 53.2-58.7%; stylet 25.5-31 p. 5 dd L = 0.848-0.950 mm; a = 24.5-33; b = 5.1-5.8; c = 11-13.5; T = 51.9-58.2%; stylet = 26-33 p; spicule = 31.5-38 p; gubernaculum = 10.5-14.5 P. Female: Body cylindrical, tapering at both ends, in an open C position when relaxed by gentle heat. Lip region bluntly rounded, continuous with body contour, 6 to 8 annules. Head skeleton strongly developed. Stylet robust, 26 to 33 u long, with well developed, rounded basal knobs. Six incisures in the lateral field. Tail narrower than body 2.5 to 311 times anal body width in length, slightly clavate, with a rounded, annulated terminus. Cuticle 59

I : I I I I

50p

Fig. 13 Tylenchorhynchus icarus: A, head; B, female tail; C, male tail. T. macrurus: D, head; E, female tail; F, male tail. 60

coarsely annulated. Phasmids conspicuous, opening at middle of tail. Male:

Similar to female. Bursa envelops tail. Spicules 31.5 to 38 p long with notched distal end.

Tylenchorhynchus macrurus is very similar to T. icarus from which it was separated mainly on body size and size of stylet by Wallace and Greet (1964). It can also be distinguished from T. icarus by 1) its longer tail in relation to body length (see 'C' ratios), 2) its slender and slightly clavate tail, and 3) the annules on the tail terminus being similar in size to annules on the rest of the tail, whereas with T. icarus the terminal annules are narrower. 61

SECTIGN IV. THE FEEDING BEHi,VIOUR OF NEM-TODES ON GRISS ROOTS.

Introduction

Root-feeding nematodes can be arbitarily split into different groups depending on their mode and position of feeding in relation to the roots on which they feed: 1) ectoparasites, nematodes that remain with their bodies completely external to the root throughout feeding; 2) endoparasites, nematodes invading roots and feeding on internal tissues and 3) semi- endoparasites, species that feed with only the anterior part of their bodies embedded in the root, females becoming swollen and sedentary. Some nematodes that remain vermiform and migratory but feed with their heads embedded in the root have been termed semi or intermittent parasites (Goodey, 19+3)•

The feeding behaviour of external root-feeders, or ectoparasites, varies with different genera: some have a more involved, or advanced, relationship with the host plant, such as Hemicycliophora (McElroy and Van Gundy, 1968), while others, such as Tylenchorhynchus, Tetylenchus and Tylenchus, have been classed as browsing nematodes because of their reported short feeding times and cursory feeding behaviour (Zuckerman, 1969).

However, many generalisations have been made on the behaviour pattern of a complete genus when only a few species in the genus have been studied in detail. 62

The complete feeding process of ectoparasitic nematodes can

be divided into three distinct phases - stylet penetration, a

delay period and pulsation of the median oesophageal bulb.

Nematodes with short stylets normally only penetrate the surface

cells of the root (Khera and Zuckerman, 1962; Klinkenberg, 1963;

Krusberg, 1959; Linford et al., 1949; Rohde and Jenkins, 1957;

Zuckerman, 1962), but nematodes with long stylets can penetrate

deeper into the roots (Cohn, 1970; Fisher and Raski, 1967; Van

Gundy and Rackham 1961; Paracer et al., 1967; Pitcher and Posnette,

1963; Schindler, 1957; Sutherland, 1969).

Stylet penetration is followed by a delay period, also

called a salivation period (McElroy and Van Gundy, 1968), when

enzymes are probably secreted into the root cell. The movement

forward of what is presumably saliva to the base of the stylet

has been observed during this period (Klinkenberg, 1963; McElroy

and Van Gundy, 1968; Rhoades and Linford, 1961; Sledge, 1959).

After the delay period there follows the final phase which is the pulsation of the median oesophageal bulb in those nematodes

that possess this structure. This phase has been termed the ingestion period (McElroy and Van Gundy, 1968) when cell contents are considered to be drawn into the nematode by the pumping action of the median bulb. In this section of the thesis the feeding behaviour of seven species of the genus Tylenchorhynchus on the same host, 63

Lolium perenne, are compared. Tylenchorhynchus species show a diversity of morphological characters (Section III) and the possibility that the feeding behaviour of these species is equally diverse was investigated. Detailed studies on the feeding of only two species of the genus have been done, T. claytoni

(Krusberg, 1959; Khera and Zuckerman, 1963; Troll and Rohde,

1966) and T. dubius (Klinkenberg, 1963).

Materials and Methods

All nematodes were initially extracted from grass pasture soils, some were obtained from soil cultures established in pots planted to grass in the glasshouse. Extraction from soil was by means of the tray modification of the Baermann funnel method

(Whitehead and Hemming, 1965), and nematodes were used as soon as possible after recovery.

The feeding behaviour of the different nematode species was studied on roots of the same host, perennial ryegrass (Lolium perenne var. S.24) - an important grass in the composition of swards, growing in sterile 1% water agar. Grass seeds were dehusked and surface sterilised by immersion in 2% sodium hypochlorite solution for 15 minutes followed by 3 washings in sterilised water. Seeds were then germinated on moistened, sterilised filter paper in Petri dishes and forceps were used to gently insert the young seedlings into the agar in sterile, plastic Petri dishes when 3 or 4.days old. Nematodes were hand 64 picked from the nematode suspension and then washed twice in sterile water before being transferred to the agar with a sterile needle; further surface sterilisation of the nematodes was found unnecessary. After nematodes had been inoculated, the petri dishes were sealed with tape and maintained in the laboratory at room temperature. Nematode behaviour on the roots was observed bybey inverting petri dishes on the stage of a compound microscope after time had been allowed for the roots to grow to the bases of the dishes. The thin plastic of the dishes enabled observations of up to 400 magnifications to be made. A thousand magnifications was obtained by transferring a thin block of agar containing roots and nematodes onto a microscope slide, covering with a cover-slip and sealing with candle-wax.

A. FEEDING BEIEVIOUR OF TYLENCHCRHYNCHUS SPECIES.

Results

All species of Tylenchorhynchus moved freely in the water agar for a minimum of 2 weeks before becoming quiescent due to a deterioration of their food supply and a slight drying out of the agar. Quiescent nematodes could be revived by transferring to water or a fresh agar plate.

A summary of the length of feeding times and feeding sites is presented in Table 5. 65

Table 5. Summary of feeding observations.

Time of Time of Nematodes stylet Delay period median bulb Feeding site penetration pulsation

______"'"- ____o..-.---.~._,. ______.~ _____

Tylenchorhynchus 33 sees - 40 sec - 3 min 45 sec - Root hair & brevi dens 3 min 40 sec 5 min 30 sec 18 min 10 sec epidermal cell T. nothus- 6 sec - 33 sec - 54 sec - Root hctir & 2 min 45 sec 11 L1~li 35 sec 20 min 20 sec epidermal cell T. dubius 15 sec - 90 sec - 90 sec - Epidermal 65 sec 6 min 30 sec 10 min 5 sec cell T. m.:tximus 10 sec - 70 sec - 80 sec - Along root 50 sec 4 min 10 sec 12 min 50 sec - 95 sec - 80 sec - Root tip 2 min 45 sec 7 min 30 sec 2 hr 52 min

T. lamelliferus 20 sec + 3 min 40 sec 5 min 25 sec Epidermal ------_16 min -55 min cell T. macrurus 13 min - Along root 13 hr 30 min root tip T. icarus 73 sec - 5 min 55 sec 17 min 19 sec Along root 2 min 18 sec -15 min -5 days root tip 1) Tllenchus 15 sec - 90 sec - 2 min 25 sec Root hair filiformis 55 sec 2 min 36 min AEhelenchoidcs 3 sec - None 5 sec - Root hair & saEr0,Ehilus 14 sec 3 min 44 sec epidermal cell

Pc..r.~ tylenchus 8 hr - Epidermal microdorus 24 hr 30 min cell :!:. Erojectus 2 hr 20 min - Epidermal 24 hrs + cell

1 ) Difficulty was experienced in observing this phase of feeding with Tylenchuso 66

T. brevidens

Nematodes of this species were very active ectoparasites

and moved rapidly from one feeding site to the next. Probing of cells Was restricted to a few tentative jabs of the stylet and often there was only a fewt•seconds delay between withdrawal of the stylet after feeding and a new penetration. They were indiscriminate in their choice of feeding sites sometimes penetrating root hairs (Figs. 14 and 15) and, on other occasions, epidermal cells of., mainly, the root hair region (Fig. 16). The complete feeding process sometimes lasted for 27 mins, but normally was much shorter averaging 11 mins. Time of stylet penetration varied from 23 secs to just under 4 mins (Table 6).

Feeding time was generally shorter on root hairs than on epidermal cells. Observations at a thousand magnifications showed that slight contractions of the median bulb occurred immediately after stylet penetration. No further movements of the bulb or nematode body took place until after the delay period. There was a forward flow of globules in a tract passing through the extremity of the median bulb during this period, however, no secretions from stylet tip were observed.

Feeding was normally at a different site each time and no damage to either epidermal cells or root hairs was observed.

One female laid 11 eggs over a period of 120 hours while feeding 67

Fig. 14 TylenchorhInchus brevidens feeding on root hair.

•

Fig. 15 T. brevidens feeding on root hair. Fig. 16 T. brevidens feedint; on epidermal cell of root hair regio

Fig. 17 T. nothus feeding on root hair. 69 along the length of a young root. The newly hatched larvae fed on the same root as their parent.

T. nothus T. nothus fed in a similar manner to T. brevidens, quickly moving from one feeding locality to the next and choosing both root hairs (Fig. 17) and epidermal cells as sources of food. Nematodes often fed on the same root hair a number of times.

Total feeding time averaged 9 min 30 secs, but sometimes was as long as 27 mins. Stylet penetration and total feeding times were generally longer on epidermal cells, averaging 19 secs and

2 min 43 secs on root hairs, and 1 min 32 secs and 17 min 8 secs on epidermal cells respectively.

During the delay period an opaque, spherical mass was often observed accumulating around the stylet tip indicating the extrusion of substances by the nematode. The accumulated mass weir, gradually reduced by pulsation of the median bulb, however, the stylet was normally retracted before all the mass had been ingested. The process was seen more clearly while nematodes were feeding on root hairs. No damage to cells was observed except that cytoplasmic streaming in root hairs appeared to be reduced after nematodes had fed upon them. T. dubius

The feeding of T. dubius was confined to the epidermal cells 70 of the root hair region (Fig. 18), region of elongation and root tip. No attempt to feed on root hairs was observed. Feeding was normally restricted to a small area on the root in which individual nematodes remained for long periods. Each feed was short, averaging just under 7 mins, and there was little delay between stylet retraction and penetration of an adjacent cell.

On numerous occasions an opaque mass could be seen increasing in size around the stylet tip during the delay period. As with

T. nanus, this accumulated mass was drawn into the stylet during median bulb pulsation.

Slight mechanical damage to epidermal cells ensued when nematodes fed in one area for a number of hours but no effect on root growth was observed.

T. maximus

When suitable roots were present T. maximus fed repeatedly for many days without any noticeable rest periods. Feeding times varied from 3 mins to 3 hours depending on the region of root chosen. They demonstrated a browsing-type feeding behaviour while on epidermal cells of the root hair region and region of elongation (Fig. 19). Here they movedcontinuously from one cell to the next feeding for comparatively short times of from

3 mins to 16 mins. A different type of feeding behaviour was observed on the root tips. Here the nematodes remained for 2 or 3 days at the one site and feeding time was extended. 71

Fig. 18 Male T. dubius feeding on epidermal cell of root hair region also T. brevidens).

Fig. 19 T. maximus feeding on epidermal cell of region of elongation. 72

Marked aggregation of nematodes occurred immediately behind the

apical meristem. After a single nematode had penetrated and

commenced feeding on epidermal cells of the root tip, other

nematodes were clearly attracted to the site possibly by cell

contents seeping out of the ruptured cells. There was a gradual increase in numbers until the nematodes completely covered one

side of the root tip (Figs. 20 and 21). Each separate feed lasted for as long as 3 hours on the root tip and repeated feeds

occurred on the same or adjacent cells. Aggregation of T. maximus did not take place on other parts of the root.

