Effects of root-invading fungi on the growth of red clover (Trifolium pratense L.) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Pathology at Massey University

Copyright is owned by the Author of the thesis. Permission is given for

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private study only. The thesis may not be reproduced elsewhere without

the permission of the Author.

EFFECTS O F ROOT-INVADING FUNGI ON THE GROWTH OF RED CLOVER (Trifol ium pratense L.)

A thesis presented i n partial fulfilment of the requirements for the degree

of

Doctor of Philosophy i n Plant Pathology at Massey Unive rsity

NAN Zhi Biao 1 989

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A BSTRACT

Metho d s to monitor i nvasion of r e d clover roots by s oilb orne fungi, and assess effects on clover growth and pers istence, were d evelop e d and tested using soil from a plant breeder's red clover evaluation block at DSIR G rasslands Division, Palm erston North, which was known to contain seve ral fungal speci e s p athoge nic t o red clover. A quantitative method employing tissue maceration a n d p l ating was u s e d to determine t h e i ntern a l microflora o f r e d clover roots from the evaluation b lock. Effects of e nvironmental factors, and of application of fungicid e drenches to the soil, on root invasion under controlled e nvironment and field conditions were also studied using the root maceration method. Fungi isolated from roots of red clover from the evaluation block were tested for their effects on establishment, growth, and persistence of red clov e r in the glasshouse and in field microplots. Light microscopy and tran smission electron microscopy were used to study invasion of red clover roots by Trichocladium basico la, and the effects of the fungicides benomy l and p rochloraz on this fungus i n vitro and i n

VIVO.

The root maceration method d etected a similar range of fungi to that found by p lating 1 ^- 2 m m long s egments, but yielded more colonies and showed

less variation. Using standardised amounts of tissue and b l e nding times ( 2 g and 6 0 o r 1 20 sec.) d ifferences i n fungal populations i n roots subjected to different treatments were readily detecte d .

Vertici l l ium dahl iae, Trichocladium basicola, and Cyli ndrocladium scoparium were the major components of the root-invading mycoflora of red clov e r in the evaluation block, which consisted of 40 fungal species. O t h e r major i nvaders were Fusarium solani, F. oxysporum, Cylindrocarpon destructans, and G liocladium roseum, which are the fungi most commonly isol ate d from roots of red clover and other forage legumes worldwide.

Fungal i nvasion of red clover roots was affecte d by plant age, soil temperature and moisture. Generally, numbers of fungal colonies isolated p rogressively increased from the seed ling stage onwards and more colonies were

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isolated from roots of plants grown at 20 and 25oc than at 1 0 and lYC, and from 60 and 80% WHC than at 40% WHC.

Prochloraz was the most suitable of 1 1 fungicides tested for use as a soil drench t o study effects of root-invading fungi on red clover growth i n the field.

I t showed a broad spectrum of antifungal activity, controlled the major root­

i nvading fungi e ncountered in the experime ntal soil, was not toxic to Rhizobium trifolii, and was least retardant t o red clover growth.

The numbers of fungal colonies recovered per gram of roots was 60 - 80%

l ower from plants from field plots receiving a single application of prochloraz drench at 3 .46 g/m2 than from p lants from untreated plots. Yields from tre ated plots harvested 4 times ove r a p eriod of 45 weeks were 28 - 95 % higher than those from untreated plots.

The m ajor root-invading fungi i solated from red clover p lants grown in the plant breeder's evaluation block, �· scoparium, T. basicola, F. solani, and F . oxysporum, reduced surv ival of red clover plants in field plots and microplots by 20 -75 % , and d ry matter yield by 20 -60%, over a periods of 62 and 76 weeks. V.

dahliae, _.C.. destructans, and _.G. roseum, also reduced plant growth i n field plots but to a lesser extent. See dling e stablishment, and n itrogen fixation and n odulation, were affected adversely by some fungal isolates .

