BIOLOGY AND CONTROL OF PYRENOCHAETA LYCOPERSICI, BROWN ROOT ROT OF TOMATO by April Ghislaine Hockey Department of Biology, LiveppDol Polytechnic A thesis submitted in part fulfilment of the degree of Doctor of Philosophy of the Council for National Academi c Awards Glasshouse Crops Research Institute Department of Biology, littlehampton Liverpool Polytechni c , West Sussex December, 1984. BIOLOGY AND CONTROL OF PYRENOCHAETA lYCOPERSICI. BRfMN ROOT ROT OF TONA'rO by A.G. HOCKEY ABSTRACT A method for the isolation of grey sterile fungi from brown root rot infected tomato root systems was developed. Semi-sel ecti ve media significantly reduced the growth of Colletotriohum ooooodes and catyptetla campa~ula with little effect on the growth of grey sterile fungi • Pycni di a characteri sti c of pyrenochaeta lyoopel'sioi were formed on VS-Juice agar (VSA) by twelve of the 19 grey sterile fungal i sol ates tested. A method for the routi ne producti on of pycni di a/ coni di a was developed: P. lyoopersioi cul tures, i nocul ated onto V8A are i ncubated at 220 C with a 16h bl ack light photoperi od • No vegetable constituent of V8-Juice, tested individually, could be shown to be solely responsible for sporulation on VSA. Conidi a requi re a temperature range of 20 to 26lC, pH range 5.0 to S.O and external nutrients to achieve germination levels qreater than 9at. Conidial germination decreased with age. Incubation of P. tycopersici coni di a ina dil ute ci rrus extract and under di fferent light regimes did not affect germination. Conidia were shown to swell prior to germination and produce either single or dual germ tubes. Using mycelial i nocul urn, grey steril e fungal 1501 ates caused lesions on tomato roots in vitro and i~ vivo. Conidia of P. tyoopersici produced 1esi ons on the roots of tomatoes grown in i nocul ated steril i sed compost and unsteril ised garden soil, withi n six weeks. Brown root rot and corky root symptoms were produced on tomatoes grown comnerct all yin soil s , infested with P. tyoopersioi coni di a, for up to 21 weeks. Root 1esi ons were produced on pl ants grown in conidia infested sterilised compost and unsterilised garden soil which had been stored at room temperature for up to 510 and 150 days respectively. Conidial germination was observed in soils, on cellophane and on polycarbonate membranes. On cellophane, coni di a germinated in unsterilised and sterilised composts forming sclerotia on a II frond" type myceli urn after three to seven days i ncubati on. Conidial germination was lower in washed sand and greatly reduced in peat. Germinati on on pol ycarbonate membranes in compost was slower and sclerotial formation reduced compared to that observed on cellophane. A soil assay has been developed to estimate soil infestation 1evel s of brown root rot (BRR). Using the BRR assay, various species of bacteria and Streptomycetes were shown to reduce the number of 1esi ons on tomato pl ants grown in soil infested with brown root rot. Potenti al fungal antagoni sts , appl i ed as wheatbran cul tures, di d not sig n1fi cantl y reduce d1sease 1evel s , Bacteria and fung i increased the root dry weight of tomato plants grown in soil infested with brown root rot. Oisease i nci dence, detected usi ng the BRR assay. was di rectl y rel ated to subsequent 1evel s of brown root rot on tomatoes eight weeks after transpl anti ng into the gl asshouse. CONTENTS Page Number AbbPeviations used in this study 1 CHAPTER ONE. INTRODUCTION 2 1 •1 P7'efaJJe 2 1.2 IntPOdwnion 8 CHAPTER TWO. METHODS 27 SECTION 1. GENERALMETHODS 27 1. 1 l'PepaPation of media 27 (a) Agar media 27 (b) Solid media 28 (c) Liquid media 29 (d) Soil media 29 1.2 Maintenance md cul tuPe of isolates 31 1.3 IPPOdiation 34 1.4 P7'epaPation of conidial suspensions of Pyrenochaeta 34 1ycopersici md potential antagonists 1.5 Pl'epaPation of the optiool In-ightenBl' ~ Calcof7,1IOP 37 Jihite 1I2R 1.6 Gezemination of tomato seed 37 1.7 Assessment of the inoculum potential of soils 38 natuPally infested with lntor.M POOt POt (BRB): ~e BRB assay 1.8 Statistical analysis 38 SECTION 2. BIOLOGY OF PYRENOCHAETA LYCOPERSICI 39 2.1 Isolation of Pyrenochaeta 1ycopersici 39 (a) Development of a semi-selective medium 39 (b) Evaluation of the semi-selective medium 42 (c) Isolation of grey sterile fungi from root 42 systems showing brown root rot (BRR) symptoms 2.2 IIyCBlial pathogeni.cit,l of (ll"BlIstePi.le f""!li 43 (a) In vitrao 43 (b) In vivo 43 2.3 SJKm.tlation ard identification of gztey stePi.le fungi 44 ii Page Number (a) Induction of pycnidial production 44 (b) Effect of light on pycnidial production 44 (c) Effect of the vegetable constituents of 46 V8-Juice on pycnidial production 2.4 Gezomination of Pyrenochaeta 1ycopers i et CDrIidia in 46 vitro (a) Effect