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BioiogicaI Control of Echinochloa Species with Pathogenic Fungi
Wenming Zhang
Department of Plant Science Macdonald Campus of McGill University Montréal, Québec, Canada
March 1996
A Thesis submitted to the Faculty of Graduate Studies and Research in partial fulfilment of the requirements of the degree of Doctor of Philosophy • © Wenming Zhang, 1996 National Ubrary Bibliothèque nationale 1+1 of canada du Canada Acquisitions and Direction des acquisitions et Bibliographie Services Branch des services bibliographiques 395 Welhoglon S1reet 395. rue Wellinglon onawa Onlario Ottawa (Ontano) K1AON4 K1AQN4
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ISBN 0-612-12517-3
Canada •
Short title:
Biocontrol ofEchinochloa Species
Wenrning Zhang
• • Abstract Six pathogenic fungal species werc isolated from naturally-infeeted Echinochloa species and evaluated as biological control agents for E. crus-galli, E. c%na. and E. glabrescens in nce. Bipolaris sacchari. Cllrvularia genicuIata, and Exserohilum menoceras werc non-pathogenic ta nce and caused high mortality ofEchinochloa species. E. menoceras was selected for further study. Under regulated greenhouse conditions, an inoculum dose of 2.5 x 107 conidialm2 killed E. crus-galli and E. glabrescens seedlings while S.O x 107 conidialm2 caused 100% mortality of E. colona seedlings. The loS-Ieaf stage was the most susceptible growth stage for ail three Echinochloa species. E. glabrescens was most susceptible ta E. menoceras infection, E. crus-galli had an interrnediate susceptibility, and E. colona was least susceptible. The optimum temperature for 100% mortality was between 20 and 30 C for ail Echinochloa species. whereas the minimum dew period for 100% mortality was 16 h for E. colona, 12 h for E. crus-galli, and 8 h for E. glabrescens. Under screenhouse conditions and in the absence of an artificial dew period, over 90% ofEchinochloa seedlings were killed when inoculum was sprayed in an oil emulsion or when applied as a dry powder ta the water surface of a simulated paddy field. Maximum conidia production occurred on V-8 juice agar or centrifuged V-8 juice agar. at 28 C in the dark. No conidia were produced in liquid media. Ofvarious agricultural products tested as solid substrates, the highest sporulation (1.81 x 106 conidialg dry weight) occurred on corn leaves. Host range tests on 54 plant species in 43 genera and 19 families showed !hat Rorrboellia cochinchinensis. was also highly susceptible ta this fungus. Of ta'le crops tested, only corn seedlings werc lightly infected under optimum greenhouse conditions but no disease occurred on corn under field conditions. Bipolaris sacchari. Exserohilum menoceras. and E. oryzoe produced phytotoxins that caused 100% leaf area chlorosis and wilting of intact Echinochloa seedlings that werc placed in cell-free culture filtrates. Two phytotoxins werc isolated and • purified from E. menoceras• i • Résumé L'étude du contrôle biologique de Echinochloa crus-ga/li, E. colona et E. glabrescens dans les cultures de riz, a permis d'isoler six isolats fongiques d'Echinochloa infectés. Parmi les espèces Bipolaris sacchari, Curvularia geniculata et Exserohilum menaceras qui provoquent de haut taux de mortalité chez les espèces d'Echinachloa. l'espèce E. menaceras a été sélectionnée pour des études plus approfondies. En serre, sous conditions contrôlées, des doses de E. menaceras de 2.5 x 107 spores/rn2 et de 5.0 x 107 spores/m2 ont causé la mort des plantules de E. crus-ga/li, E. glabrescens et de E. colo.U1, respectivement. Au stade de croissance le plus susceptible (1.5 feuille), le plus haut taux de contrôle a été observé pour E. glabrescens suivi de E. crus-galli et de E. colona. En suspension aqueuse, le. conditions optimales pour obtenir 100% de mortalité se situaient de 20 à 30 C pour toutes les espèces d'Echinac/üoa. Par contre, pour un taux de mortalité équivalent, la durée ITlinimale requise pour la période de rosée variait de 8 heures pour E. glabrescens, 12 heures pour E. crus-galli et de 16 heures pour E. colona. En milieu naturel sous des cages grillagées et en absence de période de rosée artificielle, la mort de plus de 90% des plantules d'Echinochloa a été observé lorsque l'inoculum, incorporé à une émulsion à base d'huile a été pulvérisée ou lorsque l'inoculum sous forme de poudre a été appliqué à la surface des rizières. La production optimale de spores a été observée sur un mileu solide à base de jus V-8 et de V-8 centrifugé en conditions d'obscurité à 28 C. Le milieu solide Czapex-Dox a permis d'obtenir une croissance mycélienne optimale. Aucune conidie n'a été produite en milieu liquide. Parmi les substrats d'origine agricole testés, les feuilles de maïs ont soutenu laplus forte sporulation (1.81 x Hf spores/go matière sèche). Les essais portant sur les hôtes potentiels, qui regroupaient 55 espèces de plantes de 43 genres et de 19 espèces, ont permis d'établir que Rottboellia cochinchinensis était susceptible à E. menaceras. Parmi les cultures testées, seules les plantules de maïs ont montré une légère susceptibilité à des conditions optimales en serre. Toutefois, en milieu naturel et sans rosée artificielle, aucun symptôme n'a été observé. L'analyse des métabolites produits par les espèces B. sacchari, E. menaceras et E. oryzae a révélé que les trois espèces produisent des phytotoxines qui causent 100% de chlorose foliaire et le flétrissement de plantules d'Echinachloa. Une caractérisation partielle a également été effectuée sur les phytotoxines produites par E. • menaceras. ii • Acknowledgements 1 would fJrSt of alllike to thank Or. Alan K. Watson. my thesis supervisor. for his guidance. encouragement. and endless support over the course of this research. My sincere appreciation is alse exter.ded to my research advisory comminee members: Ors. Steve G.