T . basicola was found to penet rate roots of red clov e r directly and colonise tissue by means of "beade d hyphae" ( intracellular hyphae which were constricted at their septa) then "straight hyphae" ( unconstricted hyphae growing parallel to the root axis) . The fungus i s h e mibiotrophic. Invaded host cells in the epidermis and cortex of the root are appa r ently unaffe cted at first then degene rate and die.

Papillae are often found at sites of p e netration through cell walls but these rarely obst ruct fungal development. Pre-treatment of seedlings with benomyl or proch loraz re duced fungal p e n e tration and growth within tissues. Changes in fungal ultrastructure resulting from benomyl treatment were an i ncreased frequency of lomasome p roduction and occasionally a disorganisation of cell contents. Changes resulting from prochloraz treatmen t include d thickening and fragmentation of cell walls, and necrosis of hyphal cell s .

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ACKNOWLEDGEMENTS

It has been a privilege for me to study at Massey U niversity and DSIR Plant Protection. I wish to express my sincere thanks to: -

my sup e rvisors, Dr P.G. Long of the Plant Health Departme nt, Massey U n ive rsity, and D r R.A. Skipp of DSIR Plant Protection, Palmerston North, for their und erstanding and willingness to provide supervision during the d evelopmental p hase of this study, for their stimulation and wise couns e l during the experimental work, for their constructive critici s m and correction of my English during the writing of the thesis, and for their looking after my p ersonal w e lfare during my stay i n N ew Zealand. In particular, I am deep ly indebted to Dr R .A . Skipp for his persistent efforts to obtain the financial support which made this study possible, for his untiring gui dance throughout, especially during the last three months of the writing when Dr Long was o n sabbatical leave ove rseas. To m e , the three year study h as been an invaluable educational experie nce. I am p roud of my relationship with them both which is not only as sup e rvisor and s tudent, but also as intimate friends.

Prof. K.S. Milne of the Plant Health Department, M assey U n iversity, for acting as chief supe rvisor when Dr Long was away, and for his encourage ment and i ntere st during t h e study.

This study was funded by Department of Scientific and Industrial Research ( DSIR) of New Zealan d . The receipt of a D SIR p ost graduate r e search fellowship i s greatly app reciated . Travelling allowance and study l e ave giv e n by Gansu G rassland Ecological Research Institute of China i s also acknowledged. I am most grateful to: -

Dr P.J. Brook of DSIR Plant Protection, and Prof. R e n J izhou of Gansu G rassland Ecological Research Institute, China, for their encouragem e nt throughout and for the i r major role i n obtaining and administering financi al support for this study ;

Dr G.C.M. Latch for his permission t o use the DSIR Plant Protect i o n l ab oratory and facilities at t h e Palmerston North sub-station, f o r his e ncouragement, forb earance, and h e lp.

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I sincerely acknowledge the h elpful support from the staff of DSIR Plant Protection, Palmerston North. The i r co-operative manner, willi ngness to h e lp, a n d frie n dly attitude have made a substantial contribution to the success of this study . I n p articular, I wish express my thanks to : -

M r M .J . Christensen for his p e rsonal inte rest and providing support a n d assistance w h enever it was needed.

D r R.E. Falloon for his efforts to obtain funds and for his e ncourage ment w h e n h e worked at Palmerston North .

M r B.J. Tidball and Mr R . E. Hickson for field assistance .

I also wish to thank the following at D SIR Palme rston North Centre:

Mr R . H . Fletcher for statistical help.

M r D . Hop croft and M r R.J. Bennett for their s kilful p rocessing of experimen tal m aterial for e l e ctron m icroscopy studies, and for valuable comments on some TEM photo graphs.

M r P. Spring for p hotographic s e rvices.

M r S.A. Northover, Miss K. Jones, and Mrs A. A inscough for their prompt a n d efficient library servi ce .

M r J.P.M. Evans, D r J . R . Crush, D r J .R. Caradus, Dr R.J.M. H ay, D r M . P . Rolston a n d M r M .J. Hare f o r their h e l p at d ifferent stages o f the study.

I acknowledge the assistance given to me by the following at M assey U niversity : -

D r D . R . Scatter for m e asuring soil water potential.

M r H.F. Nelson and Mrs L.K. D avis for field assistance .