of temperature and incubation period 46 (b) Effect of temperature, nutrients and isolate 47 (c) Effect of pH and isolate 47 (d) Effect of light and isolate 47 (e) Effect of age, conidial washing, cirrus 49 extract and isolate (f) Effect of the optical brightener, Calcofluor 49 White M2R (g) Observations on conidial germination 50 2.5 pathogenicity of Pyrenochaeta lycopersici conidia 50 (a) Conidial pathogenicity in sterilised compost 50 (b) Conidial pathogenicity in unsterilised garden 50 soil (c) Conidial pathogenicity ina tomato crop grown 51 under commercial conditions 2.6 BehavioUl' of Pyrenochaeta lycopersici tWrIidia in soil 57 (a) Survival of conidial inoculum in sterilised 57 compost and unsterilised garden soil (b) Germination and subsequent growth of py~enochaeta 57 tycope~8ici conidia in various substrates SECTION 3. BIOWGICALCOII'rROLOF PYRENOCHAETA LYCOPERSICI 58 3. 1 DeVeloprumt of the scpeenif'lg techrriqr.uJ 58 3.2 sm-eemf'lg potential tDttag01lists agai1l8t lnem.7lt l'OOt 59 rot (BRB) disease lePels in aoila JIllttaeally infeated with Pyrenochaeta lycopersici 3.3 MeaBUl'ementof lneOWlt root: rot (BRR) diaease levela in 61 soila 1fQtUl'ally infeated with Pyrenochaeta 1ycopersici CHAPTER THREE. RESULTS 64 SECTION 1. BIOWGY OF PYRENOCHAETA LYCOPERSICI 64 1.1 Isolation of Pyrenochaeta 1ycopers1c1 64 (a) Development of a semi-selective medium 64 (b) Evaluation of the semi-selective medium 70 (c) Isolation of grey sterile fungi from root 70 systems showing brown root rot (BRR) symptoms iii Page Number 1.2 ~celial pathogenicity of gPey sterile f~i 73 (a) In vitr'o 73 (b) In vivo 73 1.3 Sptmllation and identification of gl'ey sterile 76 fungal isolates (a) Induction of pycnidial production 76 (b) Effect of light on pycnidial production 76 (c) Effect of the vegetable constituents of 86 V8-Juice on pycnidial production 1 .4 Gezomination of Pyrenochaeta lycopersici conidia 86 in vitro (a) Effect of temperature and incubation period R6 (b) Effect of temperature, nutrients and isolate R6 (c) Effect of pH and isolate 90 (d) Effect of light and isolate 90 (e) Effect of age, conidial washing, cirrus 90 extract and isolate (f) Effect of the optical brightener Calcofluor 102 White M2R (g) Observations on conidial germination 102 1.5 PathogtmWty of Pyrenochaeta lycopersici conidia 108 (a) Conidial pathogenicity in sterilised compost 108 (b) Conidial pathogenicity in unsterilised garden 113 soil (c) Conidial pathogenicity in a tomato crop grown 113 under commercial conditions 1.6 BehaDiouzoof Pyrenochaeta 1ycopers 1Cl conidia in soil 116 (a) Survival of conidial inoculum in sterilised 116 compost and unsterilised garden soil (b) Germination and subsequent growth of 122 pYr'enochaeta lycopel'sici conidia in various substrates SECTION 2. BIOLOGICAL CORrROL OF PYRENOCHAETA LYCOPERSICI 133 2.1 DeVelopment of the SCZOBBningtechnique 133 2.2 Sczoeemng potential antagonists against bPown root: 136 POt (BRR) 2.3 IleaBUl'Blllentof browra rooe zoot (BRR) disease levets 136 in soits naturatly infested with ~renochaeta lycopers 1Cl CHAPTER FOUR. DISCUSSION 147 iv Page Nu.mer ACKNOWLEDGEMENTS 187 REFERENCES 188 PERSONAL COMMUNICATIONS 206 APPENDIX 1. A ppelimif'IQ.Py"tPial testing the potential of I antagonists fop the contMl of bPm.1rt root: POt (BRB) in the glasshouse APPENDIX 2. possible use of the BRB assay to ppediet the VIII degt'ee of disease contMl and consequent yield illmeeases achieved by potential antagonists applied to glasshouse tomato meops. v AbbPeviatiOf1s used in this study Ab.alc. absolute alcohol a. i . active ingredient ADAS Agricultural Development and Advisory Service A.T.C.C. American Type Culture Collection A Ampicillin anhydrous B bacteri a BL black light BRR brown root rot caused by pypenochaeta tycopepsici Cp. catyptetta campanuta CRR catyptetta root rot °c centigrade (degrees) cm centimetre{s) C Chlorampheni col C.c. Cottetotpichum coccodes c crystal day day(s) f.sp. fopma speciatis G.C.R.I. Glasshouse Crops Research Institute 9 gram{s) GSF Grey sterile fungus (fungi) h hour(s) Kg kilogram(s) lq liquid 1 1itre{s ) MBC Methyl benzimidazol-2-yl carbamate ml mi11i1itre (s) tm1 millimetre(s) NT not tested o other fungi P Penicillin PST Penicillin/Streptomycin/Tetracycline agar PSTBay Penicillin/Streptomycin/Tetracycline/Bayleton agar Pm Pimafucin PDA potato dextrose agar RW root dry weight SW shoot dry weight SH shoot height S.D. standard deviation SED standard error differences between the means SOW sterile distilled water SME sterile malt extract S Strepto~cin sulphate TW tap water TWA tap water agar T Tetracycline anhydrous U.V. ultra violet U.K. United Kingdom V Vanco~cin VSA VS-Juice agar w.p. wettable powder white light WL- > greater than < 1ess than llm m1crometre(s) t percentage 1 QiAPTER ONE.