Hallett. TlIIl C. Paulirz. and Antonio DiTommaso for constructive suggestions on the conduct of the research and manuscript revisions. 1 am also deeply indebted to my superviser at IRRI, Or. Keith MoDdy. for allowing me to use his laboratory facilities and for bis valuable suggestions. 1am grateful ta Or. G.A. Strobel at Department ofPlant Pathology. Montana State University. Bozeman. Montana, for encouraging me ta carry out the phytotoxin research and for providing the phytotoxin standards and other pertinent reading materials. Many thanks go to the persons al IRRI who assisted me in various ways. Special
mention goes ta the members ofWeed Biocontrol Group. especially Maxima Mabbayad and Camille Yandoc. The teehnical assistance of Maria Roberta Miranda and Dany Lucillo is also acknowledged. Special thanks are extended to the students and staff of the Department of Plant Science, especially those members ofthe 'Weedies' group. secretaries (Mrs. May Couture. Roslyn James, and Carolyn Bowes), Helen Cohen-Rimmer. and Guy Rimmer for their
help and kind cooperation during my stay al Macdonald Campus of McGill University. A special thank you also goes ta Marie Ciotola and Christian Leger for the French translation of my thesis abstraeL A scholarship from IRRI and financial support from McGill University are gready appreciated. Lasdy, 1 would like ta thank my wife, Jian, and daughter, Yang-yang, for their love, loyal support, and compassionate understanding throughout this study. Thank you • from the bottom of my heart. iii • Table of Contents
Absuact 1
Résumé ii
Acknowledgements .....•...... 111
Table of Contents iv
List of Tables ...... •...... •...... XlI
List of Figures .•...•...... •..•...... •...... xiv
List of Appendices ..•...•...... •...... •...... xix
Description of Thesis Format .•...... •...... •...• xx
Chapter 1. General Introduction .... •.... " ...... •...... •..•...... 1
1.1. Absuaet ••...... •.•••...... •...•..•..• .. 1
1.2. Introduction ..•.....•...•...... ••..•.•••.•...••.•..••• 1
1.3. Echinochloa species .•..•...•.•••..••.•...... ••...... 2
1.4. Control suategies against Echinochloa species in rice ..••.••..•... 4
1.5. Biological control of weeds •.••..•..•...•...... ••.••..•..• 5
1.6. Cunent status of biological control on Echinochloa species •.•...•.. 8
17Th•• esIS' 0b'~ecn'ves •..•...•..•.••...... ••.....•...••..•. .•·9
1.8. Literature cited •••.••.•.•.••..••.•••.••..••.••..••..•. 10
Connecting Text ••.••••••.••.•••.••..••••••.••..•..••.••..•... 19
Chapter 2. Responses of Echinoch/oa species and riec (Oryza saliva) 10 • indigenous pathogenic fungi •.•••.•••.••..••.•••.•••.••..•... 20
iv 2.1. Ab~traet ...... " 20 • 2.2. Introduction 20 2.3. Materials and Methods ...... 21
2.3.1. Isolation and identification of fungi 21
2.3.2. Inoculum production ...... 22
2.3.3. Pathogenicity of the testea fungi 22
2.3.4. Comparison of disease severity 23
2.3.5. Bioassay for phytotoxin production " 24
2.3.6. Data analyses " 25
2.4. Resu1ts ...•••...•...•••.••...... •.•...•...... •... " 25
2.4.1. Isolation and identification of fungi 25
2.4.2. Pathogenicity of the tested fungi ..•...... 25
2.4.3. Comparison of disease severity 26
2.4.4. Bioassay for phytotoxin production ..•...... " 28
2.5. Discussion ...... •.••...... •.•...•.. 29
2.6. Literature cited ....••...•.....•...... ••...... 31
Connecting Text •••••.•...••...•.....•....•...... •...... 40
Chapter 3. Efticacy of Exserohilum monoceras for the control of Echinoch/oa species in nec (Oryza saliva) ••••••••••••••••••••••.••.•••••. 41
3.1. Abstract ••..•••••••••...•...... ••..•.•'. .••....•..• •. 41
3.2. Introduction •••••.•••...••..••...••...••..•....•...•. 41 • 3.3. Materials and Methods ..••••..••..•.•.••....•.•••...•." 42 v 3.3.1. Inoculum production ...... 42 • 3.3.2. Plant production ...... 43 3.3.3. General inoculation procedure 43
3.3.4. Assessment of efficacy ...•..•...... 43
3.3.5. Effect of inoculum density ...... 44
3.3.6. Effect of plant growth stage...... 44
3.3.7. Interaction between inoculum density and plant growth stage. 44
3.3.8. Effeet of formulation ...... •...... 44
3.3.9. Data analyses .•...... 45
3.4. Results ...... •...•...... •..•...... •. 46
3.4.1. Effect of inoculum density •••.•.•....•...... •..• .. 46
3.4.2. Effect of plant growth stage .••.••••••...... •.• •. 47
3.4.3. Interaction between inoculum density and plant growth stage. 47
3.4.4. Effect of formulation .•.••.•.•.••...... •••..•••.. 48
3.5. Discussion ••...•••••••.•.•••..••...••...•.•••.••••.. 48
3.6. Literature cited •.•.....••••..••••••.••...... •••• .• 51
Connecting Text ...... ••.•.••••...... •...•...... •.•...... 63
Chaptt:r 4. Effect of dew period and temperature on performance of Exserohilum monoceras for the control of Echinochloa species •. •••••.••••••.. .. 64
4.1. Abstract ••••••••••••.•..•••....••••••••••••••...... 64
4.2. Introd.uetion •..•...... •..•...... •..... M • 4.3. Materials and Methods •••••••.•••••.....•••••••••••••• .• 66 vi 4.3.1. Inoculum production ...... 66 • 4.32. Plant production ...... 66 4.3.3. General inoculation procedure 67
4.3.4. Assessment of disease development 67
4.3.5. Effect of dew period temperature , 67
4.3.6. Effect of post-dew temperature ...... 67
4.3.7. Effect of dew period duration 68
4.3.8. Interaction between dew period temperature and duration '" 68
4.3.9. Effeet of delaying initial dew period ...... 68
4.3.10. Effeet of seqloential dew periods ...... •...... 68
4.3.11. Data analyses •.•.....•....•...... •.•...... 68
4.4. Results ..••••...... ••...... 69
4.4.1. Effeet of dew period ternperature ...... •...•...... 69
4.42. Effect of d~w period duration ...... •..•...... •..... 70
4.4.3. Effect of post-dew ternperature ...... •...... ••... .. 71
4.4.4. Interaction between dew period ternperature and duration •.. 71
4.45. Effeet of delaying initial dew period .•.•••..••.....• •. 73
4.4.6. Effeet of sequential dew periods •...•...•.•...••••. .. 73
4.5. Discussion ..••..••....•....••••...••....•..••...... • 73
4.6. Literature cited .••...•..•••••.••••.•••...••...•.••...• 77
Connecting Text ....••••....•••••....•...••••.•••....•..•.•... 92
Chapter 5. Characterization of growth and conidial production of Exserohilwn • monoceras on different substrates •••••...•••.•••.•.•..••.••... 93 vü 5.1. Abstraet ...... 93 • 5.2. Introduction 93 5.3. Materials and Methods ...... 95