Grateful thanks i s exten d e d to our friends, Nitayaporn and M i chael Hare, f o r their genuine concern, care and understanding.

My h eartfel t thanks als o goes to my parents and the family, and my parents i n law for their enthusiasm and sacrifices m ade to look afte r our d aughter.

Final ly, I thank my w ife, Yan R ong, for h e r continual moral support during my first year study, for her i nv a luable company during the l ast two years while she was also studying at Massey U niversity for a masterate in the Seed Technology Centre, h e r presence has made l ife i n a foreign land much e as ie r and most i nteresting.

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TA BLE O F CONTENTS

A BSTRACT . . . . . u

ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . tv

LIST OF TAB LES . . . ^{. .} . ^{. .} . . . . ^{. . .} . . . ^{. .} . ^. . . . ^Xlll

LIST OF FIGURES . . . xvi

LIST OF PLATES . . . xvi i i CHAPTER 1 INTRODU CTION . . . . . . . . . . . . . . . . . . . . . . 1

CHAPTER 2 REVIEW O F LITERATURE . . . . . . . . . . . . . . . . . . 3

2 . 1 Introduction . . . ^. . . . ^. . . . . ^·. . . . . . . . . . . . . . . . . . . 3

2.2 R e d clover plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.2. 1 Taxonomy, o rigin and distribution . . . . . . . . . . . . . . . . . . . . . 3

2.2.2 Importance i n world agriculture . . . . . . . . . . . . . . . . 4

2.2.3 Effects of soil temperature on red clove r ^. . . . . . . . . . . . . . . . . . . 5

2.2.4 Effects of soil moisture on red clover growth . . . . . . . . . . . . 6

2.3 Root d iseases of forage legumes, and associat e d fungi . . . 8

2.3. 1 . Fungi associated with root diseases ^. . ^{. .} . . . 8

2.3.2 Seedli ng d am ping-off . . . ^. . . . ^. . . . . 1 4 2.3.3 Root rot . ^. . . . ^. . . . ^{. .} . . . ^. . 1 4 2.3.4 Wilt . . . ^. . . . .^. . . . 1 6 2 . 4 Effe cts of soil te mperature and moisture, a n d other factors o n root rot complex of forage legu me s . . . 1 7 2.4. 1 Effects o f temperature and moisture on mycelial growth of root-rotting fungi from forage legumes in culture . . . ^. . . . . 1 7 2.4.2 Effects of temperature and moisture o n the d evelopment of root rot . . . . . . . . . . . . . . . . . 1 9 2.4.3 Effects of other factors on root rot development . . . 20

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2.5 Methods used to obtain plants with d ifferent levels of root disease . . ^. . 23

2.5 . 1 Glasshouse studies ^. . ^. . . . ^. . . ^{. .} . ^{. .} . . . ^. . . . . ^{. .} . ^{. .} . ^. . . ^. . ^{. .} . . . 23

2.5 . 1 . 1 Soil (growth substratum) infestation method . . . ^{. .} . . . ^. 23

2.5 . 1 .2 Root dip method . . . 24

2.5 . 1 .3 Root-injuring method . . . 25

2.5 . 1 .4 Slant-board method . . ^{. . . .} . . . .^. . . ^. . ^. . . . ^. . . . 25

2.5 . 1 .5 Fungicide application method ^{. .} . ^. . ^. . . . ^{. . .} . ^. . . . . ^{. .} . 26

2.5.2 Field plot m e thods . . . ^. . . ^. . . . . ^{. . .} . . . ^. . . . ^. . . . 26

2.5 .2. 1 Fungicide application method ^{. . .} . ^. . ^. . . ^{. .} . ^. . ^. . . . 26

2.5 .2.2 Plant i noculation method ^. . . . ^. . . . ^. . . . ^. . 27

2.5.3 M icroplot m e t h od . . ^{. .} . ^{. . . .} . . ^. . . . ^{. .} . ^{. .} . ^. . ^. . . ^. . ^{. .} . . . ^. . ^. 28