The intensive feeding on root tips caused mechanical break-

down of epidermal cells. Nematodes continued feeding on the underlying cells until eventually cavities were formed in the side of the root (Fig. 22) into which nematodes partially or wholly entered to feed on cortical cells and undifferentiated cells of the vascular system (Fig. 21). Feeding also caused the root to bend back on itself (Fig. 24) and resulted in the cessation of growth usually within 48 hours of nematodes com- mencing to feed. When only two or three nematodes fed on root tip cells they produced slight mechanical breakdown (Fig. 23) and abnormal bending of the root. The reduction is division and elongation of cells damaged by feeding was probably the reason for abnormal bending of the roots. The cells of the primary root tips attacked by large numbers of T. maximus eventually 73

Fig. 20 Lggregation of T. maximus on root tip.

Fig. 21 T. maximus feeding' inside cavity on roct tip. 74

'RP

111

Fig. 22 T.S. of damaged root tip showing cavity formed by breakdown of cells after intensive feeding by T. maximus.

Fig. 23 Slight mechanical breakdown of epidermal cells after feeding by T. maximus. 75 became exhausted of contents and at this stage the nematodes dispersed back along the roots and often re-aggregated on the root tips of young lateral roots.

T. lamelliferus

The feeding behaviour of T. lamelliferus was similar to that of T. maximus. Feeding occurred on epidermal cells of the region of elongation and root hair region, but was mainly confined to root tip cells (Fig. 25). No stylet penetration or probing of the root hairs was observed. Feeding times varied from 10 mins to over one hour with an average of 30 mins from beginning of stylet penetration to the end of median bulb pulsation. The pumping of the median bulb was irregular at all times, alternating from rapid to slow pulsations.

Nematodes were attracted to root tip cells on which other nematodes had commenced feeding and attempted to feed on either the same cells, or adjoining cells. During feeding nematodes became firmly attached to the epidermal cells and were rarely dislodged by the vigorous movements of other nematodes attracted to the same site. Nematodes that had stopped feeding and moved short distances away from the root often returned to the same cell and began feeding again.

Large numbers of T. lamelliferus were not readily available for this study and aggregation or root tip damage was not as marked as it was for T. maximus. Six nematodes feeding on a 76

Fig. 24 Bending of root caused by feeding of T. maximus.

„lammill111

Fig. 25 Male and female T. lamelliferus feeding just behind root tip. 77

single root caused cessation of growth and bending of the root

tip was observed in some instances after feeding by 2 or 3 nematodes.

T. icarus and T. macrurus

T. icarus and T. macrurus are morphologically very similar

(Section III) as are their feeding habits. The behaviour of both species on roots differed markedly from that of the previous five species described. Both species were comparatively inactive and they moved along roots for long periods probing cells with their stylets (Fig. 26) before selecting suitable cells to penetrate.

Ectoparasitic feeding behaviour occurred on the epidermal cells of the root hair region, region of elongation, root tip, and root cap cells (Fig. 27). Stylet penetration of the cell wall was accomplished by direct thrusts of the stylet at right angles to the cell with lips pressed firmly against the wall.

The nematodes remained motionless during the following dhlay period and median bulb pulsation. Both species fed for much longer periods as ectoparasites than other Tylenchorhynchus species; feeding T. macrurus was observed/continuously for 13 hours 30 mins and

T. icarus for as long as 31 hours 45 mins on epidermal cells.

T. icarus and T. macrurus differed from all other Tylencho- rhynchus by feeding on outer cortical cells with their heads embedded in the root in the manner of semi-endoparasites (Fig. 28).

Penetration into the root tissue was achieved by continual thrusts 76

of the stylet against the epidermal cell wall at slightly

different positions. A large number of penetration holes were

produced until eventually the nematodes broke through into the

cell. Stylet thrusts were accompanied by slow side to side move-

ments of the head. Similar penetrations of the sub-epidermal cells followed until the nematode lay with its head embedded in the cortex and then feeding in the normal manner took place.

Sometimes short simple feeds occurred during this sequence.

When nematodes became embedded in the root tissues in the above manner median bulb pulsation continued for many days, 5 days 2 hours 30 reins was the maximum observed for T. Icarus.

Short delays in pulsation may possibly have occurred during these long feeds but none were observed during extensive observations on many nematodes. Nematodes became very firmly attached to the root and were not easily dislodged by other nematodes brushing against them.

Very little observable damage or host reaction was caused by even long feeds. Cells surrounding the head of an embedded nematode became darker in colour but growth of roots appeared normal. On contaminated plates bacteria quickly accumulated around a penetration hole as the cell contents were released into the surrounding agar. Fig. 26 Juvenile T. icarus probing epidermal cells of region of elongation.

Fig. 27 Male T. icarus feeding on root cap ccalr. 80

Fig. 28 T. icarus well embedded in the root during feeding. 81

Discussion

The observations have shown that feeding behaviour varies

considerably between species of the genus Tylenchorhynchus on

roots of perennial ryegrass.

Species that can be classed as browsing ectoperasites are

T. brevidens, T. nothus and, to a lesser extent, T. dubius,

because of their desultory feeding behaviour on epidermal cells

and root hairs with no apparent preference for feeding sites,

and their short term feeding times. T. dubius has been observed

feeding on root hairs of Lolium perenne, but also accumulating

around a root tip (Klinkenberg, 1963).

The feeding of T. maximus and T. lamelliferus was mainly

confined to root tip cells. T. maximus initially migrate along

the roots feeding at different sites for short periods until

they reach the root tip where individual feeds are prolonged

and nematodes remain for many days. The feeding behaviour

exhibited by these nematodes cannot be termed 'browsing' because

they remained at one locality for long periods, and, although

feeding was always on exposed root cells, the breakdown of surface

cells by aggregations of the nematodes enabled them to feed on

the cells of the cortex and differentiating vascular cylinder.

T. maximus can be described as 'intermittent feeders', a phrase coined by Goodey (1943) in another context. Selection of root tips as the main source of food cells has been demonstrated 02

for a number of different ectoparasitic genera including Xiphinema

(Fisher and Roski, 1967; Sutherland, 1969) Hemicycliophora

(Klinkenberg, 1963; McElroy and Van Gundy, 1968) and Longidorus

(Cohn, 1970). McElroy and Van Gundy (1968) observed that

Hemicycliophora avenaria 'sampled' cells at sites other than the root tip, but the complete feeding process (which continued for up to 6 days) only occurred on the root tip.

T. icarus and T. macrurus differ from the other five species of the genus studied by having long feeding times either external to the root on epidermal cells, or embedded in the root on cortical cells. Both species are able to actively penetrate the outer root cells and to feed with their heads embedded in the outer cortex. Goodey (1943) found an adult female of Anguillulina macrura (Tylenchorhynchus icarus) with its anterior oesophageal region embedded in the cortex of Lolium perenne and he called it, as mentioned above, a "semi or intermittent parasite". However,

suggest that 'migratory semi-endoparasite" is a more appropriate and descriptive term to describe the feeding of T. icarus and T. macrurus.

The feeding of most Tylenchorhynchus species had little effect on root growth, but aggregation and repeated feeding of

T. maximus caused cell destruction and cessation of root growth and in so doing destroyed the supply of food cells normally within

2 to 3 days of initial feeding. Destruction of the feeding site 83

on grass is not necessarily inhibitive to the survival of T. maximus because the adequate production of new roots enables a continuous food supply to be available. The very large nematode population that would be required to directly cause the death of established seedlings does not appear to build up under grass pasture. In contrast to feeding causing destruction of cells some ectoparasitic nematodes produce specific; feeding sites by alterations of the host's cellular metabolism. Examples of these nematodes are:- Dolichodorus heterocephalus (Paracer et al., 1967),

Hemicycliophora arenaria (Van Gundy and Rackham, 1961), Longidorus africanus, L. brevicaudatus (Cohn, 1970), Xiphinema diversicaudatum

(Schindler, 1957) and X. index (Fisher and Raski-, 1967). Most nematodes that produce specific feeding sites have in common a very long, continuous feeding time and a very long stylet that permits feeding on inner root tissues with nematodes remaining completely outside the root.

The morphological differences between the seven species in feeding behaviour studies are outlined in Section III. T. brevidens and T. nothus are comparatively small nematodes resembling some members of the Tylenchinae with their tapering bodies, comparatively weak stylets and indistinct head skeletons. At.. the other extreme are T. icarus and T. macrurus, both large nematodes with strong, robust stylets and heavy head skeletons that seem more closely akin to members of the Hopolaiminae. The simple cursory feeding 84 behaviour of T. brevidens and T. nothus contrasts sharply to the more sedentary behaviour of T. icarus and T. macrurus. Although it is unwise to make generalisations with such a small represent- ation of the whole genus, there does appear to be a relationship between morphology and feeding behaviour in the genus

Tylenchorhynchus. From this evidence it might be suggested that several genera are represented by the species that are now included in the genus Tylenchorhynchus.

B. FEEDING BEHAVIOUR OF OTHER NEMATODES

Not all genera of root feeding nematodes move freely in an agar medium and of those used here, Criconemoides, Longidorus,

Xiphinema and Helicotylenchus became inactive in the 1%. agar within a very short time. Paratylenchus and Pratylenchus occasionally survived in the medium for longer periods.

The length of feeding times and feeding sites for the species observed are presented in Table 5.

Tylenchus filiformis

Males, females and juveniles of T. filiformis confined all their feeding activities to the root hairs of perennial ryegrass

(Fig. 29) and no attempts were made by the nematodes to feed on, or probe, cells of other parts of the root. Total feeding time varied from 4 mins 15 secs to over 38 mins with a mean of 13 min 19 secs, nematodes remaining completely motionless throughout. -5

Fig. 29 Tylenchus filiformis (male) feeding on root hair.

Fig. 3Q Tylenchus filiformis on root hair showing accumulation of cell contents around stylet tip during feeding. 86

There was usually a considerable delay between retraction of the

stylet from the root hair and commencement of another feeding

sequence. No secretions into the root hair from the stylet

were observed during the delay period, however, cell contents

quickly accumulated around the stylet tip after the median bulb

had begun pumping (Fig. 30).

No external symptoms of damage by the nematodes was

observed, but the flow of cellular contents appeared to be

reduced and, in some cases, vacuoles were present in the root

hairs after feeding.

Aphelenchoides saprophilus

Only two A. saprophilus were observed on roots in the agar feeding very rapidly on either root hairs (Fig. 31) or epidermal cells without any apparent selection of feeding sites. Stylet probing of cells was not commonly observed and nematodes did not discern root cells from the base of the petri dish; stylet penetration (as witnessed by rapid thrusts of the stylet) was attempted whenever the nematodes came into contact with a resistant surface. Total feeding times were very short varying from 8 secs to 4 mins with an average of 40 secs. The feeding differed from all the other genera studied by having no delay period between stylet penetration and beginning of median bulb pulsation. Paratylenchus microdorus and P. projectus

P. microdorus and P. projectus, although observed Fig. 31 Aphelenchoides saprophilus feeding on root hair.

Fig. 32 Paratylenchus microdorus on epidermal cell during stylet penetration. 88

infrequently on roots, had similar feeding habit::. Both species

fed ectoparasitically on epidermal cells of the root hair region

(Fig. 32) for long periods. The observed feeding times for

P. microdorus varied from 8 hours to 24 hours 30 mins, and

similar times of 2 hours 30 mins to 24 hours were recorded for

P. projectue. Timed observations were begun after the median

bulb had commenced pumping and feeding times for both species

were probably much longer. Females of both species laid eggs

while feeding on the roots.