5.3.1. Pathogen isolation and culture maintenance ...... 95
5.3.2. Grov.th and conidial production on standard agar media 95
5.3.2.1. Effect of nutrient media on mycelial growth
and conidial production 95
5.32.2. Effect of temperature on mycelial growth,
conidial production, and conidial gennination ...•. 96
5.32.3. Effect of light, dark, and NUV on mycelial
growth and conidial production ...... •...... 97
5.32.4. Effect of Echinochloa leaf decoction on
conidial production and genmnation ...... •... 97
5.3.3. Conidial production on liquid media ••...... •••.. .. 97
5.3.4. Conidial production on agriculturally-based solid substrates .. 98
5.3.4.1. Evaluation of solid substrates ••••••...... •••. 98
5.3.4.2. Effect of incubation period on conidia!
production on corn leaves .•.....••••...... • 99
5.3.4.3. Effect of moisture content and substrate
quantity on conidia! production on corn leaves ...• 99
5.35. Effect of conidial production methods on conidial
production, gennination and virulence •••••...••••.... 99
5.3.6. Data analyses ••.••.•..••.••.•...... ••••••.. .. 100 • 5.4. Results .•.•••••••••••••..•.••••••••.•...•..•••••. .. 101 viii 5.4.1. Growth and conielial production on standard agar media ... 101 • 5.4.1.1. Effect of nutrient meelia on mycelial growth and conielial production 101
5.4.1.2. Effect of ternperature on mycelial growth.
conielial production, and conielial gennination .... 101
5.4.1.3. Effect of light, dark. and NUV on mycelial
growth and conidial production 101
5.4.1.4. Effect of Echinochloa leaf decoction on
conielial productiun and gennination 102
5.4.2. Conielial production on liquid media ...... •...... 102
5.4.3. Conielial production on agriculturaIly-based solid substrates. 102
5.4.3.1. Evaluation of solid substrates ..•...... 102
5.4.3.2. Effect of incubation period on conielial
production on corn leaves ...... •.•...•... .. 103
5.4.3.3. Effect of moisture content and substrate
quantity on conielia! production on corn leaves ..• 103
5.4.4. Effect of conidia! production methods on conidial
production, germination, and virulence .. .. 103
5.5. Discussion ••...••...•....•.•••..•...•.•...... 104
5.6. Literature cited ..••...... •.•..•..••.•.•...... •...... 106
Connecting Text .••••.••••••••..••...•..•.••..••..••..•..•... 119
• Chapter 6. Host range of Exserohilum monoceras •••••••••.••••••.••••• 120 ix 6.1. Abstraet ...... 120 • 6.2. Introduction 120 6.3. The organisms 122
6.3.1. The target weeds: Echinochloa species...... 122
6.3.1.1. Echinoch/oa crus-galli (L) Beauv. 122
6.3.1.2. Echinochloa colona (L.) Link...... 123
6.3.1.3. Echinoch/oa glabrescens Munro ex Hook. F. 123
6.3.2. The biocontrol agent: Exserohilum monoceras
(Drechsler) Leonard & suggs 123
6.4. Materials and Methods ...... 124
6.4.1. Inoculum production ...... ••...... 124
6.4.2. Plant production ...... 125
6.4.3. General inoculation procedure ...... •• 125
6.4.4. Disease development on trap plants •...... •.•..... 127
6.4.5. Field inoculation of corn •. .•...... 128
6.5. Results ..•••.•..•...... •••.•...... ••.•...... 128
6.5.1. Host range screening ...... •...... • •. 128
6.5.2. Disease development on trap plants ...... •. 129
6.5.3. Field inoculation of corn .• •..•..••...... •.. .. 129
6.6. Discussion ..••••••....•...•...... 129
6.7. Literature cited .....•••••...... •••••••••...... ••.•. 131 • COMectïng Text ••••.•••.••...... ••..•••...... ••.•...... 142 x Chapter 7. Isolation and partial characterization of phytotoxins • produced by Exserohi/um monoceras •...... 143 7.1. AbstraCt ...... 143
7.2. Introduction 143
7.3. Materials and Methods ...... 144
7.3.1. Culturing ...... 144
7.32. Isolation and purification of toxins 144
7.3.3. Leaf bioassay .....•...... •...... 145
7.3.4. Isolation of toxins produced in vivo 145
7.3.5. Host specificity of toxins , 146
7.3.6. Root growth inhibition .....•...... 146
7.3.7. Comparison of toxins with standards of
bipo1aroxin and exserohilone ...... •..•...... , 147
7.4. Results .•....•...••..••...... •....•...... •...... 147
7.4.1. Isolation and purification of toxins "•...... •. .. 147
7.42. Phytotoxicity of toxins .....•.•.•...... •...... 148
7.4.3. Specificity of toxins .•.....•...•....•..••...... 148
7.4.4. Root growth itthibition .••..•....•.•...... •.. 148
7.5. Discussion ••...•...... •...••...... •.. 148
7.6. Literature citee! .•••.••..•...•..•••.•. ...•..•...••.. .. 150
Chapter 8. General Conclusions •••.••..••.••...... •.••..•...•. .. 155
Chapter 9. Contribution ta Knowlcdgc ••••••••••••••••••••••••••.••• 156 • Appendices •••...•••.••...••••••.••..••.•.•.•••.•...•.•... •• 157 xi • List of Tables 2.1. Pathogenieity of six fungi on various Echinochloa spccies and riee (Oryza saliva) .' ...... •..•.•....•....•....•...••..•...•..•..• •. 35
2.2. Arca of living leaf tissue of three Echinochloa spccies 48 h after roots were placed in ee11-free ftItrates of six fungi 36
3.1. Exserohilum monoceras inoculum doses causing 50% and 90% dry weight reduction of three Echinochloa species ...... •...... 56
3.2. Estimated regression parameters and associated statistics for the regression of arc sine-transformed mean percent mortality and dry weight reduction of Echinochloa crus-ga/li, E. colona, and E. glabrescens at various growth stages as a funetion of the log inoculum density 10 days after inoculation. .. 57
3.3. Percent mortality (M) and dry weight (DW) of Echinochloa spccies and riee seedlings 14 days airer being treated with water, Exserohilum monoceras at a rate of 5 x 10' eonidialm2 in oil emulsion, or with the oil emulsion aIone 58
3.4. Percent mortality (M) and dry weight (DW) of Echinochloa species and riee seedlings 20 days after being treated with water, a dry powder formulation 2 of Exserohilum monoceras at a rate of 5 x 10' conidialm , or with a dry powder formulation not containing conidia ••••....••.•••...•..... •• 59
4.1. Effect of sequentiaI dew periods on mortality and dry weight reduction of Echinochloa crus-galli •.•.•••••..•..••••....••.••..•..•••.• •. 82
5.1. Effect of nuttient media on Exserohilum monoceras myceliaI growth and conidiaI production •.••••••.•...... •••..••.••.•••.••••.••.. 111
5.2. Effect of light, daIk, and ncar ultraviolet light (NUV) on myceliaI growth and conidiaI production of Exserohilum monoceras grown on V-8 juice agar 112
