14IAITE INSTITUTE q' as NEH LIBRÀRY". 7 IWESTIGATIONS TO DEVELOP METHODS TO CONTROL THE NEIi{ATODE ASSOCIATED WITH ANNUAL RYEGRASS TOXICITY by A.C. !{cIGY, B. Àgr. Sc. (MeIb) DeparÈnent of Plant Pathology, Waite Agricultural Research InsÈitute' The University of Adelaide, SouEh AusEralia A thesls submitted to the university of Adelaide in partial fulfilmenÈ of Èhe reguirements for the degree of Doctor of Philosophy. q'at^-t"L/ 2o -C -f \' (January f985) TABLE OF CONTENTS Paqe SUMMÀRY i STATETT{ENT v ACKNOWLEDGETT{ENTS vi GENERAL INTRODUCTION (a) Etiology and dispersal of annual ryegrass Èoxicity I (b) Symptons of annual ryegrass toxicity 2 (c) The toxins 4 (d) The bacter iu¡n 6 (e) The nematode 9 (f) Growth and development of annual ryegrass 13 (g) Controlling annual ryegrass toxicity t5 CTIAPTER 1 ECOTOGICAL STUDIES OF THE NEI{ATODE Introduction L7 Materials and Methods (a) Experimental design I8 (b) Details of sites l9 (c) AssessmenÈs of nematode and ryegrass developnent 20 (d) Statistical analYsis 2L Results (a) Nematode movement into the plant and tiller 25 producÈion (i) Climatic conditions and tiller production 25 (ii) Nematode movement inÈo tiller 27 (iii) Grazing 30 (b) Nematode and planÈ development after gaII induction 30 (i) Development of the nemaÈode in the egg 3I (ii) Hatching of J2s 3I (iii) Association between haÈching of J2s in the gall and Plant develoPment. 34 (iv) Assessment of variaÈion in tiller development when 2t had reached the sEage of head fully emerged or anthesis 36 (v) Grazing 38 Discussion (a) Nematode movement into Plant 4t (b) Plant and nematode development after gall initiation 42 (i) Development of the nematode in the egg 43 (ii) Hatching of J2s 44 (iii) AssociaÈion between hatching of J2s and plant development 45 (c) Suggested methods of control 46 CHAPÎER 2 EVALUATION OF I4ETHODS TO CONTROL THE NEII{ÀTODE Introduction 49 MaÈerials and llethods 49 (a) Experinent 1: Effect of herbicides applied before gall initiat,ion 50 (b) Experiment 2: Comparison of herbicides and ne¡natode parasitic fungus applied before gall iniÈiation with mechanical topping afÈer gall initiation 51 (c) Experiment 3: Comparison of mechanicat topping, a desiccant herbicide and a systemic nematicide applied after gall iniÈiaion 52 Results (a) Experiment l: Effect of herbicides applied before gall initiation (i) Nematode gall production 54 (ii) Pasture production s6 (b) Experiment 2: Comparison of herbicides and nematode parasitic fungus applied before gall initiation with mechanical topping after gaII inÍtiation (i) Nematode gall production 57 (ii) Pasture production 57 (c) ExperimenÈ 3: Comparison of mechanical topping, a desiccant herbicide and a systemic nematicide applied afÈer gaII initiaion (i) Nematode gall production 6l (ii) Pasture production 63 Discuss ion 63 CHAPTER 3 BIOLOGICAL CONTROL OF THE NEITIATODE fntroduction 68 Materials and Methods (a) Corynebacterium sp. 68 (b) Dilophospora alopecuri 69 (c) Verticillium sp. 70 Results (a) Corynebacterium sp. 70 (b) Dilophospora alopecuri 7L (c) Verticillium sp. 74 Discussion (a) Corvnebacterium sp. 74 (b) Dilophospora alopecuri 75 (c) verticillium sp. 78 CHAPTER 4 MODEL OF POPULATION DYNA¡{ICS OF THE NEI{ÀTODE fntroduction 79 Materials and tleÈhods (a) Materials 81 (b) ExperimenÈ design 82 (c) Assessments 83 Results (a) Weekly assessments of tiller, nematode and gall numbers per plant 86 b Production of ryegrass heads 88 c Model of gall production 89 d Proportional change in gall nunbers 92 e Proportion of galls colonised by bacteria 93 (f) ProportÍon of heads with bacterial slime 96 Discussion (a) Model of gall production 98 (i) Developing Èhe models 99 (ii) Possible Explanation for the trends in gall production as initial gall density increased 100 (iii) Possible explanat.ion for the trends in galt production as plant density increased J-O2 (iv) Source of bacterium 104 (v) Possible environmental effects on gaII production (f) Effects of time of nematode movement 105 (2') EffecÈs of early germination of ryegrass 109 (3) Effects of late germination of ryegrass II0 (b) Implications for management of the nematode population tlt CHÀPTER 5 DISPERSAL OA THE NEITIÀTODE AND FREQUENCY WITH T{HICH PADDOCKS BECOME TOXIC Introduction I15 Materials and Methods rt6 Results (a) Distribution Il7 (b) Toxic paddocks r18 (c) Paddocks infested with the nematode 119 (d) Livestock losses rt9 Discussion t2l GENERÀL DISCUSSION ]-24 APPENDICES Appendix Ia L29 Àppendix Ib r30 Appendix IIa r31 APpendix IIb L32 Appendix III 133 Àppendix rv r38 BIBLIOGRAPHY t4s i SUMMÀRY The seed gall forming nematode, Ànguina funesta, was studied in field experimenÈs to determine Èhe most practical method of control. Thls nematode is the essential vector of a bacterium, Corvnebacteriu¡t sp. which produces toxins in the seedheads of annual ryegraes. l{hen consumed by grazing animals, Èhese toxins cause an often fatal dÍsease of livestock known as annual ryegrass toxicity. Movement of nematodes