Helicotylenchus varicaudatus

Helicotylenchus species did not adapt well to the agar

medium and most specimens inoculated onto the plates became

inactive within a few hours. H. varicaudatus was occasionally

observed feeding ectoparasitically on epidermal cells of the

root hair region, and was also seen repeatedly penetrating cell

walls with its stylet in a cutting or perforating action with

no apparent feeding taking place as though attempting to enter

the root tissue.

Pratylenchus crenatus

P. crenatus was never observed feeding externally on the

roots, but was often seen attempting to enter the roots by

repeated stylet penetration of the epidermal cell walls.

Attempted entry into the roots was by continuous thrusts of the stylet against the cell wall in a definite cutting action that

involved the gradual inclination of the nematode's head. A

straight cut was produced in the wall of the epidermal cell

through which the nematode could begin entry into the root.

although complete invasion was not observed. No pulsation of

the median bulb occurred during this sequence.

Discussion

Investigations on the biology of Tylenchus species (Khera

and Zuckerman, 1963; Sutherland, 1967) have suggested that they

can be classed as browsing ectoparasitic nematodes because of

their relatively short feeding times. Observations of T. filiformis

support this view, as feeding times were no longer than 38 mins

and nematodes continually moved from one site to another.

Feeding of T. filiformis was restricted to root hairs which is

probably related to the comparatively weak stylet that species

of the genus possess. Khera and zuckerman (1963) observed that

T. bryophilus and T. agricola fed more readily on root hairs than on other cells.

Aphelenchoides saprophilus fed for very short periods on root cells and feeding contrasted to other species by the absence of a delay period between stylet penetration and median bulb pulsation. The simple mechanical removal of cell contents has also been shown for related species, Aphelenchoides bicaudatus (Siddiqi and Taylor, 1969) and Aphelenchus avenae

(Fisher and Evans, 1967; Linfords 1937) on fungal and algal hosts. 90

Siddigi and Taylor (1969) showed that feeding of A. bicaudatus

lasted for 2 to 90 secs on fungal hyphae and 2 to 10 secs on

algae, and suggested that the duration of feeding was dependent

on the volume of food available in a cell; however, the same

argument cannot apply to the short feeding periods of A. saprophilus

on root cells. A. saprophilus is probably a mycophagous nematode

in the soil as it can be easily cultured on agar plates with

fungus (Franklin, 1957).

Prolonged feeding periods were observed for both

Paratylenchus microdorus and P. projectus with the nematodes

remaining external to the roots, and support observations on

other species, P. minutus (Linford et al., 1949), P. projectus

and. P. dianthus (Rhoades and Linford, 1961), that Paratylenchus

are relatively sedentary ectoparasitic nematodes remaining at

feeding sites for long periods feeding continuously.

A number of hypotheses have been put forward to differentiate

between ectoparasitic nematodes on the basis of their feeding

habits. McElroy and Van Gundy (1968) have suggested that there

exists a developmental trend in parasitic specialisation within

the ectoparasitic nematodes related to feeding behaviour and morphology. On their assumption that long feeding times and

enlargement of the median oesophageal bulb are concurrent with greatest parasitic development, the Criconematidae represent the extreme in ectoparasitic evolution and members of the Tylenchidae, 91

with comparatively small median bulbs and short feeding times, are considered to be the most primitive ectoparasites.

Paracer -et al. (1967) and Zuckerman (1968) proposed that ectoparasitic nematodes could be divided into groups on the basis of feeding habits and extent of symptoms they induce in host tissues: 1) primary or severe pathogens - cause extensive local- ised host reaction, have long feeding times and a moderate degree of host specificity; 2) weak pathogens - produce only slight host reactions, feed for short periods and have a very wide range of hosts. Tylenchorhynchus and Tylenchus are included in the latter group. This is a welcome approach to the problem of defining ectoparasitic behaviour, but it is an over-simplif- ication of a complex situation. For example, Tylenchorhynchus icarus and Paratylenchus both have long feeding periods but are placed in separate groups or at different ends of the evolutionary scale; Tylenchorhynchus maximus have relatively short individual feeding times but they remain at one location for long periods and are capable of feeding on internal root tissues. Zuckerman

(1969) supports the view that there is a gradual evolutionary progress towards endoparasitism and if this is true the migratory semi-endoparasitic feeding habits of T. icarus and T. macrurus represent an advance towards this goal.

Further observations on a wider range of nematodes and possibly physiological studies of ectoparasites may give a clearer 92

understanding of the parasitic relationships involved with these

nematodes.

Endoparasitic nematodes spend part of their lives completely

within the roots and they are here defined as nematodes which

are able to actively penetrate into the root, that is, by the

deliberate breakdown of epidermal and sub-epidermal cell walls

to gain access to the internal tissues. This definition is

restricted to root feeding nematodes as it does not take into

account those nematodes that invade aerial parts of plants

through stomata, such as, Aphelenchoides ritzemabosi (Wallace,

1959), A. fragariae (Klinger, 1970). Active penetration of the

roots can be achieved by repeated stylet thrusts against the

cell wall as observed for T. Icarus, or the more deliberate

perforation of the cell walls by a series of stylet punctures in a straight line thus causing a slit as was observed for

Helicotylenchus and Pratylenchus and is the method by which

Heterodera larvae emerge from the egg (Doncaster and Shepherd,

1967). 93

SECTION V

RELATIONSHIPS OF MIGRATORY NEMATODES (MAINLY TYLENCHORHYNCHUS AND PARATYLENCHUS) TO GROWTH OF PERENNIAL RYEGRASS.

Introduction

Investigations have shown that some species of Tylencho-

rhynchus and Paratylenchus may adversely affect the growth of

various grasses. Powell (1964) showed that declining lawn grasses

(mainly fescues and Kentucky Blue Grass) were greatly improved by'

the application of Nemagon, and he suggests that the moderately

high populations of Tylenchorhynchus maximus in the soil were a

major contributing factor to the decline. T. cylindricus can be

pathogenic to Agropyron cristatum under greenhouse conditions

(Havertz, 1957) and T. martini can cause reduced growth of

Bermudagrass (Johnson, 1970). Tylenchorhynchus spp. may cause stunting of roots and/or top growth of other plants, for example,

T. dubius on cotton, Tempary bean (Reynolds and Evans, 1953) and potatoes (Kyrou, 1969), T. martini on sugar cane (Birchfield and

Martin, 1956), T. claytoni on tobacco (Graham, 1954), azaleas

(Barker and Worf, 1966; Sher, 1958) and maize (Nelson, 1956), and T. maximus on field corn (Griffin, 1964). Few examples of

Paratylenchus spp. giving rise to damage of grasses can be found in the literature with the exception of the stunting of tall fescue by P. projectus (doursen and Jenkins, 1958). In all natural soils many different nematode genera and species are represented and damage to plants may be caused by the action of 94

two or more species possibly acting synergistically. Some nematodes

can cause reduction or alteration of root growth in the absence of

other pathogenic organisms, however, damage to roots dces not

necessarily produce reduction of top growth.

The survey of grassland soils (Section II) showed that

Tylenchorhynchus and Paratylenchus species were two of the most

abundant genera associated with grass and their relationships to

growth of the widely used pasture grass, perennial ryegrass,

were therefore studied. Three experiments were conducted in

different environmental conditions with mixtures and pure pop-

ulations of nematodes. The first two experiments were concerned

with both Paratylenchus and Tylenchorhynchus inoculated into soil

around roots of grass seedlings grown in pots in the glasshouse

under fluctuating temperature conditions, and in glass tubes at

constant temperature in a controlled temperature room. The

effects of nematodes on perennial ryegrass growth were assessed

by comparison of shoot and root weights. For the third experiment,

grass seedlings were grown on agar plates inoculated separately

with two species of Tylenchorhynchus and effects estimated by

measuring root lengths.

a) Glasshouse experiments with Paratylenchus microdorus and a mixture of plant nematodes.

Materials and Methods

Clay pots (3il- inch in diameter) were each filled with

250 ml of steam sterilised soil that had been allowed to aerate 95

for 8 weeks before use. Seeds of Lolium perenne var. S24 were germinated in petri dishes on moist filter paper and 3 seedlings per pot were planted out when 13 days old. Nematodes were extracted from grass pasture soil collected from Lime Kiln field,

Grasslands Research Institute, Hurley, and high, almost pure, populations of Paratylenchus microdorus were obtained from the

15-20 cm soil depths (see Section IIB). A mixture of plant parasitic nematodes that excluded the bulk of P. microdorus and contained the majority of other migratory nematodes present, namely, Tylenchorhynchus (T. dubius, T. maximus, T. icarus, T. microdorus, T. brevidens), Helicotylenchus (H. vulgaris, H. varicaudatus, E. digonicus) and Tylenchus (3 spp.), was obtained from the 0-10 cm soil depth in Lime Kiln. Ten pots were each inoculated with a water suspension of 36,000 P. microdorus, and ten pots were each inoculated with a mixture of 13,000 nematodes of which plant parasites constituted 57.5% of the total: soil extract water was added to ten additional pots as controls.

Pots were arranged in a completely randomised design in the glasshouse. The experiment was terminated after 7 weeks and the fresh weights of roots and foliage were taken together with number of tillers. Nematode populations were estimated by extraction from

100 ml of soil per pot using the tray extraction method. 96

Results Initial and final nematode populations are given in Table 6. High numbers of both Paratylenchus microdorus and Tylenchorhynchus spp. were present at the start of the experiment but pocuiations

declined over the seven,week period.In pots containing P. microdorus

Table 6. The number of plant parasitic nematodes per pot of soil in which Lolium perenne was grown for 7 weeks.

Mixture of Nematodes P. Helico- Tylench- Tylenchus Total Para- microdorus tylenchus orhynchus spp. tylenchus spp. spp. microdorus

Initial pop. 5,020 440 2,040 120 7,630 36,000

Final pop.1) 1,263 30 1,874 95 3,28o 7,160

% of initial 25.2 6.8 91.8 79.2 43 19*9 pop.

1) Means of 10 replicates

numbers decreased to 19.9% of the original population, and, in the soil containing the mixture of nematodes, P. microdorus,

Helicotylenchus spp. and Tylenchus spp. decreased markedly while Tylenchorhynchus decreased only slightly and one species, T. dubius, increased during the experiment. There were no significant differences between root weights 97 foliage weights or number of tillers of plants in treated pots and those of the control (Table 7).

Table 7. Fresh weights of shoots and roots, and number of ' .iump,,,zan_n_2 inoculated with a mixture of plant parasitinematodes and Paratylenchus microdorus alone

Fresh wts. (gm) No. of tillers

Shoots Roots per pot

Control 4.45 1.59 17.8 Mixture of 5.42 1.80 19.2 nematodes

P. microdorus 4.99 1.46 16.9

1) Means of 10 replicates 2) Each pot contained 3 plants.

b) Controlled temperature experiments with two species of Tylenchorhynchus and Paratylenchus microdorus.

Materials and Methods Glass tubes, measuring 4 x 20 cm, sealed at their bases with perforated aluminium foil, were each filled with 140 ml of steam sterilised soil. Seeds of Lolium perenne var S.24 were germinated on moist filter paper as before and single seedlings were planted into each tube when 13 days old. Two species of Tylenchorhynchus and Paratylenchus microdorus were obtained from 98 pure cultures that had been built up in pots under grass in the glasshouse. Tubes containing soil and grass seedlings were each inoculated with nematodes as follows: 8 with 63,000 P. iaicrodorus, 8 with 200 T. icarus and 8 with 340 T. maximus per tube. Soil extract water was used for the control, and tubes were plunged into sterilised sand in deep plastic bowls and placed in a 20°C controlled temperature room with lights set for a 14 hour day.

After 13 weeks nematodes were extracted from the soil of each tube by spreading both soil and roots carefully on tissue of the tray extraction method and leaving for 24 hours.

Following this soil was washed off the roots and the clean roots were oven-dried before weighing. Dry weights of shoots were taken and number of tillers per seedling noted.