5.3. Effect of various agricu1turaIly-based prodUClS as solid substrates on conidiaI • production of Exserohilum monoceras •••••••.••••••••••••••••• •. 113 xii 5.4. Effect of substrate quantity and moistur'e content on conidial production of • Exserohilum monoceras on corn leaves o...... 114 5.5. Effect of conielia! production rnethods on Exserohilum monoceras conielial production, gerrninatitm, and virulence 115
6.1. List of test plant species used for host-specificity screening of Exserohilum monoceras against Echinochloa species o. 137
6.2. Results of host-specificity screening for Exserohilum monoceras - e1isease severity 140
6.3. Results of host-specificity screening for Exserohilum monoceras - sporulation ...... 141
7.1. Host specificity of Exserohilum monoceras and its associated toxins 152
• xiii • List of Figures 2.1. Disease development (expressed as the mean percentage of leaf area damage (%LAD)) of severa! pathogenic fungi on Echinochioa species subjected to 12 h and 24 h dew periods following inoculation. Seedlings of E. crus-galli, E. colona, and E. glabrescens al the 2-leaf stage were 6 inoculated with 1 x lOS - 1 X 10 conidia/ml of Exserohilum monoceras, Bipolaris sacchari, Curvularia geniculata, and Dactylaria dimorphospora, respectiveIy. Data from two triaIs were not pooled because variances were heterogenous, but trends for the two aiaIs were sirnilar. Data represent four replicatel. of one triaI. The 5% LSD between dew periods, fungi, and weeds are 8.5%, 8.9%, and 8.7%, respectively...... 37
2.2. Disease development (expressed as the standardized area under disease progress curve (SAUDPC)) of severa! pathogenic fungi on Echinochloa species subjected to 12 h and 24 h dew periods following inoculation. Data represent means of four replicates. Note DO = Dactylaria dimorphospora, CG =Curvularia geniculata, BS =Bipolaris sacchari, and EM = Exserohilum monoceras. ••...•••••••••••.•••..••...... 38
2.3. MortaIity of Echinochioa species at different leaf stages caused by pathogenic fungi for either a 12 h or 24 h dew period. Seedlings of E. crus-galli, E. colona. and E. glabrescens al the 2-leaf stage were inoculated 6 with 1 x lOS - 1 X 10 conidia/ml of Exserohilum monoceras, Bipolaris sacchari, Curvularia geniculata, and Dactylaria dimorphospora, respectiveIy. Data from two triaIs were not pooled because variances were heterogenous, but trends for the two triaIs were similar. Data represent four replicates of one triaI. The 5% LSD between dew periods, fungi, weed species. and leaf stages are 16.9%, 16.9%, 17.0% and 17.6%, respective1y. •.•...•••.•••. 39
3.1. Effect of inoculum density on the control of Echinochioa species by Exserohilum monoceras, expressed as percent mortaIity and reduction in dry weight 10 days after inoculation. Seedlings of E. crus-ga/li, E. colona, and E. glabrescens al the 1.5-1eaf stage were inoculated with 0, 0.16 x 10', 0.31 X 10', 0.63 X 10', 1.25 X 10', 2.50 X 10', 5.00 X 10', or 10.00 x 10' conidia/m% and provided with a 24 h dew period. Observations from two triaIs were poo1ed because variances were homogenous. Each data point • represents the mean of eight replieates. The relationships of mortaIity and xiv % reduction in dry weight versus log inoculum density are described by logistic equations generated from actual data. A. B. and C: Percent mortality • versus log inoculum density for E. crus-galli. E. colona. and E. glabrescens. respectively; D. E, and F: Percent dry weight reduction versus log inoculum density for E. crus-galli. E. colona. and E. glabrescens. respectively. 60
3.2. Effect of plant growth stage on the control of Echinochloa species by Exserohilum monoceras. expressed as percent mortality and reduction in dry weight 10 days after inoculation. Seedlïngs of E. crus-galli. E. colona. and E. glabrescens at 0.5-. 1.0-. 1.5-. 2.0-. and 3.0-leaf stages were inoculated with 5.0 x 107 conidialm2 and provided with a 24-h dew period. Data from two trials were pooled becal:se variances were homogenous. Data points represent the mean of eight replicates. The relationships of mortality and % reduction in dry weight versus leaf stage are described by polynomial equations from actual data. A. B. and C: Percent mortality versus leaf stage for E. crus-galli. E. colona. and E. glabrescens. respectively; D. E. and F: Percent dry weight reduction versus leaf stage for E. crus-galli. E. colona, and E. glabrescens. respectively. ...•...... 61
3.3. Effect of Exserohilum monoceras inoculum density and plant growth stage on the control of Echinochloa species, expressed as percent mortality and reduction in dry weight 10 days after inoculation. Seedlïngs of E. crus-galli. E. colona, and E. glabrescens at the 0.5-, 1.0-, 1.5-, 2.0-, and 3.0-1eaf stages 7 7 7 were inoculated with 1.25 x 10 , 2.5 x 10" 5.0 X 10 , and 10.0 x 10 2 conidialm , respectively, and provided with a 24-h dew period. Data from two trials were pooled because variances were homogenous. Data represent the mean of eight replicates. A, B, and C: Percent mortality for E. crus-galli, E. colona, and E. glabrescens, respective1y; D, E, and F: Percent dry weight reduction for E. crus-galli, E. colona, and E. glabrescens, respectively. ... •. 62
4.1. Effect of dew temperature on disease development caused by Exserohilum monaceras on three Echinochloa species, expressed as percent leaf area damage (% LAD). Seedlings at the 1.5-1eaf stage were sprayed with a 7 2 conidial suspension of E. monoceras at a rate of 5 x 10 conidialm • Dew period duration was 24 h. Data from two trials were pooled because variances were homogenous. Data represent means of eight replicates. For comparative pmposes, the disease progress eurves over time were summarized by using the standardized area under the disease progress • eurve (SAUDPC) (Figure 4.2). 83 xv 4.2. Effect of dew pcriod temperature on e1isease development caused by Exserohilum monoceras on three Echinochloa species, expressed as the • standardized area under the e1isease progress curve (SAUDPC). Data represent means of eight replieates. Bars within each temperature treatrnent having the same letter are not significantly clifferent accorcling to DMRT at the 5% significance level. 84