2.6 Effects of root diseases on growth of forage legumes . ^{. .} . . . ^. . ^{. .} . . . . ^. . 29

2.6. 1 Effects of d i s e ases on e stablishment ^. . . . . ^. . . ^. . ^. . ^. . . . . ^{. .} . ^. . . 2 9 2 . 6 . 2 Effects of d i s e ases o n y i e l d . ^{. . . .} . ^. . . ^{. .} . . . ^. . ^. . ^{. .} . ^. . . ^. . ^. . ^. . . 3 1 2.6.3 Effects of d i s e ases on n itrogen fixation and nodulation ^. . . ^{. . .} . 3 3 2 . 6 . 4 Effects of d i s eases on p e rsistence . . . ^{. .} . . . . ^{. .} . . ^{. .} . ^{. . .} . . . ^. . . 33

2. 7 Light and electron m i croscopy studies of root invasion by l.'richocladium basicola and of effects of fungicides o n fungal growth in vitro and i nvasion of host tissue . ^{. .} . . . . ^{. . .} . . ^{.. . .} . . . ^. . ^. . . . ^. . . ^. . 34

2. 7. 1 U ltrastructure of T. basicola . . . ^. . ^. . . ^. . . . . ^{. . .} . . ^{. .} . ^. . ^. . . . 34

2. 7.2 Root i nvasi o n by T. basicola ^{. .} . ^. . . ^. . ^. . . . . ^{. .} . . ^. . ^{. .} . ^. . ^{. .} . . . ^. 35

2. 7.3 Effects of benomyl on ultrastructure of fungi grown in culture and in hosts ^{. .} . . ^{. .} . ^. . ^{. .} . ^. . ^. . ^{. . .} . . . ^{. .} . ^. . . ^. . . ^. . ^. . . . ^{. . . .} 37

2.7.4 Effects of e rgosterol b iosynthesis i n h ibiting fungicides on ultrastructure of fungi grown i n culture and i n h osts . . . . ^{. .} . . ^. 3 8 CHAPTER 3 GENERAL MATERIALS AND METHODS ^. . ^. . ^{. .} . . . . ^{. . .} . . . ^. . ^. . 40

3 . 1 Seed ^{. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .} 40

3.2 Experimental sites ^{. . .} . ^{. . .} . . . ^. . . ^. . . ^{. .} . ^. . ^{. . . . . .} . ^{. .} . ^{. .} . ^{. . .} . . ^{. . . . . . .} 40

3.2. 1 Plant breeder's evaluation block ^{. . . .} . ^. . . ^{. . . . .} . . ^. . ^{. . .} . ^. . ^{. .} . 4 0 3.2.2 Pasture block ^{. . . . .} . ^{. . . . . .} . . ^{. .} . ^. . ^{. . . .} . ^{. . .} . . ^. . . ^{. . . . .} . . . ^{. .} . 4 1 3.3 Soil p rocessing for pot exp e ri m ents ^. . ^. . ^{. .} . . ^. . . . . ^{. . .} . ^{. .} . . ^. . . . ^{. . .} . ^. . 42

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3.4 Determination of soil wate r holding capacity . . ^{. . .} . . . ^{. . .} . . . ^. . . . ^. . ^. 42

CHAPTER 4 Q UANTITATIVE ASSESSMENT OF ROOT INVASION BY SOILBORNE FUNGI UNDER CONTROLLED ENVIRONMENT AND FIELD CONDITIONS . . . ^. . . . . ^. . . . ^{. .} . . . 44 4. 1 Introduction . . . . ^. . ^. . . . ^. . . ^. . . . ^. . . . ^. . . . . ^. . . . 44 4.2 Experi ment one: assessment of a root mace rati on method for studying

invasion of red clover roots by soilborne fungi ^. . . . ^. . . . ^. . . . . 45 4.2. 1 Introduction . . . ^. . . ^. . . . ^. . . ^{. . .} . . . . 45 4.2.2 Materials and methods . . . ^. . . . ^. . . . ^{. .} . . ^. . . . . 46