Results

Populations of P. microdorus decreased from a high initial population of 63,000 to 23,360, and very few T. icarus remained in the soil at the end of the 13 week period. However, T. maximus numbers increased tenfold from 340 to a mean of just under 4,000 per tube (Table 8). No observable effects on growth of nematode-infested grasses was apparent, and shoots and roots of these grasses appeared as healthy as those of controls.

Statistical analysis on the data indicated that there were no significant differences between shoot and root weights, or 99

number of tillers, of controls and infested grasses (Table 8).

Table 8. Number of nematodes per tube and dry weights 017 shoots and roots of Lolir perenne inoculated with t])- 7,e nematode species

No. of nematodes Dry wts (gm) No. of Initial pop. Final pop. Shoots Roots tillers

Control - - 0.63 0.84 6.9 P. microdorus 63,000 23,360 0.65 0.80 4.8 T. maximus 340 3,968 0.70 0.77 7.5 T. icarus 200 28 0.70 0.69 7.8

1) Mean of 8 replicates

c) Root growth experiments with Tylenchorhynchus maximus and T. icarus on agar plates. Materials and Methods Feeding observations (Section IV) have shown that Tylenchorhynchus species remain active in agar medium and it was possible to use them for short term root growth experiments by following the method described in Section IV for the culture of grass seedlings and nematodes on 1% sterile agar. Seeds of Lolium perenne were dehusked, surface sterilised in 2% sodium hypochlorite for 15 mins, washed in sterile water and then germinated on sterile, moist filter paper in petri 100

dishes. After 4-5 days, following emergence of the radicle,

single seedlings were carefully pushed into the agar of each

plastic petri dish using sterile forceps. Nematodes were washed

in sterile water after extraction from the soil and hand-picked

onto agar plates, ten plates were inoculated with 50 T. maximus

and ten plates were inoculated with 50 T. icarus. Nematode-

infested agar plates were compared to controls containing

seedlings only. Petri dishes were sealed with tape after

inoculations had been completed and maintained on the laboratory

bench in lighted conditions.

Root lengths were measured at the outset of the experiment

and subsequent root growth was recorded each day for a period of

14 days. Measurements of root lengths were made with lead fuse

wire laid on the bases of the petri dishes and both total root

growth and lateral root growth were measured for each seedling.

Results

Seedlings continued growing on the agar plates for over 2

weeks and shoots remained healthy during this period. Feeding of both T. maximus and T. icarus on roots was observed, T. maximus aggregated on root tips mainly in the first 6 days

(Appendix Table 11) and behaviour was similar to that described in Section IV, no aggregation of T. icarus occurred. Damage to roots was evident with seedlings inoculated with T. maximus and

5 to 14 nematodes feeding on the root tips caused reduction or

101

cz>" -cs :t10 • • • 8 • •

"r, 2 6— ▪c_ • ns E • 15 4

-9 2 • • • • • • • 1 1 0 2 4 6 8 10 12 14 No. of nematodes aggregating on root tips

Fig. 33 Main root growth related to numbers of T. maximus aggregating on root tips. 102

cessation of main root growth (Fig. 33) and a characteristic bending of the root tip (Fig. 34). No damage to roots was observed on plates inoculated with T. Icarus. The results of root growth on the agar plates over 0,)- 14 day period are presented in Table 9. The total root growth after 14 days was not significantly different between seedlings infested with either T. maximus or T. icarus and controls, however, lateral root growth was significantly greater with T. maximus at the 5% level. Increased lateral root growth occurred on roots damaged by the feeding of T. maximus (Fig. 34) in comparison to normal growth of controls of the same age without lateral root development (Fig. 35).

Table 9. Total and lateral root growth of seedlings on agar plates inoculated with T. maximus and T. icarus.

1) Root growth (cm) Total Lateral only Days after inoculation 4 9 14 4 9 14

Control 4.00 9.15 21.14 0.10 1.30 5.40 T. icarus 4.82 12.67 24.94 0.07 1.03 3.92 T. maximus 4.20 10.75 31.39 0.10 4.45 12.27

1) Mean of 10 replicates

Significant at the 5% level. LSD0.05 = 5.60 cm

ISO 10:-5

Fig. 34 Increased lateral root growth on roots damaged by feeding of T. maximus (arrowed).

Fig. 35 Nematode-free seedling at same stage of growth as Fig. 34. 104

Discussion

The results have shown that the migratory nematodes

Paratylenchus microdorus, Tylenchorhynchus icarus and T. maximus do not cause any reduction in top growth or any noticeable reduction of root growth of perennial ryegrass when cultured in sterilised soil even when populations of all three nematode species were equivalent to, or greater than field populations.

P. microdorus was found in very high numbers under grass (Section

II) and perennial ryegrass is a favourable host (pot cultures in the glasshouse increased from initial population of 10 to over one million in an 18 month period) but the high populations used in the experiments had no apparent effect on the grasses.

The observations of seedlings growing on agar plates showed that root growth was decreased or halted by the feeding of T. maximus and it appears that aggregation and intensive feeding on the root tip is necessary to produce this effect by nematodes that cause mainly mechanical damage. Individual feeding by other nematode species, such as, T. icarus, along the roots does not produce similar reduction in growth (Section IV). Pitcher

(1967) observed that aggregation of Trichodorus on the zone of elongation of apple roots caused similar cessation of root growth although over a longer period. The reduction in main root growth by T. maximus resulted in increased lateral root growth which possibly explains why total root growth in all three experiments 105

with T. maximus was not significantly difff.ront from controls.

Increased lateral root production seems to be a normal host

reaction to damage of the root tip to compensate for loss in

main root growth. Increase in plant growth has been shown by a

number of workers to be induced by the presence of Tylenchorhynchus

in the soil. In some instances top weights of cereals and grasses

were found to be greater in soil supporting populations of T.

claytoni than in nematode-free soil (Krusberg, 1959;, Troll and

Rohde, 1966) and Chapman (1959) showed that T. martini could

increase top growth of both red clover and alfalfa plants.

Examples of other nematodes that produce increased plant growth

are, Paratylenchus projectus that has been shown to cause

increased tillering and root proliferation of tall fescue

(Coursen and Jenkins, 1958) and Heterodera rostochiensis in small

numbers increased root growth of potato (Peters, 1961). Wallace

(1963) points out that the production of numerous small roots

at the expense of the larger more deeply penetrating roots increases the susceptibility of plants to wilting and reduces

their ability to absorb nutrients.

The use of sterile conditions in my experiments would exclude

many of the naturally occurring soil pathogenic organisms. Even

though T. maximus caused serious damage to individual roots in vitro this was not reflected as reduced total root growth,

however, in field conditions it is possible that these nematodes 106 may act as incitants of disease allowing the entry of other pathogens into the roots. Sturhan (1966) considers that

Tylenchorhynchus species are more important in association with fungi than alone, and Zuckerman (1968) suggests that other microorganisms in the soil play a dominant role in the cause of diseases by Tylenchorhynchus. In theory ectoparasitic nematodes such as

T. maximus are capable of causing reduction in growth of grasses in the absence of other organisms, but the numbers that would be required to cause serious damage are not found in pasture soil

(see Section II) because of the rapid and prolific root growth by grasses. Further studies are required to consider the inter- relations between T. maximus and, especially, root pathogenic fungi of grass, and also to elucidate the effects of T. maximus on other plants were root growth is not as prolific as it is in grass. 107

SECTION VI.

SOME ASPECTS OF THE BIOLOGY OF TYLENCHORHYNCHUS MAXIMUS AND T. ICARUS

Fecundity and Hatching of T. maximus

T. maximus do not require males for reproduction which

enables them to be used in simple fecundity experiments.

Plastic petri dishes filled with a thin layer of sterile 1%

water agar and planted with perennial ryegrass seedlings were

used as previously described. Twenty mature female T. maximus

were washed in sterile water and a single female was hand-picked

onto each agar plate using a sterile needle. The petri dishes

were sealed with tape and maintained at room temperature.

Observations were made every day for the period of the experiment

and the number of eggs laid by each female was noted.

Of the total nematodes 5 either died, or became quiescent

within the first few days. The remaining 15 females laid eggs from the first day and continued laying for 19 days along roots or on the surface cif the agar. After this period 95 eggs altogether had been laid averaging more than 6 eggs per female and one nematode had produced 14 eggs in the time. Eggs hatched 17 to 19 days after they were laid. Nematodes became quiescent after 4 weeks on the agar and most eggs that contained fully developed larvae failed to hatch after this period, but the same eggs normally hatched within a few hours when transferred from the agar to a drop of water or a fresh agar plate suggesting that water 108

stress was responsible for the failure to hatch.

Emergence from the egg

The final stages of emergence from the egg of T. maximus

larvae were observed by transferring developing eggs from agar

plates to thin agar squares on a microscope slide which were

covered with a coverslip and sealed with candle wax.

Three days prior to hatching fully formed T. maximus larvae

are in a state of continual motion within the egg performing

figure-of-eight movements both in the forward and reverse

directions similar to that described by Wallace (1968) for

Meloidogyne javanica. The egg shell remains rigid until the

last stages of development, but in the 12 hours before hatch the

shell softens and the egg then becomes larger; movements of the

nematode produce distortions of the wall and distinct bulges are

caused by the nematode forcing its head against one or other end

of the egg. After a number of hours the nematode begins thrusting its stylet at one spot on the end wall of the egg at the same

time pressing with its head until the stretched wall, when

penetrated by the stylet, ruptures and the larva emerges. Emergence from the egg of T. icarus was identical to that of T. maximus. Figure 35 shows bulging of the egg wall immediately prior to hatch with the nematode thrusting its stylet against the wall and Figure 36 is the same nematode emerging from the egg one to two seconds later after the egg wall has been penetrated. _ 109

Hatching of both T. maximus and T. icarus is similar to that

described for Meloidogyne javanica (Jallace, 1968) and

Pratylenchus penetrans (Edwards, 1960), but is in contrast to

Heterodera rostochiensis which escapes from the egg by making a line of perforations in the rigid egg wall (Doncaster and Shepherd, 1967). 110

Fig. 36 Bulging of the egg wall immediately prior to hatch of Tylenchorhynchus icarus.

Fig. 37 Emergence of Tylenchorhynchus icarus from the egg after rupture of the egg wall. 111

GENERAL DISCUSSION 112

SECTION VII.

GENERAL DISCUSSION The results from the survey of grassland pasture soils show that- many different plant parasitic nematode genera are found associated with grasses and certain genera, namely, Tylenchorhynchus, Paratylenchus, Helicotylenchus and Tylenchus occur more commonly than others. The occurrence of some plant parasitic nematodes under grass may be of considerable economic importance in leys and pastures, and nematodes are also considered as damaging to golf courses and putting greens in America (Nutter and Christie, 1958;

Taylor et al., 1963; Troll and Tarjan, 1954). Nematode population levels varied in the grassland soils and did not always appear to have reached high proportions in view of the long standing and undisturbed grass cover. Paratylenchus spp., when present, occurred in the highest numbers under grass and very high populations of nematodes of this genus have been recovered from other soils (Linford,Oliveira and Ishii, 1949; Jenkins, 1956; gouts, 1966) but the levels attained in field soil are often lower than those reached in 'greenhouse cultures. P. microdorus increased from initial inocula of 10 to over 1 million in 18 months in pots in the glasshouse planted to perennial ryegrass, and P. hamatus has been shown to increase from 100 to nearly 46,000 in pots over

5 months (Raski and Radewald, 1958). Similarly, potential population levels of some Tylenchorhynchus species are not reached in field soil and T. maximus, for example, builds up to far greater population levels in sterile soil in controlled environmental 113

conditions. Fluctuating physical factors in the field are likely

to adversely affect the survival and reproduction of nematodes even though food supply and vegetative cover remain comparatively stable. The ceiling for population increase in the field may also be affected by competition between nematodes for habitable space and

feeding sites, or by predators, such as protozoa and tardigrades (Doncaster and Hooper, 1961) and other nematodes (Linford and Oliveira, 1937), and by other microorganisms in the soil, for example, nematophagous fungi (Duddington, 1960). In the genus