4.3. Effect of dew period duration on e1isease development caused by Exserohilum monoceras on three Echinochloa species, expressed as percent leaf area damage (% LAD). Seedlings at the 1.5-1eaf stage were sprayed with a conielia: suspension of E. monor:eras at a rate of 5 x 107 2 conielialm • Dew temperature was 25 C (in dark). Data from two trials were pooled because variances were homogenous. Data represent means of eight replicaœs. For comparative pl1IpOses, the e1isease progress curves over time were summarized. by the standarclized area under the clisease progress curve (SAUDPC) (Figure 4.4). ..•....•...... •...... • 85
4.4. Effect of dew pcriod duration on e1isease development caused by Exserohilum monoceras on three Echinochloa species, expressed as the standardized area under the e1isease progress curve (SAUDPC). Data represent means of eight replicaœs. Bars within the same dew period duration having the same letter were not significantly clifferent accorcling to DMRT at the 5% significance level. ..•.••.•••....•....•..•••.. 86
4.5. Effect of post-dew temperature on cIisease development caused by Exserohilum monoceras on three Echinochloa species, e.'qlressed as percent plant mortality 10 days after inoculation. Seedlings at the 1.5-leaf stage were sprayed with a conielial suspension of E. monoceras at a rate of 5 x 107 conielialm2 and given a 12 h or 24 h dew periocl. Data from twO trials were not pooled because variances were not homogenous. Data represent means of four replicaœs. Bars within each temperature treatrnent in a dew period having the same letter are not significantly clifferent accorcling to DMRT at the 5% significance leve1. .•••••.••...•••...• 87
4.6. Interaction effect between dew period temperature and duration on cIisease deve10pment caused by Exserohilum monoceras on three Echinochloa species, expressed as percent plant mortality 10 days after inoculation. Data from two trials were poo1ed because variances were homogenous. • Data represent means ofeight replicaœs. •••.•••••••..••••••.....•• 88 xvi 4.7. Mortality response (arc sine-transfonned) of (A) Echinoch/oa crus-ga//i, (B) Echinoch/oa colooo, and (C) Echinoch/oa g/abrescens 10 days after • Exserohi/um monoceras inoculation to increasing dew period duration at each of five temperatures. The response was calculated by fitting a linear regression 10 the means ...... 89
4.8. Effect of dew period temperature on the predicted dew period duration required for 100% disease severity of three Echinochloa species by Exserohi/um monoceras expressed as percent plant mortality 10 days after inoculation. The relationship was best described by the following equations: 1) Echinoch/oa crus-ga/li: ln D = (M+15.1039)/(-36.1946 + 6.5039T-o.1323Tl>, (p=O.OOOl, r=O.7213); 2) Echinoch/oa colona: ln D = (M+33.45)/(-47.14+7.44T-o.14ST"J, (p=O.OOOl, r=O.7828); 3) Echinoch/oa glabrescens: ln D =(M+3.7930)/(-35.0961+6.5739T -o.l344Tl>, (p=O.OOOl, r=O.9254). Where M = mortality, D = dew period duration, and T = dew temperature. Percent mortality data were arc sine- transfonned before analysis. .. .•••....••...... •...... 90
4.9. Effect of de1aying initial dew period on disease development caused by Exserohi/um monoceras on three Echinoch/oa species, expressed as percent plant mortality 10 days after inoculation. Seedlings at the l.5-leaf stage were sprayed with a conidial suspension of E. monoceras at a rate of 5 x 107 conidialm2. Data from two trials were not pooled because variances were not homogenous. Data represent means of four replicates. 91
5.1. Effect of temperature on radial mycelial growth (A), conidial production (B), and conidial gennination (C) of Exserohi/um monaceras grown on V-8 juice agar plates. The predicted equations are: (A) GROW1H = exp(7.28 + 3.00lnT' + 1.45ln(1-T'), R2 = 0.93; (B) PRODUCTION = exp(14.83 + 8.49lnT' + 8.67ln(1-T'), R2 = 0.99 (temperature range from 20 to 35 C); and (C) GERMINATION = -92.41 + 16.43T - 0.3~,R2 = 0.82, where T =temperature and T' =(T-T...,.)/(T....-T...,). .••.....••...... ••.. 116
5.2. Effect of Echinoch/oa leaf decoction on conidial production (A), germination rate (B), and genn tube length (C) of Exserohi/um monoceras grown on V-8 juice agar (VA), lima bean agar (LBA), and potato dextrose • agar (PDA). •••.•••••.•••.•••..••••..•.••.•••.•••...•.... 117 xvü 5.3. Effect of incubation time on Exserohilum T7UJnoceras conidia production on corn leaves. One gram dry corn leaves were placed in 250-1lÙ Erlenmeyer • flasks, moistened with 10 llÙ distilled water, and autoelaved twO limes for 15 min (100 kPa and 121 C). Afrer cooling, flasks were inoculated and incubated at 28 C in the dark ...... 118
7.1. Effect of Exserohilum T7UJnoceras spores and toxin 1 on Echinochloa crus-galli leaves, in situ, 48 h after treatment. Detached leaves were wounded with a glass capillaIy tube. Spore application was carried out using 50 J.Ù of E. T7UJnoceras at a rate of 5 x 10' conidialllÙ in 2% aqueous ethanol and 0.05% Tween 20. Toxin 1 was applied using 50 !Ù of toxin 1 solution in 2% aqueous ethano1 and 0.05% Tween 20. The toxin solution was composed of 50 Ilg of pure toxin 1/1lÙ. The control treatment consisted of applying only 50 J.Ù of 2% aqueous ethanol and 0.05% Tween 20 solution. ••...... •....•...... 153
7.2. Inhibition of seedling root growth of rice and Echinochloa species by toxin 1produced by Exserohilum T7UJnoceras. •.••..•••••••••...••••••• 154
• xviü • List of Appendices 1. A comparison of Exserohi/um numoceras characteristics reponed in different countries 157
2. Effect of Exserohi/um monoceras conidial field application on severa! Echinochloa crus-ga/li growth parameters 28 days after tteatment ...... 158
3. Effect of an oil emulsion fonnulation having a different ratio of oil: water and Exserohilum monoceras conidia on monality of Echinochloa crus-gal/i and riec (Oryza sativa) under greenhouse conditions .....•...... 159
4. Effect of an oil emulsion fonnulation having a different ratio of oil: water and Exserohilum monoceras conidia on the plant height of Echinochloa crus-galli and riec (Oryza sativa) under greenhouse conditions •...... 160
5. Effect of wetting agents and chemical herbicides on disease severity caused by Exserohilum monoceras under greenhouse conditions 161
6. Effect of suspension media on Exserohilum monoceras conidial viability in freeze-dried tteatment ..••..••...... •....••...... 162