4.2.2. 1 Comparison of root segment and root mace ration

methods . ^{. .} . . ^. . . . ^. . . . ^. . . . ^. . . . ^{. . .} 47 4.2.2.2 Effects of the quantity of roots blend e d and blending

time on the number of fungal colonies isolated ^. . ^{. .} . . . . 48 4.2.2.3 Effects of the quantity of roots blended and blending

time o n percentage of root tissue mace rated . . . ^. . ^. . . 49 4.2.2.4 Calculation of maximum number of colonies . . . 49 4.2.2.5 Statistical analyses . . . ^. . . . ^. . . ^. . 49 4.2.3 Results ^. . . . ^{. ·} . . . ^{. .} . . . ^. . . . 5 0

4.2.3 . 1 Comparison of the root segme n t and root maceration methods . ^. . . ^{. .} . ^. . . . ^{. .} . ^. . . . ^. . ^. . . . 50 4.2.3.2 Effects of the quantity of roots blended and blending time

o n the number of colonies isolated . . . ^{. .} . . ^. . . . 5 0 4.2.3.3 Effects of the quanti ty of roots blended and blending

time o n the pe rcentage of root tissue macerated . . . 5 1 4.2.3.4 M ax i mu m numbe r of colonies per gram fres h roots ^{. .} . 5 1 4.2.4 Discussion . . . ^. . . . ^. . . . 5 6 4.3 Exp eriment two : quantitative appraisal of t h e effects of soil

temperature and moisture on i nvasion of re d clov e r roots by

soilborne fungi . . ^. . . . ^. . . . ^. . . ^. . . . ^. . . 5 8 4 .3 . 1 Introducti o n . ^. . . ^. . . . ^. . . . . ^. . . . ^. . . . ^. . . 5 8 4.3.2 Mate rials and m e t h o d s . . . ^. . . . ^. . . . ^. . . . ^. . . 5 9 4.3.2 . 1 Glasshouse trial . . . ^. . ^{. .} . . . ^. . . . ^. . . . ^. 5 9 4.3.2.2 F i e l d trial ^. . . . . . . . . . . . . . . . . . . . . . 6 1 4.3.2.3 Numbers of e merged seedlings . . . ^{. . .} . ^. . . . ^. . 6 1 4.3.2.4 Shoot and root fresh weight ^. . . . ^. . ^. . . . ^. . ^. . . . 6 1 4.3.2.5 Isolation of root-invading fungi . . . ^. . ^. . . . ^. . ^{. .} . . . ^. 6 1

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4.3.2.6 Calculation of parameters of fungal invasion . . . 62

4.3.2. 7 Field soil moisture and tempe rature· . . . 63

4.3.2.8 R e lationship between soil water content and matric pote ntial . . . ^. . . . 63

4.3.2.9 S tatistical analysis . . . ^. . . . 63

4.3.3 Results . . . 64

4.3.3 . 1 G lasshouse t rial ^. . . . ^. . . . ^. . . . 64

4.3.3.2 Field trial . . ^. . . . 70

4.3.3.3 Field soil tempe rature and moisture . . . . ^. . . . 73

4.3.3.4 R e lationships between soil water content and matric potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

4.3 .4 Discussion . . . 73

CHAPTER 5 A STUDY OF EFFECTS OF DISEA S E S ON THE GROWTH OF RED CLOVER BY USE OF FUNGICI DES DRENCHES . . . 79

5 . 1 Introduction . . . . ^. . . . ^. . . . ^. 79

5 .2 Experiment three : effects of fungicides on growth of root-invading fungi, Rhizobium trifolii, and red clover see d lings in the laboratory . . 80

5 .2. 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

5 .2.3 Materials and meth ods . ^. . . . ^. .^.. . . ^. . . 8 1 5 .2.3 . 1 Fungicides ^. . . . ^. . ^. . . 8 1 5 .2.3.2 Fungi and r hizobia . . . ^. . ^. . . ^{. .} . . . 8 1 5 .2.3.3 Effe cts o n mycel ial growth and spore germination . . . . 82