Tylenchorhynchus, the reproduction.of T. martini has been shown to be reduced by the activity of other nematodes in mixed cultures (Johnson, 1970), and Hollis and Johnson (1957) suggested that the numbers of T. martini and T. acutus are reduced by microorgansims under unsterile conditions. The reasons for the distribution of nematodes at different depths in the soil cannot be fully explained. Of the nematodes I have studied, Tylenchorhynchus, Longidorus and Paratylenchus species occur in highest numbers at different depths and this distribution remains constant throughout the year. Root distribution, aeration, moisture and soil structure may all affect the distribution of nematodes in the soil profile as I have mentioned in the discussion in the relevant section, but other factors may also be important. It is possible that competition between nematode species in naturally occurring mixed populations may affect their vertical distribution in the soil. Some species of 114

Lorlsidorus, for example, may be poor competitors and consequently

only reach high population• levels in the absence of other nematodes in the lower soil depths; Paratylenchus microdorus similarly occurs in highest numbers at soil depths where numbers of other ectoparasitic nematodes are low. It is evident that many different factors, environmental and otherwise, may be responsible for the vertical distribution of nematodes in the soil to which certain species or genera react in dissimilar ways. My studies on nematodes in the genus Tylenchorhynchus have

established that species differ considerably in their morphology• and feeding behaviour. Morphological variations are marked even between the 10 species found in grassland soil and these represent a small percentage of the total species in the genus. They range from small nematodes with tapering tails, weak head skeletons and small, delicate stylets, such as, T. brevidens, T. nothus and T. microdorus, to the large species T. Icarus and T. macrurus with cylindrical bodies, strong head skeletons and robust, well developed stylets. One species, T. lamelliferus, has a long slender stylet, unique male and female tails, and prominent longitudinal striations on the cuticle. T. maximus and T. dubius both have post anal intestinal sacs. The stylet is one of the most variable characters amongst nematodes, it is also the structure that has a major role in a parasitic mode of life. It is, perhaps, unwise to theorise on the feeding behaviour of a species by reference to stylet structure, but it does appear that this type 115

of relationship occurs in certain Tylenchorhynchus species. Paramonov (1962) in his elaborate explanations of phylogenetic links between plant parasitic nematodes postulates that one of the most important characters of evolution of, what he terms, the

"ectoparasitic perforatOrs" of higher plants is the progressive development of a strong stylet with strongly developed basal knobs. Certain nematodes, such as those in the Criconematidae, have evolved long stylets amply suited for deep penetration of root

tissues while remaining completely ectoparasitic. According to

Paramonov, nematodes in the Hoplolaimidae with shorter more robust stylets are those nematodes which are attempting "to pass over to endoparasitism", and some of the hoplolaimids have become, what he terms, "endoparasitic erratic perforators", intermediate between ectoparasites and true endoparasites. Tylenchorhynchus species have been classified in various ways by different workers: browsing ectoparasites (Paracer et al., 1967;

Zuckerman, 1968), ectoparasitic perforators (Paramonov, 1962), migratory semi-endoparasites (Winslow, 1960) and intermittent parasites (Goodey, 1943). My observations on seven species of Tylenchorhynchus indicate that these species and possibly others within the genus can be placed into different groups mainly on the basis of their mode of feeding and also, in some cases, on their morphological characteristics. Some species, such as T. brevidens and T. nothus, have a simple cursory or browsing ectoparasitic feeding habit moving from one feeding site to the next and feeding 116

for short periods at each site. They have short, delicate

stylets and with their other morphological characters they resemble some members of the Tylenchinae. In a second group can be placed species, such as T. lamelliferus and, in particular, T. maximus that feed as ectoparasites mainly on root tips in aggregations, their individual feeding times are relatively short but they feed in the one locality for many days causing destruction of the root cells. This second group can be termed intermittent feeders. A third group includes the distinctive species T. icarus and T. macrurus, with large, well developed stylets and heavy

head skeletons closely allied to the hoplolaims. Both T. icarus and T. macrurus have a sedentary ectoparasitic feeding behaviour remaining for many hours or days feeding continuously at the one locality, and they have also developed what can be termed a migratory semi-endoparasitic behaviour enabling them to penetrate roots and feed on internal tissues. A fourth group is a matter of

conjecture, but may include other species with very long stylets, such as T. superbus and T. conicus that may feed as sedentary

ectoparasites similar to the Criconematidae, their long stylets enabling them to penetrate cells deep within the root. The demarcation between groups is not sharp and there will be a certain amount of overlapping. One example is T. dubius which I observed

to have a cursory type of feeding behaviour, but it has been reported feeding on root tips in small aggregations (Klinkenberg.

1963) and even recovered from root tissue (Hopper, 1959; Reynolds 117

and Evans, 1955). It is possible that some species of Tylenchorhynchus are endoparasitic; Steiner (1937) reported that T. claytoni was endoparasitic in tobacco roots, and Hopper (1959) suggested that the larval stages of T. ewingi were endoparasitic in pine seedlings after he recovered numerous nematodes by incubation of the root systems. On the last point concerning endoparasitism in my discussion on feeding behaviour I defined root endoparasites as nematodes that are capable of penetrating the external cell layers to gain acoess into the root although they do not always enter in this manner. Many different nematode species are to be found within roots, they may be plant parasitic, mycophagous or saprobes, a large majority of them will have entered through breaks in the root epidermis and their presence within the root is not always indicative of an endoparasitic habit.

It has been suggested that Tylenchorhynchus is made up of a number of different genera (Allen, 1955; Siddiqi, 1970; Sher, personal communication) because of the wide differences in morphological characters, and my observations on the differences to be found in the feeding behaviour of species within the genus lend support to this suggestion.

Results from pot experiments to test the pathogenicity of Tylenchorhynchus Icarus, T. maximus and Paratylenchus microdorus on perennial ryegrass produced no observable effects on root or top growth of the grasses. Experiments of this nature are not 118

truly representative of natural conditions because an attempt is

made to produce optimum levels of moisture, heat and lighting in

controlled conditions. The experiments on agar plates showed that

T. maximus at numbers equivalent to 500 per 200 ml of soil can

cause complete cessation of main root growth which results in the increase of lateral root growth. In optimum conditions this

damage to the root system would not produce any observable effects

on plant growth, however, in adverse conditions growth may be

retarded. If the feeding of nematodes has caused damage to the

nutrient and water absorbing area of the roots then the plant is

less able to withstand adverse environmental conditions suxh as

dry periods or nutrient deficiencies. Wallace (1963) reasons that reduction in the more deeply penetrating main roots increases the

susceptibility of plants to wilting. Nematodes will also be

affected during dry periods and it has been demonstrated that

populations of a number of different Tylenchorhynchus species are

suppressed by drying out of the soil (Johnston, 1958; Mukhopadhyaya

and Prasad, 1968; Wallace and Greet, 1964). A number of people (Reynolds and Evans, 1953; Sturhan, 1966;

Winslow, 1960; Zuckerman, 1969) have suggested that the majority

of Tylenchorhynchus species are not severe pathogens, however,

they also conclude that?the damage caused to plants by Tylencho- rhynchus is difficult to ascertain. The predominance of Tylencho- rhynchus in many grassland soils and around roots of other planlls,

such as, rice and sugar cane (Fielding and Hollis. 1956; Parsons, 119

1970), corn (Nelson, 1956), tobacco and soybean (Holderman, 1956)

and wheat (Nukhopadhyaya and Prasad, 1968) is not necessarily

indicative of their economic importance, but it suggests that

Tylenchorhynchus may cause more damage, either directly or

indirectly, than has so far been discovered. The feeding of

Tylenchorhynchus on healthy root tissues may produce infection

courts for other organisis especially soil pathogenic fungi, and

as such act as incitants of a disease, as suggested by Sturhan

(1966) and Zuckerman (1968). This would apply more to species that penetrate deep within the root by various means, such as, T. maximus and T. icarus, than to species that only cause superficial damage to epidermal cells. However, Holdeman (1956) has shin in glasshouse experiments that T. claytoni, described as a browsing ectoparasite by Zuckerman (1969), is capable of increasing the incidence of Fusarium wilt in tobacco and he suggests that the nematode may be one of the factors responsible for the erratic behaviour of tobacco wilt in the field. The comprehensive reviews on interrelationships of nematodes and other organisms (Christie,

1960; Pitcher, 1965; Powell, 1963) make it obvious that many plant parasitic nematodes are involved in interactions with pathogenic soil organisms and there is a likely possibility that these organisms play an important part in diseases with which Tylencho- rhynchus species are associated. 120

REFERENCES ALLEN, M.W. 1955. A review of the nematode genus Tylenchorhynchus. Univ. Calif. Publ. Zool. 61: 129-166. ANONYMOUS 1970. Ministry of Agriculture, Fisheries and Food, 1970. Agricultural Statistics - England and Wales - results of June 1969 census. Published annually. BAKER, A.D. 1953. Rapid method for mounting nematodes in glycerine. Canad. Ent. 12: 77-78. BAKER, H.K. 1962. A survey of English grasslands. In Proc. VI British Weed Control Conference: 23-30. BANAGE, W.B. 1962. Some nematodes from the Moor House National Nature Reserve, Westmorland. Nematologica 7: 32-36. BARKER, K.R. and G.L. WORF. 1966. Effect of nutrients on nematode activity on azalea. Phytopathology 56: 1024-1027. BIRCHFIELD, W. and W.J. MARTIN. 1956. Pathogenicity on sugar cane and host plant studies of a species of Tylenchorhynchus. Phytopathology 46: 277-280. BREESE, E.L. 1968. Improving Britain's grassland output. Span 11: 162-165. CHAPMAN, R.A. 1959. Development of Prat7lenchus penetrans and Tylenchorhynchus martini on red clover and alfalfa. Phyto- pathology 49(6): 357-359. 121

CHRISTIE, J.R. 1960. Some interrelationships between nematodes and other soil-borne pathogens. In Nematology: Fundamentals and Recent Advances with Emphasis on Plant Parasitic and Soil Forms. Ed. Sasser, J.N. and Jenkins, W.R. Univ. N. Carolina Chapel Hill Press. 1960, 480 pp. COHN, E. 1970. Observations on the feeding and symptomatology of Xiphinema and Longidorus on selected host roots. J. Nematology 2: 167-173. COURSEN, B.W. and 4.R. JENKINS. 1958. Host-parasite relationships of the pin nematode, Paratylenchus prolectus, on tobacco and tall fescue. Pl. Dis. Reptr. 42: 865-872. DONCASTER, C.C. and D.J. HOOPER. 1961. Nematodes attacked by protozoa and tardigrades. Nematologica 6: 333-335. DONCASTER, C.C. and A.M. SHEPHERD. 1967. The behaviour of second- stage Heterodera rostochiensis larvae leading to their emergence from the egg. Nematologica 13: 476-478. DUDDINGTON, C.L. 1960. Biological control - predacious fungi. In Nematology fundamentals and recent advances with emphasis on plant parasitic and soil forms. Ed. J.N. Sasser and W.R. Jenkins. Chapel HillL Univ. North Carolina Press, 480 pp. EDWARDO, A.A., DI. 1960. Time-lapse studies of movement, feeding and hatching of Pratylenchus penetrans. Phytopathology 2,2:570. FIELDING, M.J. and J.P. HOLLIS. 1956. Occurrence of plant parasitic nematodes in Louisiana soils. P?. Dis. Reptr. 40: 403-405. 122