7. Percent mortality and reduction in fresh and dry weight between paired flats of Echinochloa crus-gal/i seedlings treated either with Exserohilum monoceras plus an adjuvant, or with the adjuvant alone. using different sprayers under greenhouse conditions ....••...•.... .•...• .. 163
8. Shelf life of Exserohilum monoceras conidia harvested from corn leaves .• .• 164
9. Disease severity between paired flats of Echinochloa seedlings treated either with Exserohilum monoceras plus an adjuvant, or with the adjuvant alone under greenhouse conditions .•.•••.•...•.•..•...••..•...••... •• 165
la. Conidial production ofBipo/aris sacchari on different substrates ...•..• •. 166 • xix • Description of Thesis Format This thesis is comprised of original papers that have becn and will he submitted to appropriate scientific journals for publication. In accordance with part B, section 2 of the "Guidelines Conceming Thesis Preparation" from the Faculty ofGraduate Studies and Research, McGiII University, 1 quote the entire text that applies to this format:
H2! Manuscripls and auPwrship: Candidates have the option. su/;ject to the approval oftheir Department. ofincluding. as pan oftheir thesis. copies ofthe text ofa paper(s) submittedfor publication, or the clearly-duplicated text ofa published paper(s). providing that these copies are bound as an integral pan of the thesis. Ifthis option is chosen, ronnecting texts, providing logical bridges between the different papers are mandatory. The thesis must still conform to all other requirements ofthe HGuidelines Conceming Thesis PreparationHand should be in a literary form that is more than a mere collection ofmanuscripts published or to be published. The thesis must include, as separate chopters or sections: (1) a table ofcontents, (2) a general abstract in English and French, (3) an introduction which clearly states the rationale and objectives ofthe study, (4) a comprehensive general review ofthe background Iiterature to the subject ofthe thesis, when this review is appropriate, and (5) ajinal overall conclusion and/or summary. Additional material (procedural and design data, as weil as descriptions ofequipment used) must be provided where appropriate and in sujJicient detail (e.g. in appendices) to allow a clear and precise judgement to he made ofthe imponance and originality ofthe research reponed in the thesis. ln the case ofmanuscripts, co-aurhored by the candidate and others, the candidate is required to make an explicit statement in the thesis ofwho contributed to such work and to what ex/ent; supervisors must attest to the accuracy ofsuch claims at the Ph.D. Oral Defence. Since the task ofthe • examiners is made more dijJicult in these cases, it is in the candidates's xx interest to make perfectly c/ear the responsibi/ities of the different allthors of • co-allthored papers." In order for this thesis to be consistent with the above statement. it is structure
The various rnanuscript chapters are 1Ù'.ked via connecting texts 50 as to establish logical bridges between the different papers. A general discussion and synthesis of the major conclusions of the thesis are presented in Chapter 8. The main conttibutions to knowledge ofthis research are outlined in Chapter 9. An Appendices section which presents results from ex~ents conducted but not presented in the relevant chapters is also included. Manuscripts from chapters 1, 3, 4, 5, 6, and 7 are co-authored by Dr. A.K. • Watson. The candidate (Wenming Zhang) performed aU the cxperimental research, xxi e· statistical analyses, and is the primaI)' author of all five manuscripts. Dr. A.K Watson provided supervisory guidance and assisted in manuscript preparation. The manuscript from chapter 2 is co-authored by Ors. A.K Watson and K Moody. The candidate (Wenming Zhang) perforrned all the experimental research, statistical analyses, and is the primaI)' author ofthis manuscripL Dr. A.K Watson and K Moody provided supervisory guidance and assisted in manuscript preparation.
• xxii • Chapter 1. General Introduction 1.1. Abstract A number of Echinochloa species constitute a serious weed problem in most rice growing areas of me world and as such are prime wgets for biologicaI control. The findings ofsevera! researcb. programs which have been initiated to examine me possibility -0, of utilizing indigenous fungal pamogens ta control EchinochIoa species will be outlined.
Moreover, me specific objectives of Ù1ÏS mesis will aIso be presented.
1.2. Introduction Rice (Oryuz sativa L) is me moS( important food crop in me worlcl, particularly in Asian countries where 92% of me world's rice is produced and 87% is consumed (Holm et aI., 1977; IRRI, 1989; Gohbara & Yamaguchi, 1994). Unfortunately, weeds still constitute one of me major biologicaI constraints to greater rice yields and huge resources are consumed for meir control C'Naterhouse, 1994). Weeds commooly cause rice yield losses of 10% to 40% and, occasionaIly, losses of 100% C'Natson, 1994). EchinochIoa species are me most serious weed species in rice (Holm et al., 1977; Moody, 1983; Smim, 1983; Zhang, 1989). Various management strategies are available to control mese weeds wim me greatest emphasis on me use of synmetic chemical herbicides (Gupta & Q'Toole, 1986). Herbicides are often me most rapid and effective solution ta these weed problems. However, me use ofherbicides is not the ooly means or necessarily the moS( appropriate memod to control weed infestations C'Natson, 1994). Increasing herbicide resistance in weeds, me necessity to reduce input costs, and widespread concems about environmental and social issues, have placed greater pressure on farmers ta reduce me use of chemical herbicides. The new realities have necessitated me discovery and development of new weed control technologies and/or me improvement ofexisting weedcontrol technologies that are economica\ly and ecologica\ly sustainable C'Natson, 1994)• • To date, me moS( biologica\ly effective and extensively evaluated alternatives ta 1 chemicals for weed control are plant pathogens. in particular. plant pathogenic fungi • (Boyette et al.• 1991). The use of plant pathogens to control weeds offers an exploitable bioteehnology that is an effective supplement to conventional weed control (Charudattan. 1991).
1.3. Echinochloa species
Echinochloa belongs ta the tribe Paniceae. subfanûly Panicoideae. Poaceae Family. Cyperales Order (Cronquist, 1981; Gould & Shaw. 1983).