5 .2.2.4 Effe cts on growth of rhizobia . . . . ^. . . . ^. . . 83

5 .2.2.5 Effe cts on g e rmination and s e edling growth of red clover . . . ^{. .} . . . ^. . . . 83

5 .2.2.6 Statistical analysis . . . ^. . . . 84

5 .2.3 Results . . . ^. . . ^. . . . ^. 84

5 .2.3 . 1 Effe cts on mycelial growth and spore germination . ^. . . 84

5 .2.3.2 Effe cts on R h izobium growth . . . ^. . . . ^. . ^. . ^. . . . . 85

5 .2.3.3 Effects on germi nation and seedling growth of red clov e r . . . ^. . . . ^. . . . ^. . . . 87

5 .2.4 Discussion . . . . ^. . . . ^. . . . ^. . . . 92

5 .3 Experiment four : effects of prochloraz dren ches on populations of soil micro-organisms, i nvasion of roots by fungi, and growth of red clov e r seedlings u n d e r glasshouse conditions . ^. . ^{. .} . . . ^. . ^{. .} . ^. . . . . ^{. .} . ^. 94

5 .3. 1 Introduction . . . . ^{. .} . . . ^. . ^. . . . ^. . . . . ^. . . . . ^{. .} . . . ^{. .} . ^. . ^. . . . 94

5 .3.2 Materials and methods . . . ^. . . . ^{. .} . . . ^{. .} . . . ^. . . ^. . . . 94

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5 .3.2. 1 Microbial population of the s oil ^{. . . . . .} . ^{. .} . . ^{. . .} . . ^{. . . .} 95

5 .3.2.2 Se e d germination and seedling growth of red clov e r ^. . ^. 96

5.3.2.3 Root-invading fungi . . ^{. . . . .} . ^{. . . . .} . ^{. .} . ^{. . . . . . . . . . . . .} 96

5 .3.2.4 Statistical analysis ^. . ^{. . . . .} . . ^{. . . . . . . .} . . ^. . ^{. .} . . ^{. . . .} . ^. 97

5 .3.3 Resu lts ^{. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .} 97

5 . 3 .3 . 1 Pop ulations of b acte ria and fungi in the soil ^{. .} . . ^{. . . . .} 97

5.3.3.2 See d ling emerge nce and growth of red clover . ^. . ^{. .} . ^. 1 00 5 .3.3.3 Root-invading fungi ^{. . . . . . . . . . . . . .} . ^. . ^{. . . . . . .} . ^{. . . .} 1 02 5 .3.4 Discussion ^{. . . . . . .} . ^{. . . . . . . . . . . . . . . . . . . . . . .} . ^{. . . . . . . . . . . .} 1 05 5 .4 Exp e riment five : effects of fungicidal drenches on root-invading fungi and growth of red clover under fiel d conditions ^{. . . . .} . . ^{. . . . . .} 1 08 I I 5 .4. 1 Introduction ^{. . . . . . . . . .} . ^{. . . . . . . . . . . . . . . .} . ^{. . . . . .} . ^{. . . . . . . .} 1 08 5 .4.2 Mate rials and methods ^{. . . . . . . . . . . . . . . . . . . . . . . . . . .} . ^{. . . . . .} 1 09 5 .4.2. 1 A ssessment of plant growth ^. . ^{. . . . . .} . . ^. . ^{. . .} . . ^{. .} . . ^{. .} 1 09 5.4.2.2 Isolation of root-invading fungi ^{. . . . . .} . ^. . ^{. . . . . . .} . ^{. .} 1 1 0 5 .4.2.3 Statistical analysis ^{. .} . ^{. .} . ^{. . . . . . . .} . ^{. . . .} . ^{. . .} . ^{. . . . . .} 1 1 0 5 .4.3 Results ^{. . .} . ^. . ^{. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .} . ^{. . . . . . .} 1 1 1 5 .4.3 . 1 See d ling emergence and plant growth . ^{. . . . . .} . ^{. . . . .} . 1 1 1 5.4.3.2 Root-invading fungi ^{. . . . . . . . . . . . . . . . . .} . ^. . ^{. . . . . . . .} 1 1 4 5 .4.4 Discussion ^{. . .} . ^{. . . . .} . ^{. . .} . ^{. . . .} . ^{. . . . . . . . . . . . . . . .} . ^{. . . . . . . .} 1 1 8