FISHER, J.M. and A.A.F. EVLDTS, 1967. Penetration and feeding of Aphelenchus avenae. Nematologica 12: 425-428. FISHER, J.M. and D.J. RASKI. 1967. Feeding of Xiphinema index and X. diversicaudatum. Proc. helminth. Soc. Wash. 74: 68-72. FLEGG, J.J.M. 1967. Extraction of Xiphinema and Longidorus species from soil by a modification of Cobb's decanting and sieving technique. Ann. appl. Biol. 60: 429-437. FLEGG, J.J.. M. 1968. The occurrence and depth distribution of Xiphinema and Longidorus species in southeastern England. Nematologica 14: 189-196. FRANKLIN, M.T. 1957. Aphelenchoides composticola n.sp. and A. saprophilus n.sp. from mushroom compost and rotting plant tissues. Nematologica 2: 306-313. GAIR, R. and E.T.ROBERTS. 1969. Pest and disease control in grass and fodder crops. In Insecticide and Fungicide Handbook: 138-158. Edited by H. Martin, 387 pp. GERAERT, E. 1967. Results of a study on the oecology of plant parasitic and freeliving soil-nematodes. Ann. Soc. Roy. Zool. Belgium 27,: 59-64. GOODEY, J.B. 1959. Gall forming nematodes of grasses in Britain. J. Sports Turf. Res. Inst. 10: 1-7. GOODEY, T. 1943. A note on the feeding of the nematode Anguillulina macrura. J. Helminth. 21: 17-19. 123

GRAHAM, T.W. 1954. Plant abnormalities caused by parasitic nematodes. Pl. Dis. Reptr. Supp. 227: 83. GRIFFIN, G.D. 1964. Assoniation of nematodes with corn in Wisconsin. P1. Dis. Reptr. 48: 458-461. GUIRAN, G. De. 1967. Description de deux especes nouvelles du genre Tylenchorhynchus Cobb, 1913 (Nematoda: Tylenchinae) accompagnee d'une cle des femelles, et precisions sur T. mamillatus Tobar-Jimenez, 1966. Nematologica 13: 117-230. HARRISON, B.D. and R.D. WINSLOW. 1961. Laboratory and field studies on the relation of arabis mosaic virus to its nematode vector Xiphinema diversicaudatum (Micoletzky). Ann. appl. Biol. 49: 621-633. HAVERTZ, D.S. 1957. Nematode pathogenicity to crested wheat grass, Agropyron cristatum (L.) Gaertn, at U.S. Forest Service Experiment Station, Benmore, Utah. University of Utah, Ph.D. Thesis. HEALD, C.M. and H.D. WELLS. 1967. Control of endo- and ectoparasitic nematodes in turf by hot-water treatments. P1. Dis. Reptr. 11: 905-907. HOFF, J.K. and W.F. MAI. 1964. Influence of soil depth and sampling date on population levels of Trichodorus christiei. Phytopathology 21.: 246. HOLDEMAN, Q.L. 1956. The effect of the tobacco stunt nematode on the incidence of Fusarium wilt :7'ne-curnd tobacco. Phytopathology 46: 129. 124

HOLLIS, J.P. and T. JOHNSTON. 1957. Microbiological reduction of nematode populations in water-saturated soils. Phytopathology 47: 16 (abstr.). HOPPER, B.E. 1959. Three new species of the genus Tylenchorhynchus (Nematoda: Tylenchida). Nematologica 4: 23-30. JENKINS, W.R. 1956. Paratylenchus projectus, new species (Nematoda: Criconematidae) with a key to the species of Paratylenchus. J. Wash. Acad. Sci. 46: 296-298. JOHNSON, A.W. 1970. Pathogenicity and interaction of three nematode species on six Bermudagrasses. J. Nematology 2: 36-41. JOHNSTON, T. 1958. The effect of soil moisture on Tylenchorhynchus martini and other nematodes. Pro. La. Acad. Sci. 20: 52-55. JONES, F.G.W., D.W. LARBEY and D.M. PARROT. 1969. The influence of soil structure and moisture on nematodes, especially Xiphinema, Longidorus, Trichodorus and Heterodera spp. Soil Biol. Biochem. 1: 153-165. KHERA, A. and B.M. ZUCKERMAN. 1963. In vitro studies of host- parasite relationships of some plant-parasitic nematodes. Nematologica 9: 1-6. KLINGER, J. 1970. The reaction of Aphelenchoides fragariae to slit-like micro-openings and to stomatal diffusion gases. Nematologica 16: 417-422. 125

KLINKENBERG, C.H. 1963. Observations on the feeding habits of Rotylenchus uniformis, Pratylenchus crenatus, P. penetrans, Tylenchorhynchus dubius and Hemicycliophora similis. Nematologica 9: 502-506. KRUSBERG, L.R. 1959. Investigations of the life cycle, reproduction, feeding habits and host range of Tylenchorhynchus claytoni Steiner. Nematologica 4: 187-197. ” KYROU, N.C. 1969. Tylenchorhynchus dubius (Butschi, 1873) Filipjor; 1936: an injurious root ectoparasite of potatoes? Eur. Potato J. 12: 215-218. LEWIS, S. and D. WEBLEY. 1966. Observations on two nematodes infesting grasses. Pl. Path. 12: 184-186. LINFORD, M.B. 1937. The feeding of some hollow-stylet nematodes. Proc. helminth. Soc. Wash. 4: 41-46. LINFORD, M.B. and J.M. OLIVEIRA. 1937. The feeding of hollow spear nematodes on other nematodes. Science q2: 295-297. LINFORD, M.B., J.M. OLIVEIRA and M. ISHII. 1949. Paratylenchus minutus, n.sp., a nematode parasitic on roots. Pacific Sci. 111-119. LOOF, P.A.A. 1959. Miscellaneous notes on the genus Tylenchorhynchus (Tylenchinae: Nematoda). Nematologica 4: 294-306. LOOF, P.A.A. and M. OOSTENBRINK. 1962. Rotylenchulus borealis n.sp. with a key to the species or Rotylenchulus. Nematologica 7: 83-90. 126

McELROY F.D. and S.D. VAN GUNDY. 1968. Observations on the feeding processes of Hemicycliophora erenaria. Phytopathology 1558-1565. MacFAYDEN, A. 1957. Animal Ecology. Aims and methods. Sir Isaac Pitman & Sons Ltd., Lond.;: 264 p. DE MABSBNBER, J. 1963. Okologische BeobaGhtungen an Freilebenden Nematoden in Weiden. Nematologica 2.: 255-261. MUKHOPADHYAYA, M.C. and S.K. PRASAD. 1968. Population dynamics of Tylenchorhynchus. Nematologica 14: 404-418. NELSON, R.R. 1956. Resistance to the stunt nematode in corn. P1. Dis. Reptr. 40: 635-639. NORTON, D.C. and J.E. SASS. 1966. Pathological changes in Agropyron smithii induced by Anguina agropyronifloris. Phytopathology 56: 769-771. NUTTER, G.C. and J.R. CHRISTIE. 1958. Nematode investigations on putting green turf. Proc. Florida State Hort. Soc. 71: 445-449. PhRACER, S.M., M. WASEEM and B.M. ZUCKERMAN. 1967. The bioirlzy and pathogenicity of the awl nematode, Dolichodorus heterocephalus. Nematologica 13: 517-524. PARAMONOV, A.A. 1962. Plant-Parasitic Nematodes. Vol. 1. The origin of nematodes. Ecological and morphological characteristics of plant nematodes. Principles of taxonomy. Editor K. Skryabin. Jerusalem: Israel Program for Scientific Trurls- lations (for the U.S. Department of Agriculture and ',,he National Science Foundation, Washington, D.C.), 390 -pp. (Translated from the Russian) 127

PARSONS, H.N. 1970. Preliminary investigations on the incidence and control of plant nematodes in a new sugar-cane estate in Nigeria. Trop. 24riculture, Trin. 47: 103-113. PEPPER, E.H. 1963. Nematodes in North Dakota. P1. Dis. Reptr. 47: 102-106. PERRY, V.G. 1958. A disease of Kentucky blue grass incited by certain spiral nematodes. (Abstr.). Phytopathology 48: 397. PERRY, V.G., H.M. DARLING and G. THORNE. 1959. Anatomy, taxonomy and control of certain spiral nematodes attacking blue grass in Wisconsin. Research Bulletin University of Wisconsin, No. 207, 24 pp. PETERS, B.G. 1961. Heterodera rostochiensis population density in relation to potato growth. J. Helminth. R.T. Leiper Suppl: 147-150. PITCHER, R.S. 1965. Interrelationships of nematodes and other pathogens of plants. Helm. absts. 34: 1-17. PITCHER, R.S. 1967. The host-parasite relations and ecology of Trichodorus viruliferus on apple roots, as observed from an underground laboratory. Nematologica 13: 547-557. PITCHER, R.S. and A.F. POSNETTE. 1963. Vascular feeding by Xiphinema diversicaudatum (Nicol.). (Abstr). Nematologica 9: 301-302. POIIELL, N.T. 1963. The role of plant-parasitic nematodes in fungus diseases. Phytopathology 22: 28-34. 128

POWELL, 1964. The occurrence of Tylenchorhynchus maximus in Georgia. Pl. Dis. Reptr. 48(1): 70-71. RASKI, D.J. and J.D. RADEWALD. 1958. Reproduction and symptomatology of certain ectoparasitic nematodes on roots of Thompson seedless grape. Pl. Dis. Reptr. 42: 941-943. REYNOLDS, H.W. and EVANS. 1953. The stylet nematode, Tylenchorhynchus dubius, a root parasite of economic importance in the south west. P1. Dis. Reptr. 37: 540-544. RHOADES, H.L. 1962. Effects of sting and stubby-root nematodes on St. Augustine grass. P1. Dis. Reptr. 46: 424-427. RHOADES, H.L. and Mb. LINFORD. 1961. A study of the parasitic habit of Paratylenchus projectus and P. dianthus. Proc. helm. Soc. Wash. 28(2): 185-190. ROHDE, R.A. and W.R. JENKINS. 1957. Host range of a species of Trichodorus and its host-parasitic relationships on tomato. Phytopathology 47: 295-298. SCHINDLER, A.F. 1957. Parasitism and pathogenicity of Xiphinema diversicaudatum, an ectoparasitic nematode. Nematologica 2: 25-31. SIDDIqI, M.R. 1970. On the plant-parasitic nematode genera Merlinius gen. n. and Tylenchorhynchus Cobb and the classific- ation of the families Dolichodoridae and Belonolaimidae n. rank. Proc. helm. Soc. Wash. 37: 68-77. 129

SIDDIQUI, I.A. and D.P. TAYLOR. 1969. Feeding mechanisms of Aphelenchoides bicaudatus on three fungi and an alga. Nematologica 15: 503-509. SLEDGE, E.B. 1959. The extrusion of saliva from the stylet of the spiral nematode, Helicatylenchus nannus. Nematologica 4: 356. SHER, S.A, 1958. The effect of nematodes on azaleas. Fl. Dis. Reptr. 42: 84-85. SOUTHEY, 1969. A gall-forming nematode (Anguina sp.) parasitic on cocksfoot grass. Pl. Path. 18: 164-166. STEINER, G. 1937. Tylenchorhynchus claytoni n. sp., an apparently rare nemic parasite of tobacco plant. Proc. helminth. Soc. Wash. 4: 33-39. STOLZY, L.H., S.D. VAN GUNDY, C.K. LABANAUSKAS, and T.E. SZUSZKIEWICZ 1962. Influence of soil-oxygen diffusion rates and temperature on the citrus nematode and sweet orange seedlings. Phyto- pathology 2: 754. STURHAN, D. 1966. Uber Verbreitung, Pathogenitat and Taxonomie der Nematodengattung Tylenchorhynchus. Mitt. biol. Bund Anst. Ld-u. Forstw. 118: 89-99. SUMNER, D.R. 1967. Nematodes in bluegrass. P1. Dis. Reptr. 22: 457-460. SUTHERLAND, J.R. 1967. Parasitism of Tylenchus emarginatus on conifer seedling roots and some observations on the biology of the nematode. Nematologica 22: 1' -36. 130