into the plant was dependent on suitable ¡noisture conditfons. The Eime of this movement varied considerably and was difficult to predict. Inside the plant the nemaÈodes congregated near the apex, and nere protecÈed from external factors by the leaf sheaths. l{hen the nemaÈodes moved into the plant early in the season, many left the tillers before they could initiate a gall in a developing ovary primordiun. This may have been due to crowding at Èhe apex or they nay have been excluded from Èhe apex and carried out of the plant by newly initiated leaves. Nematodes Èhat left tillers and those in tillers that died, were apparently unable Èo iniÈiaÈe a gall in anoÈher tÍller. rnside the gaII the nematodes pass Èhrough three moults to become adults. These mate and the female lays eggs. The nematode moults once inside Èhe egg and hatches as a second stage juvenile. AII the stages of nematode development excepÈ Èhe second sÈage juveniles are susceptible to desiccaÈion. The first nematodes hatch aa the first tillers reach anthesis. Às the ryegrass maÈures and dries in late springr Ehe second stage juvenÍIes enter a guiescent state to survive the dry autnmer months. rn the field, few of these nematodes survive more Èhan one growing season, so prevenÈing nematode reproduction for one season should greaÈIy reduce the population. 1I A number of different, methods were very effective in controlling the nematode ¡rcpulation. Before gall initiation, controlling Èhe ryegraas wiÈh the herbicides diclofop methyl and paraguat gave good control of the nematode, although lack of ryegrasa caused a shortage of fodder in winter when feed tyas usually scarce. Diclofop methyl was the preferred herbicide because it did not damage legunesr but Ít. was less active on ryegrass under stress or on large planÈs. Àfter gaII initiation, mechanically topping the ryegrass or spraying it with paraguat as the firsÈ t.illers reached anthesis or earlier r also gave good control of the nematode population. Provided the ryegrass is tall enough to cut¡ mechanical topping has the advantage of not killing the plantr so new uninfested tillers can be initiaÈed and set seed. The advantages of paraguaÈ are that the heighÈ of the pasture Ís not important and large areas can be Èreated quickly. However there 1s tÍttle regrowth and the treated ryegrass tends to breakdown quickly. Grazing afÈer gaII initiation can also give good conÈrol of the nemaÈode population, provided the ryegrass is consumed before the galls mature and shatter to the ground. Grazing before gall initiation can increase the production of galls, probably by maintaining a short canopy and reducing the ¡norÈaliÈy of ryegrass t,illers. A promising biological control agent of the nenatode was found in the Mid North region. The fungus, Dilophospora aropecuri is a plant parasite which is transported into the plant by the nematode. The spores can rnultiply by budding while on the nematode and can spread to other nemaÈodes on contacÈ. rnside Èhe gall¡ Èhe spores produce hyphae and colonise Èhe gall, destroying it before the nematode can reproduce. ttt A model of gatl production, developed from data obtained from smalt plots, indicated that the abitity of the nematode Èo produce galls was dependent on the initial gall and ryegrass plant densities. The experiment was made more complicated by the developne nt of Corynebacterium sp. which varied depending on the initial gall and plant densitles. The multiplicaÈion rate of the nematode was decreased and became less than one as the initial nematode density increasedr ând rose to a maximun and then declined as planÈ density Íncreased. The ¡naximum increase in gall number r greater than 100 fold, occurred at low initial nematode and moderate plant densitiesr ând the greatest decline' about 99t, occurred at high Ínitial nematode and low plant densities. In this experiment the bacteria were nore likely to colonise galls and produce sline on infested seedheads as Èhe fnitial nemaEode density increased and plant denslÈy decreased. The develo¡xnent. of sli¡ne aPpeared Èo have reduced Èhe ability of the nematode to init,iate galls but increased Èhe risk of toxicity. At low nematode populations the bacterial poPulation increased more slowly than ÈhaÈ of the nematode. Therefore reducing Èhe nematode population Eo a low level by either controlling the ryegrass ¡npulation or by using the Èreatments mentioned earlier r would probably make the pasÈure safe to graze for a number of successive seasons. The results of a survey of farmers revealed that 808 of the paddocks thaÈ had been toxic' were toxic only oncer âDd only 2t of the non-Èoxíc paddocks that were found Èo be infested wíth the nematode had subseguenÈly become toxic. It Èherefore aPpears that ARGT, is a relatively easy disease to control. The maÍn problem now is that farmers cannoÈ determine if they have successfully controlled 1V the nemaÈode and bacterial populations untÍl the naEure pasture has been grazed.