13.1. Echinochloa crus-ga/li (L.) Beauv. E. crus-ga/li is believed native to Europe or India (Holm et al.• 1977; Maun & Barrett, 1986). It is now widely cosmopolitan and occurs throughout the tropical and
temperate regions of the world from latitude 50 N ta 40 S. E. crus-gal/i is an erect,
clumped, C•• annual grass growing up ta 15 m high. with branching stems near the base and rooting when decumbent. Leaves are fiat, tapering to a point, hair1ess. or with a few hairs on the margins near the broad base. Inflorescences typically consist of 15 greenish (often tinged with purple) spikelets. Flowering occurs year-round in the Philippines. with
each plant producing al 1east 200 seeds. Reproduction is exc1usive1y via seed. Sorne seeds germinale immediate1y. although others remain viable in sail for several years. E. crus gal/i is a very morphologically variable species with numerous ecotypes present around the world. E. crus-ga/li is ranked as the world's third worst weed (Holm et al. 1977). It is a common and serions agricu1tural weed in many areas of the world as is shown by the faet that it constitutes a problem in 36 different crops in 61 countries. particularly in rice (Holm et al. 1977). E. crus-ga/li is the most cosmopolitan of the Echinochloa spccies flourishing in aImOst every rice-growing country and is a particu1ar1y serions weed in irrigated lowland rice and rain fcd lowland rice (De Dana, 1981; Zhang. 1989). At carly growth stages. E. crus-ga/li and rice have a similar appearance and their seedlings are • often mistakenly transplanted at the sarne lime. In direct-seedcd riec, E. crus-ga/li also 2 genninates at about the same time as rice (5 to 6 days) or/and grows at a similar rate for • the f1I'St few weeks, but cventually E. crus-ga/li grows tal1er. The fibrous root system of E. crus-ga/li provides a competitive advantage for nutrient uptake compared to rice (Holm et al., 1977). In fac!, dense stands of E. crus-ga/li can remove nearly 60 to 80% of the available nitrogen from a given area. Under heavy competition from E. crus-gal/i, tillering in rice is reduced by up to 50% (Holm et al., 1977). Similarly, season-long competition by E. crus-gal/i can reduce rice grain yields by up to 70% (Smith, 1968; Zimdahl, 1980).
132. Eclùnochloa colona (L.) Link. E. colona is native to India but its present range extends from latitude 45 N to 40 S (Holm et al., 1977). This annual, C. grass has prostrate seedlings which may attain 70 75 cm in height at maturity. E. colona closely resembles E. crus-ga/li, however, E. colona individuals do not possess a ligule, have red-purple tinged leaf sheaths and blades, typical1y possess awnless spikelets, and have smaller seed (caryopsis). E. colona can be best distinguished from E. crus-ga/li by the absence of awns on its spikelets. E. colona, junglerice, is ranked as the world's fourth worst weed and is listed as a principal or most serious weed in 35 different crops in more than 60 countries. E. colona occurs almost everywhere rice is grown but tends to be equatorially distributed and is a major weed of upland rice (Holm et al., 1977; De Datta, 1981; Moody, 1989; Moody, 1991). Rice yield reductions of25.2% are typically observed at E. colona density of 80 plantslm2 during the initial 40 days of growth with this crop (Mercado & Talatala,
1977). Although E. colona is a less vigorous competitor than E. crus-galli, E. colona tan
pose a serious problem in rice systems because typical populations of this weed tan greatly exceed the 80 plantslm2 critical density.
133. Echinochloa glabrescens Munro ex HookE. E. glabrescens is widespread from the Indian subcontinent through mainland • Southeast Asia, China to Korea and Southem Japan (pancho, 1991). It has also been 3 recorded in Togo. West Africa (pancho. 1991). E. glabrescens is very similar ta E. crus • galli but only grows 0.5-1 m in height. The leaf blade is acuminate. Leaf sheaths are almost closed and often fIattened. Awns, ifpresent, are shoner than those ofE. crus-galli (about 1 cm long). The seed (caryopsis) of E. glabrescens is larger than those ofE. crus ga/li. E. glabrescens is also an important weed in irrigated lowland rice. especially in tropical regions (De Dana, 1981; Smith, 1983; Moody. 1989). The rice yield losses due ta this weed are similar ta E. crus-ga/li (Krishnamurthy et al., 1989). Average rice yield reductions from transplanted E. glabrescens ranged from 6% at the 5% infestation level ta 73% at the 40% infestation level (Rao & Moody. 1992).
1.4. Control strategies against EchinochWa species in rice Various management strategies are available for the control of Echinochloa species, including cultural measures. hand weeding. mechanical control. and chemical herbicides (Matsunaka. 1983). Each method has its advantages and limitations. Cultural measures such as watermanagement, soil preparation (mcluding tillage). and temporal and spatial planting arrangements can be used ta reduce Echinochloa infestations (Nada, 1977; Stauber et al. 1991). but these measures are only applicable in sorne rice systems and control is not always consistent. Hand weeding is a common method ta control Echinochloa species but is expensive and time consuming. Mechanical weeding can reduce labor costs, but yields are often reduced. Moreover. this method is not appropriate
ta broadcast-seeded rice systems. Numerous herbicides. such as quinclorac (3.7-dichloro 8-quinolinecarboxylic acid). molinate (S-ethyl hexahydro-1H-azepine-I-carbotlùoare), butachlor(N-(buthoxymethyl)-2-chloro-N-(2,6-diethylphenyl)acetamide),andpropanil flV (3,4-dichlorophenyl)propanamide), ete. can provide effective control of Echinochloa species in mast rice systems (Tlatlg, 1989; Eang et al, 1992; Zhang, 1990a,b). Recently, reduced availability and higher cost of water and labor resources make the control of Echinochloa species difficult, resulting in an increased emphasis on chemical herbicides • and these changes are also bringing about modifications in the way rice is grown (IRRI, 4 1994). Large production areas are shifting from transplanting to direct seeding, resulting • in increased weed populations and chemical herbicide use (IRRI. 1994). This greater dependency on herbicides will likely accelerate the development of herbicide resistance in weed populations as weil as increase environmental and social concems. The use of biological control agents is an alternative or complementary tactic to reduce herbicide inputs. however, this strategy has received limited attention in the major rice producing areas of Asia (Watson. 1991).
1.5. Biological control of weeds Biological weed control is the deliberate use of living organisms to suppress the growth or reduce the population of a weed species (Watson, 1991). Insccts, mites, nematodes. plant pathogens, and aquatic and terrestrial herbivores have becn used as biotic agents in biological weed control prograrns. Biocontrol methods include the classical approach (inoculative), the inundative approach (including bioherbicides and augmentation), and herbivore management (Wapshere, 1982).