SUTHERLAND, J.R. 1969. Feeding of Xiphinemu bakeric Phytopathology 22: 1963-1967. TARJAN, A.C. 1964. A compendium of the genus Tylenchorhynchus (Tylenchidae: Nematoda). Proc. helminth. Soc. Wash. 31: 270-280. TAYLOR, D.P., M.P. BRITTON and H.C. HECHLER. 1963. Occurrence of plant parasitic nematodemin Illinois golf greens. Pl. Dis. Reptr. 47: 134-135. THOMAS, P.R. 1969. Crop and weed plants compared as hosts of viruliferous Longidorus elongatus (de Man). Pl. Path, 18: 23-28. THORNE, G. and R.B. MALEK. 1968. Nematodes of the Northena Great Plains. Part 1. Tylenchida (Nemata: Secementea). Agr. Exp. Stn. S. Dakota Univ., Tech. Bull. 31. TROLL, J. and R.A. ROHDE. 1966. Pathogenicity Of Pratylenchus penetrans and Tylenchorhynchus claytoni on turfgrasses. Phytopathology 995-998. TROLL, J. and A.C. TARJAN. 1954. Widespread occurrence of root parasitic nematodes in golf-bourse greens in Rhode Island. P1. Dis. Reptr. 2E1: 342-344. TSENG, S-T., K.R. ALLRED and G.D. GRIFFIN. 1968. A soil population study of Ditylenchus dipsaci (Kuhn) Filipjev in an alfalfa field. Proc. helminth. Soc. Wash. 22: 57-62. 131

VAN GUNDY, S.D. and R.L. RACKHAM. 1961. Studies on the biology and pathogenicity of Hemicycliophora arenoria. Phytopathology 51: 393-397. VAN GUNDY, S.D., L.H. STOLZY, T.E. SZUSZKIEWICZ and R.L. RLCKHAM. 1962. Influence of oxygen supply on survival of plant- parasitic nematodes in soil. Phytopathology .5.a: 628-632. VAN GUNDY, S.D. and L.H. STOLZY. 1961. Influence of soil oxygen concentration on the development of Meloidogyne jawilnica. Science 134: 665-666. VEGTE. F.A. VAN DER and J. HEYNS. 1963. A report on nemacodes found in soil and root samples from the Kruger National Park. Koedoe. Journal for Scientific Research in the National Parks of South Africa, 6: 134-136. WALLACE, H.R. 1956. Soil aeration and the emergence of larvae from cysts of the beet eelworm, Heterodera schachtii Schm. Ann. appl. Biol. 44: 57-66. WALLACE, H.R. 1959. Movement of eelworms. V. Observations on Aphelenchoides ritzemabosi (Schwartz, 1912). Steiner, 1932 on florists' chrysanthemums. Ann. appl. Biol. 47: 350-360. WALLACE, H.R. 1962. Observations on the behaviour of Ditylenchus dipsaci in soil. Nematologica 7: 91-101. WALLACE, H.R. 1963. TheBioloofPlanLy - ::aslti...... saen22-todes. E. Arnold Ltd., London, England. 132

WALLACE, H.R. 1968. Undulatory locomotion of the plant parasitic nematode Meloidogyne l[vanica. Par-Isitology 58: 377-391.

WALLACE, H.R. and D.N. GREET. 1964. Observations on the taxonomy and biology of Tylenchorhynchus macrurus (Goodey, 1932) Filipjev, 1936 and Tylenchorhynchus icarus sp. nov. Parasitology 54: 129-144. WHITEHEAD, A.G. and J.R. HEMMING. 1965. A comparison of some quantitative methods of extracting small vermiform nematodes from soil. Ann. appl. Biol. 5_2: 25-38. WILLIAMS, H.T. 1966. The changing pattern of agriculture, J. Agr. Soc. U.C.W., Aberystwyth 47: 25-33. WINCHESTER, J.A. and E.O. BURT. 1964. The effect and cou:Irol of sting nematodes on Ormond Bermuda grass. P1. Dis. Reptr. 48: 625-628. WINSLOW, R.D. 1960. Some aspects of the ecology of free-living and plant parasitic nematodes. In Nematology: Fundamentals and Recent Advances. Eds. J.N. Sasser and W.R. Jenkins. Univ. North Carolina Press, Chapel Hill, North Carolina, USA. WOUTS, W.M. 1966. Paratylenchus halophilus (Nematoda: Criconemat- idae) a new species from New Zealand. New Zealand J. of Science 9: 281-286. YUEN, P.H. 1966. The nematode fauna of the regenerated woodland and grassland of Broadbalk wilderness. Nematologica 12:

195-214. 133

ZUCKERMAN, B.M. 1962. Parasitism and patht,:;enesis of the cultivated highbush blueberry by the stubby root nematode. Phytopathology 52: 1017-1019. ZUCKERMAN, B.M. 1968. Caribbean Symposium on Nematodes of Tropical Crops, Trinidad 1968. Special Report. Helminth. abst. 2z: 185-189. ZUCKERMAN, B.M. 1969. Specialised adaptations to parasitism among ectoparasitic plant nematodes. In: Nematodes of tropical crops. Ed. J.E. Peachey. Tech. Commun. Commonw. Bur. Helminth. No. 40, 355 pp.

134

Appendix Table 1. a) Vertical distribution of nematodes in Highfield on 22/10/68. Number of nematodes/ 200 ml soil. Means of two subsamples.

CO CO E 4 ta a) 0 0 .1 0 El

O spp. 4 0 11) ;-1 0

0 0 hus 0 CO 0 0 H 0 0 0 $-1 c-3

0 lenc o •r-I 1 t 0 O Co •r1 Ty rn E-1 E-1 0 0-5 2530 84o 58o 430 ?0 30 1401 3 # 8180 5-10 7430 117o . 1170 350 50 10 410, - 4 15290 10-15 688o 670'; 300 120 10 20 2901 1 1156o 15-20 8690 450 32o 50 20 230 5 2 12710 20-25 5790 56o 5o - 90 10 loo ; 8 3 8420 25-3o 2750 400 - 130 40 16 4 4990 30-35 85o 17o - 190 40 1 24 2 1690 35-4o 78o 120, - 350 101 3 1580 a. 0

b) Highfield 22/10/68

Soil Soil moisture Root content gm Temp. depth content c;c, dry (dry wt/100 gm o (cm) wt. of soil of soil) C

0-5 26.90 1.1329 14.o 5-10 20.77 0.1699 13.5 10-15 21.33 0.1714 13.5 15-20 20.47 0.1962 13.0 20-25 19.19 0.2470 12.5 25-30 19.12 0.0911 12.5 30-35 17.51 0.1391 12.5 35-40 16.55 0.0503 12.5

135

Appendix Table 2. Vertical distribution of nematodes in Lime Kiln, Grasslands Research Institute. No. of nematodes/200 ml soil. Means of two subsamples. a. Date sampled 22/1/69

I 0 0 O 0›g *IC U, t g .4 I CI 4 0 co 0 •ri rd 0 .14 o .0 0 0 0 0 TS 0 r.,. a I)-1 a cl d Pi I H g-r Cl)m 0CO '00' 0 Pi Z 0 ED o j0 Sy >a o g g 0 4-1 0 0 id .... a g g• a H -P.Cl) 0 0 tiO 0 0 •ri F.1 S-/ D H H 0 0 0 'd O 0 0 cii0 0 0 g ,o d 0 0 ›., >a .1-1 g Ho • 9-1 d O > •ri 0 •rrg 0 m z 0 .ri p• .p • .4_, f.4- 0 • o >. 0 .H 0.0 g-r .H 1 05 .r.1 • 0 4-1 ^d .1-1 0 •0 d Pi COS r---; g4 _d P4 0 $.4 0 H 24' H k g-i Pi F-I $-1 ›) Pi Pi Pi rj Cdi II rr-J or:"63 z2 ci .1-i Pi 0 P-1 0 El O <4 U) ;4 H C.) 0 ..4 Mcl) E(3 E • . AC E-7 E- - I E-. I .I

0-5 780 355 235 525 785 15 65 - 54o 25 4. 4. 6750 5-10 12325 345 310 475 325 65 - 395 10 - - - 8015 10-15 14240 45o 105 6o 110 5 38o 58o 5 - 9405 15-20 ;6030 575 15 55 300 445 - 11065 20-25 13330 455 24o 5 190 6o 1 - 6360 25-301145 185 16o 5 3o 20 1 14 244o 3o-35 I 48o 40 ••• 85 5 2 21 1080 35-4o I 14o 4o 45 10 5 2 33 46o

b. Date sampled 4/3/69

0-5 1175 1215 135 520 615 15 65Q 15 - - - 11045 5-10 1820 710 1105 95 27o 30 395 - 6620 10-15 ,2860 430 4o 30 loo 160 370 2 6550 15-20 3215 450 - 15 ••• 250 38o 5 - - 1 6405 20-25 1975 715 5 - 150 5 8o 5 - 1 7 4405 25-30 46o 65 7o 25 15 12 1265 30-35 160 50 1144. 95 20 15 - 3 33 685 35-40 190 5 OMR 55 10 - 2 22 545 40-45 41.44 15 5 1 26 14o 45-5o 5 4444 41. - 4 18 110 50-55 5 - 1 17 3o 55-6o 5 7 20

136

Appendix Table 2. cont. c. Date sampled 29/4/69

U m 0 m •-• , r0 a) co

a 0 des o . (0 .1:1 0 CO Ti 0 CO ...... ' 0 o 0 o o vi TS 0 g 0 to 0 0 •r1 21 ...0 , 0 0 4 4 d 0 0 •ri 0 43 , 0 0 H ▪$ -1 0 (0 '0 'd 0 0 RI 0 .0 0E 0 0 CO ;.I %a 0 0 0 0 eri 0 0 0 0 C.) Q p. . n 00 Et0 ,0 0 0 cd 4 0o o ,d ::-2 'ZS 0 0 0 Xt 0 Q 0 r-I H 0 0 rd 0 0 0 rcS 0 nema -P 0 0 Z :--i Z 0 .ri $4 P. -r-4 0 .1-1 0 0 sr! 0 l H Cl S-1 • 0.H .7:1 -rI „to 4-5 • 4-) 0 a) • 40 • • 0 > 0 •ri Et° $4 - ' S-i 0 H PI 0 p., 0 0 r-I P4 A Pi r4 $4 -r1 H 0 0 ta o 4 d •H 0 124 •-a rj _ • $4 fai 1-1 $4 •-h P-i pi p4 0 0 ;-I 0 0 0 i E ...74 0 E-4 5, E4 Er ps r-2; pi 0 04 0 E-i V/ 0 ..." c-1 0 d 14 To cr/ 4 cl '0-5 410 100 90 425 395 5 15 56o 5 - 10580 5-loi 88o 50 80 190 350 25 330 - - 5900 10-15;1895 220 8o 35 135 135 205 - - - 5740 15-20;3200 220 15 5 50 85 195 - - - 7070 20-25:3380 160 - 5 95 10 as, 1 6 637o 25-30f1075 100 95 55 1 11 2645 30-35 665 10 15 20 10 1 12 1050 35-4o; 325 10 20 ••• 10 11.11, 15 1 14 625

d. Date sampled 25/6/69

0-5 ! 84o 170 270 790 610 35 - 66o 5 - 12850 5-1011115 180 go 225 345 95 340 - 6555 10-1511680 210 35 15 95 411.1.1 4o 5 300 - 4705 15-2012510 250 5 5 20 8o 190 1 2 4770 20-2512880 160 - - 5 Oa 6o 10 190 1 4 4980 25-3011280 130 5o 5 3o 14 2250 30-351 800 6o 25 10 20 30 1 25 1 65 35-401 725 30 4o 5 5 3o - 14 109 e. Date sampled 16/12/69

0-5 12750 335 570 1910 1260 35 4o - 510 5 - - 34650 5-10 13700 375 545 870 450 - 90 - 370 5 - - 25050 10-15 33450 425 100 50 75 - 170 - 32o - - - 41600 15-20,32850 445 20 5 20 - 200 - 17o - 4 43050 - 195 - 150 5 70 4 21050 20-25 15160 485 25-30 3120 400 5 ••• - 145 - 25 10 45 1 23 6190 3o-35 2230 80 - 135 - 3o - 45 1 46 3995 .35-40 885 50 85 - 25 5 - 4 29 2040 Sampling dates 10-15 Soil 15-20 depth 30-35 35-40 25-30 20-25 ( 01 5-10 0-5 - 1) 22/1/69 7 .4. 8.1 8.1 7.9 7.3 7.7 8.3 8.5 - 4/3/69 3.0 2.9 3.0 2.8 2.8 2.9 2.8 3.2 Soil temperature 29/4/69 11 .3 10.5 10.0 8.8 8.8 8.9 9.0 9.5 - 25/6/69 16.5 15.5 16.0 16.0 15.5 15.5 15.5 15.5 ° C 1 6/12/69 3.1 3.7 3.5 3.5 3.3 4..0 4 4.5 , 3 2 18.90 33.78 16.21 16.49 2 15.94. 22.10 22.63 21.72 / 1 % drywt.ofsoil /69 Moisture • 29/4/69 18.83 16.69 17.78 1 7.78 14.68 13.64. 13.89 27.99 cont ant 1 6/12/6 17.24. 1 8.20 15.20 16.01 26.98 20 .12 20.12 20.33 ( dry Root contentgin - rit/i 00gmofsoil) 1 6/12/69 0 .5369 0.2407 0 .1022 0 .1112 0.2396 0.1005 0.1316 0 .1061

, H)

0 ? aTquI xTpuedd,,

138

Appendix Table 3. Fresh weights of shoots and roots and number of tillers of Lolium perenne per pot inoculated with a mixture of nematodes and Paratylenchus icrodorus alone (each pot contained 3 plants).