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CHAPTER 6 A STUD Y OF THE EFFECTS OF D ISEASES ON THE

I

I

I

GROWTH OF R E D CLOVER BY USE OF I NOCULATION METHODS 1 2 1 6. 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 1 6.2 Exp e riment six : effects of root- i nvading fungi o n pe rsistence and growth of r e d clover plants in fie ld plots . . ^. . . ^. . ^. . . ^. . ^{. . . . . . . . . . . .} 1 22 6 .2. 1 Introduction ^{. . . . . .} . ^{. . . . . . .} . ^{. . . . . . .} . ^{. . . . . . .} . ^{. . . . .} . ^. . ^. . . ^. 1 22 6 .2.2 Materials and meth ods . . . ^. . ^. . ^. . . . ^. . ^. . ^. . ^. . ^{. . . .} . . ^{. . . .} 1 22 6.2.2. 1 Ass essment of symptoms and isolation of fungi from the r oots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

6.2.2.2 M e asurement of survival and growth of p l ants ^{. . .} . ^{. . .} 124

6.2.2.3 Soi l temperatures and rainfall ^{. . .} . . . ^. . . . ^. . . . ^{. .} 1 24 6.2.2.4 Statistical analysis ^{. .} . ^. . ^{. .} . ^{. . . . . . . . . .} . ^{. .} . . . . ^{. . . . .} 124

6 .2.3 Results . . ^. . ^. . ^. . . . ^{. .} . . ^. . ^{. .} . ^. . ^{. . . .} . ^. . . ^{. . .} . . ^{. .} . ^{. . . .} . ^. . . . . 125

6.2.3 . 1 Root symptoms and fungal r eisolation . . . . ^{. . . . .} . . ^{. .} 125

6.2.3.2 Plant survival ^{. . . .} . ^. . . ^{. . .} . . ^. . ^{. .} . . . ^. . . . ^{. . . . .} 126

6.2.3.3 Plant growth ^{. . .} . . . ^. . . . ^{. . . . . .} . . ^{. .} . ^. . ^{. . .} . . . ^. 126 6.2.3.4 Soi l tempe ratures and rainfall ^. . ^{. .} . ^. . ^{. .} . . ^. . ^{. .} . . . ^. . 1 3 0

x i

6 .2.4 Discussion ^{. . . . .} . . . ^. . ^{. . . .} . ^. . . . ^{. .} . ^{. .} . ^. . ^. . . . ^{. .} . ^{. . . . . . . .} 1 3 0 6.3 Experiment seven : effe cts of root-invading fungi o n t h e growth o f red clover p lants in t h e glasshouse and microplots ^{. .} . . ^{. . .} . ^. . ^{. .} . ^{. .} . . .^. . ^. . ^. . ^. . ^{. . . . . .} . ^. 1 32

6.3 . 1 Introduction ^{. .} . . ^. . ^. . . . . ^. . . ^. . ^{. .} . . ^. . ^{. . .} . . . . ^. . ^. . ^{. . .} . ^{. .} . ^{. . .} 1 32

6.3.2 Mate rials and method s ^. . . ^{. . . . .} . ^{. . .} . ^. . . . ^. . ^. . . ^{. .} . . . ^. . ^. . . . ^. 133

6.3.2. 1 Glasshouse studies ^{. .} . . . ^{. . .} . ^. . . . ^{. . .} . . . . ^{. . .} . . . . 133

6.3.2.2 Fie ld microplot studies ^{. . . .} . ^. . ^{. .} . ^. . ^{. . .} . . . ^{. .} . ^{. . . .} . 135

6.3.2.3 Statistical analysis ^. . ^. . . . ^{. . . .} . ^. . ^{. .} . . ^{. . . .} . . ^{. . .} . ^. . . 140

6.3.3 Results . ^. . . . . ^{. .} . . ^{. .} . ^. . ^. . . ^{. . . . .} . ^. . ^. . . ^. . . . ^{. . .} . . . ^{. .} . ^{. .} 140

6.3.3 . 1 Glasshouse studies ^. . . . . . . . . . . . . . . . . . 140

6.3.3.2 Field microplot studies . . . ^. . . ^. . . . . ^{. .} . . ^{. . . . .} . ^. . ^. 1 43 6.3.4 Discussion . ^. . ^{. .} . . . ^{. .} . ^{. .} . ^{. .} . ^{. .} . . . ^{. . .} . . ^{. . .} . . ^. . . ^. . ^. . ^{. .} . 1 5 1 CHAPTER 7 Experiment eight: ligh t and elect ron microscopy studies on invasion of red clov e r r oots by'Yrich ocladium basicola, and effe cts of benomyl and p rochloraz ^{. .} . . . ^. . . . ^{. . .} . . . ^{. . . .} . . . ^{. .} . ^. . ^{. . . .} . . 1 5 6 7. 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 6 7 . 2 Materials a n d methods . . . ^{. . .} . ^{. . .} . ^{. . .} . ^{. .} . . ^{. . . . .} . ^{. .} . ^{. . .} . . . ^{. . .} . . 157

7.2. 1 Fungus . ^. . . . ^. . ^. . . ^{. . . .} . ^. . . ^{. · . . . . . .} . . . ^{. .} . . . ^{. . . . . . . . . .} 1 5 7 7.2.2 Fungicide s . . . ^{. . . . . . . . . . . . . .} . ^{. . .} . ^{. .} . ^. . ^{. .} . ^{. . . . . . . .} . . . ^{. . .} 1 5 7 7.2.3 See dlings and inoculation . . . ^. . ^. . ^{. .} . . ^. . . ^. . . . ^. . . 1 5 7 7.2.4 Effects of the fungicides on the fin e structure of the fungus grown in culture ^{. . . . .} . . ^{. . . . . . .} . . . ^. . ^. . . ^. . . . ^. . ^{. .} . . ^. . . 1 5 8 7.2.5 Assessment of dise ase symptoms ^. . ^{. . . . . .} . ^{. . .} . ^. . ^{. . .} . ^{. . . . . .} 158

7.2.6 Sampling for histological studies ^. . ^. . . . ^{. . . . . . .} . ^. . ^{. .} . ^{. . .} . . ^. 1 5 8 7.2.7 Ligh t microscopy ^. . . .^{. . .} . ^{. . . . . . .} . . ^{. . . . . .} . ^{. .} . ^. . ^. . ^{. . . .} . ^. . 1 5 9 7.2.7. 1 Conidial germination a n d penetration . ^{. . . . . . . . . . . .} 159

7.2.7.2 Fungal d evelopment within the host ^. . ^{. .} . . . ^{. .} . . . . ^. . 1 5 9 7.2.8 Transmission e lectron microscopy . ^{. . . .} . ^{. . . . . . . . .} . ^. . . ^. . . . ^. 1 6 0 7.2.9 Statistical analy sis . . ^{. . .} . . . ^{. . . . . .} . ^{. . . .} . ^. . ^{. .} . ^{. .} . ^. . . . . ^. . . . . 1 6 1 7.3 R e sults ^{. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .} 1 6 1 7.3. 1 Invasion of red clover roots by T . basicola and effects o n host cells ^. . . .^{. .} . ^. . ^{. . .} . . ^{. . . . . .} . ^{. . .} . ^{. . . . . . . . . . . . . .} . . . ^. . ^{. . .} . ^. 1 6 1 7.3. 1 . 1 Course of infe ction . . . . ^{. . . . . .} . ^{. . . . . . . . . .} . . ^{. . .} . ^{. . .} 1 6 1 7 .3 . 1 .2 Reaction of host cells t o fungal invasion ^{. . . . . . .} . ^{. . .} . 1 6 9 7.3.2 Effects o f fungicides on r e d clover see dlings a n d T . b asicola ^. . . . . . . . . . . . . . . . . . . . . . . . . . . 178