Fresh wts. (gms) Replicates No. of tillers Roots Shoots

Mixture of 1 0.7658 5.2704 23 nematodes 2 2.0685 6.1435 25 3 1.9992 7.2444 21 4 1.4652 5.6744 22 5 1.3656 4.0890 18 6 3,4142 8.2630 28 7 1.074 3.6463 18 8 1.264o 2.9172 16 9 3.5567 5.6503 21 10 1.1168 5.3133 16

Paratylenchus 1 2.6867 7.5800 24 microdorus 2 1.7782 5.0974 -20 3 0.8607 3.6894 16 4 1.5104 6.1270 22 5 2.2074 4.1o56 12 6 1.5190 5.0112 16 7 0.6185 2.7167 12 8 1.9490 6.5683 18 9 1.7926 5.1897 19 10 1.6007 3.8151 19

Control 1 0.9304 4.o832 14 2 0.8172 3.0614 11 3 1.3972 4.5972 19 4 2.6832 7.0472 21 5 1.2124 4.7822 13 6 1.5522 2.354o 14 7 2.3590 5.4172 22 8 2.7944 5.0172 25 9 2.1722 4.7092 15 10 1.5072 3.4772 15 139

Appendix Table 4. Nematodes recovered from soil in pots planted to perennial ryegrass. a. Paratylenchus microdorus

Nos. of nematodes Replicates per pot (250 ml soil)

1 9,000 2 6,500 3 7,250 4 9,500 5 18,250 6 11,250 7 4,50o 8 9,500 9 4,250 10 8,000 b. Mixture of nematodes

ez u) I 0 0 ill 0 0 0 0 0 4-) 4 0 Pi 0 $4 0 0 0 0 0 o 4 .1-1 o 0 a) co 4 c-J a) 0 F4 1 F-4 4 Z m rd "0 0 0 ,I 4-3 tes 0 0 -,, co O Z £1 0 0 •,-1 4 51 114.ri H rd -1-) 0 .0T) .r1 ;-1 Fi 5 0 a) CO 0 0 4 C.) N m 0 0 0 • H • -1-) F-1 0 0 0 m co 0 •,-1 F-1 m p. P1 71 lica d ri 0 0 4 0 4-1 0 4 t—I 04 -I-) P4 -p tit $4-I-1 0 . H 0 r-4 0 ui u) 0 Pi 0 a) ri '-a 0 P F-t P Rep P4 .-r-, P E-91 ET E-.4 &.1 tai 1, 2375 25 2400 325 25 175 100 5o 5475 2 1500 25 600 150 50 25 50 2400 3 2025 5o 1800 30o 125 - 75 25 440o 4 1025 5o 2550 45o 300 125 15o 25 4675 5 1125 25 65o 225 25 25 75 215o 6 1625 - 1825 525 325 5o 100 25 4475 7 850 50 625 225 175 25 100 2050 8 475 25 1275 325 75 25 25 50 2275 9 1050 5o 145o 325 150 50 175 - 3250 10 575 - 750 100 125 - 100 - 165o 140

Appendix Table 5. Pot experiments in the glasshouse. Analyses of variance.

df SS MS Observed Required3 F F 5% 1%

Root weights Total 29 27.990 Treatment 2 0.616 0.308 0.304 3.35 5.49 Error 27 27.375 1.014

Shoot weights Total 29 38.017

Treatment 2 5.000 2.500 2.044 3.35 5.49 Error 27 33.017 1.223

No. of tillers Total 29 1128.97

Treatment 2 26.87 13.44 0.329 3.35 5.49 Error 27 1102.10 40.82

141

Appendix Table 6. Dry weights of shoots and roots of Lolium perenne in tubes inoculated with three nematode species (one seedling per tube).

Dry wts. (gms) Replicates No. of tillers Roots Shoots

Paratylenchus 1 1.0889 0.4254 4 microdorus 2 0.4411 0.6560 8 3 1.0686 0.4631 4 4 0.7870 0.6384 4 5 0.5651 0.6488 5 6 0.5403 0.7706 5 7 0.7252 0.5556 4 8 1.1714 0.4372 4

Tylenchorhynchus 1 1.1673 1.3049 15 icarus 2 0.6250 0.8124 10 3 0.5503 0.5708 7 4 1.1196 0.4744 7 5 0.6295 0.7685 8 6 0.7408 0.4772 5 7 0.4567 0.8231 7 8 0.2565 0.4031 3

T. maximus 1 1.1196 0.7854 5 2 0.7907 0.5515 5 3 0.4973 1.0565 10 4 0.7126 0.8383 11 5 0.5298 0.7673 12 6 1.4850 0.3992 4 7 Q,6423 0.5914 8 8 0.3577 0.6137 5

Control 1 2.0428 0.4749 11 2 0.5952 0.3523 3 3 0.6819 0.5049 6 4 0.6233 0.6529 6 5 0.6116 1.1077 11 6 0.8351 0.8627 5 7 0.5736 0.4902 6 8 0.7574 0.5563 7 142

Appendix Table 7. Nematodes recovered from soil in tubes planted to perennial ryegrass.

Nos. of nematodes Species Replicates per tube (140 ml soil)

Paratylenchus 1 25,200 microdorus 2 55,000 3 21,100 4 18,900 5 31,400 6 19,300 7 13,400 8 21,600

Tylenchorhynchus 1 10,110 maximus 2 6,800 3 4,000 4 5,200 5 4,200 6 6,600 7 1,270 8 1,500

Tylenchorhynchus 1 20 Icarus 2 3o 3 10 4 80 5 4o 6 10 7 6o 8 3o 143

Appendix Table 8. Tube experiments at controlled temperature. Analyses of variance.

•

df SS MS Observed Required F F 5% 1%

Root weights Total 31 3.990 Treatment 3 0.092 0.031 0.221 2.95 4.57 Error 28 3.898 0.139

Shoot weights Total 31 1.088 Treatment 3 0.036 0,012 0.318 2.95 4.57 Error 28 1.052 0.038

No. of tillers

Total 31 272.470 Treatment 3 44.595 14.863 1.827 2.95 4.57 Error 28 227.875 8.138

144

Appendix Table 9. Total root growth of perennial ryegrass seedlings on agar plates (ems).

Replicates Days after inoculatiOhwith nematodes 2 4 6 9 14

Control 1 0.90 3.50 6.20 9.85 20.50 2 1.35 3.50 6.10 9.50 19.35 3 1.90 4.75 7.90 13.40 25.20 4 2.5o 4.95 7.30 14.90 23.80 5 1.20 2.45 3.30 4.00 4.00 6 1.10 2.65 3.25 4.15 6.05 7 1.60 4.10 8.4o 15.4o 29.95 8 3.05 5.5o 8.55 15.45 33.00 9 1.90 4.85 6.15 11.20 27.35 10 2.35 5.00 6.10 6.8o 6.8o

Tylenchorhynchus 1 1.90 3.40 7.20 20.10 42.25 maximus 2 1.25 2.70 6.55 13.30 28.00 3 2.30 5.15 8.65 16.20 33.05 4 2.5o 5.45 9.5o 18.75 35.55 5 0.55 3.20 6.15 12.25 23.35 6 2.10 2.55 6.05 14.90 34.10 7 1.65 3.50 5.55 7.70 19.10 8 2.65 5.55 9.85 16.50 35.85 9 2.5o 4.75 7.30 15.00 30.95 10 2.7o 6.90 10.50 17.30 31.70

T. icarus 1 0.80 2.20 4.4o 8.3o 14.10 2 1.50 3.8o 8.8o 15.6o 29.95 3 2.75 7.65 14.30 23.35 43.80 4 1.95 4.95 9.75 17.90 35.35 5 2.20 4.65 5.50 5.80 6.3o 6 2.15 5.3o 8.55 11.55 16.95 7 0.70 1.85 3.50 6.00 6.57 8 2.4o 6.10 10.35 15.80 34.55 9 2.05 3.90 6.05 10.80 18.00 10 2.85 6.90 11.05 22.15 43.80 145

Appendix Table 10. Lateral root growth of perennial ryegrass seedlings on agar dates (cms).

Replicates Days after inoculation with nematodes 2 4 6 9 14

Control 1 - - - 1.45 5.65 2 - - - 0.30 2.95 3 - - - 0.55 3.55 4 - - - 2.7o 15.5o 5 - - - 0.80 6 - - - - - 7 _ _ 0.60 1.6o 3.20 8 - - - 2.95 11.8o 9 _ 0.60 0.75 2.6o 9.60 10 - 0.50 0.60 0.95 0.95

Tylenchorhynchus 1 - 1.45 10.60 25.90 maximus 2 - - 0.90 3.65 10.95 3 - 0.85 4.95 13.35 4 .M. ••• - 2.60 7.70 MO 5 - 1.55 5.55 6 - 0.10 2.55 9.30 22.60 7 _ - 0.25 1.75 8 _ - 0.65 2.65 15.75 9 - 0.20 1.50 6.55 14.80 10 - 0.70 1.15 2.50 4.'15

T. icarus 1 - - - la 2 - o.40 1.80 11.3o 3 - - _ 0.05 1.,3 4 - 0.50 5.30 5 - - - - - 6 _ _ - 0.30 7 - - _ - - 8 _ 0.70 10,45 9 - - - 1.00 2,,85 10 - 0.70 1.15 6.25 1.T,.35 146

Appendix Table 11. Numbers of nematodes feeding on roots of perennial ryegrass (50 nematodes/agar plate).

Replicates Days after inoculation 2 4 6 9 14

Tylenchorhynchus 1 12 12 7 5 maximus 2 1 1 1 3 3 10 14 8 4 4 10 5 2 2

5 13 24 14 7 ONO 6 16 15 11 3 7 3 3 3 3 8 7 3 2 1 9 12 18 7 5 4 4 1

T. Icarus 1 3 6 6 7 2 4 3 3 3 3 2 4 1

5 4 5 6 411.0 6 7 5 2 3 7 1 5 6 4 8 1 2 1 1 9 2 1 10 2 3 1 2 147

Appendix Table 12. Agar plate experiments. Aanalyses of variance of root lengths after 14 days growth.

df SS MS Observed Required F F 5% 1%

Total root growth Total 29 4061.09 Treatment a 537.01 268.30 2.057 3.35 5.49 Error 27 3524:08 130.52

Lateral root growth Total 29 1411.68 Treatment 2 396.53 198.27 5.273 3.35 5.49 Error 27 1015.15 37.60

Significant at the 5% level

LSD0.05 = 5.60 cm LSD = 7.56 cm 0.01