15.1. ClassicaI approach The basis of the classical approach is the introduction of natural enemies from which the exotic weed has escaped and relies on self-sustaining epidemics of introduced organisms ta control the target weed at acceptable leve\s (Huffaker, 1957; Wapshere, 1982; Watson, 1991). Used first and still the most widespread technique, the classical
approach bas provided sorne notable weed control successes including: prickly pear cactus (Opuntia spp.), St. Johnswort (Hypericurn perforat:un L.), ske\eton weed (Chondrilla
juncea L), and water rern (SaIvinia molesta O.S. Mitchell) (Julien, 1992). The vast majority of agents used in classical biological control have becn phytophagous insccts (Wapshere et al., 1989). Oassical biological weed control is not generally applicable in highly disturl:led vegetation systems (Le. annual cropping systems) and cannot be employcci where immediate weed control isrequired, although sorne exceptions have been • reported (Wapshere, 1982; Schroder, 1983; Watson, 1991). 5 152. The inundative approach • The inundative (augmentation) approach involves the production cflarge quantities of inoculum and the timely application of the inoculum to specific areas in volumes and dosages that achieve control of the target weed before economic losses are incurred (Olarudattan, 1991). Mass rearing and periodic release of insccts such as Bactra verutana Zeller for the control ofpurple and yellow nutsedge (Cyperus rotundus L and C. esculentus L) (Frick et al., 1983) and the nematode, Ditylenchus phyllobia (Thome) Brzeski, for suppression of silverleaf nightshade (So/anwn elaeagnifoüwn Cav.) (parker, 1986) are two examples of the augmentation approach. The "bioherbicide" approach is a more recent development and involves the application of inoculum of a weed pathogen in a manner analogous to chernica1 herbicide applications (Templeton, 1982; TeBeest & Templeton, 1985). The bioherbicide approach involves three major phases or stages: discovery, development, and deployment (Templeton, 1982). Bioherbicides have becn proven to be effective in rapidly providing a high degree of weed control in cultivated crops (TeBecst & Templeton, 1985; Watson, 1989; Charudattan, 1991; TeBeest et al., 1992). To date, two fungal plant pathogens have becn registered as bioherbicide weed control products in the United States and one registered in Canada. DeVine·, a liquid formulation ofPhytophthora pa/mivora (Butler) Butler was registered in 1981 for control of sttangler-vine (Morrenia odorata (H. & A.) Lindl.) in Florida citrus graves (Ridings, 1986). COLLEGO·, a dry power formulation of Colletotrichwn g/oeosporioides (Penz.) Sace. f.sp. aeschynomene was registered in 1982 for the control of northern jointveteh (Aeschynomene virginica (L) B.S.P.) in rice and soybean in Arkansas, Louisiana and Mississippi (TeBeest & Templeton, 1985). BioM~, a dry formulation ofCo//etotrichwn g/oeosporioides f.sp. malvae was registered in 1992 in Canada for the control ofround-leaved mallow (Ma/va pusma Smith.) in wheat and lentils (MOrtensen, 1988; Makowski & Mortensen, 1992). Another product, Lubao 1 Sn (Co//etotrichwn g/oeosporioides f.sp. cuscutae), is being used in China for the • control of dodder (Cuscuta chinensis and C. austra/is) on soybean (Wan et al, 1994). 6 Cha.l1danan (1991) provides a comprehensive review ofbioherbicide researcil worldwide. • The use of the bioherbicide approach is based on the fundamental principles of epidemiology. It is different from the epidemiology of crop disease. which is based on prevention of epidemics by determining critical factors favouring an epidemic. Weed biocontrol is based on the induction or enhancement of an epidemic by determining and manipulating epidemic constraints (reBeest et al.• 1992). Pathogen virulence and
fastidious environmental requirements are the IWO restraints to bioherbicide development most often cited (reBeest & Templeton. 1985; Watson. 1989; Charudanan. 1991). Host specificity is another important consideration in the bioherbicide approach (Watson. 1985). However. optinùzation of spore production ("fermentation") and formulation and application of a bioherbicide product are often critical aspects in determining the success or failure of a bioherbicide prospect (Watson & Wymore. 1990; Boyene et al.• 1991). Cunent research using the bioherbicide approach has mainly focused on dicotyledonous weeds. In contras!, few studies on graminaceous weeds have been carried out (Evans, 1991; Julien, 1992). Of the 69 species of weeds studied as targets for potential biocontrol in recent years. only nine weed species are graminaceous. The limited interest in the biocontrol ofgrass weeds is due, in part, to the concern that pathogens and insects on associated with these weeds may also anack desirable grass crops because grass weeds are commonly crop mimics. The host range of graminicolous fungal pathogens varies considerably but genus. species or subspecies-specific fungi have been reported (Chiang et al., 1989). ThUs, it is possible to fmd host specific agents for the control of selected grass weeds.
1.5.3. Herbivore management The use of herbivore management to control weeds bas been very limited, especially in cultivated crops (Wapshere, 1982).
1.5.4. B:orational approach • Phytotoxins derived from pathogens and other microorganisms are also useful for 7 weed control (Duke, 1986; Hoagland, 1990; Strobel et al., 1992). Traditionally. research • on phytotoxins bas been limited to products produced by plant pathogens of crop plants. These phytotoxins have proven useful as tools for screening plants for toxin insensitivity (disease resistance) and as probes of normal physiological plant functions (Strobel et al., 1992). Weed pathogens have had a long period to coevolve with their hosts and devise biochemical mechanisms to weaken them or influence their gross physiology (Strobel,
1982; Strobel et al., 1992). Hence, there is the potential 10 use natural compounds produced by plant pathogens as herbicides or to utilize them as building blocks for novel herbicides (Duke, 1986; Duke & Lydon, 1987; Kenfield et al., 1989; Kennedy et al., 1991). Herbicidal activity has been demonstrated in a number of species of the actinomycetes Streptomyces genus. and two herbicides, NK-ü49 (methoxyphenone) and bialophos have been developed from microbial metabolites (Duke, 1986; Watson, 1993). Other microbial toxins, including maculosin, a host-specific phytotoxin for spotted knapweed (Centaurea macuIosa Lam.), isolated from liquid cultures of Alternaria alternata (Fr.) Keissier (Stierle et al., 1988) and fumonisin BI, a broader-spectrum phytotoxin isolated from Fusarium moniliforme Sheldon (Abbas & Boyette, 1992), have
been suggested 10 have utility in weed control (Watson, 1993).
1.6. Current status of biological control on Echinochwa species Research on the biological control of Echinochloa species has been limited. Two herbivorous fish, the grass carp (Ctenapharyngodon idella) and red tilapia (Tilapia mossambica x T. nilotica-aurea) have been shown to control Echinochloa species in rice but they are not selective grazers (Itoh, 1991). Recently, 32 insects, 10 fungi, 3 nematodes, and 1 bacterium which would
possibly be classical biocontrol agents have been reported 10 be natural enemies of E.
crus-galli (Waterhouse, 1994). However, with 50 few natural enemies reported from its
presumptive vast area oforigin (Europe 10 India), it is not yet possible 10 postulate on the prospects ofsuccessfully controlling this weed viaclassical biocontrol strategy. Recently, • a stem boring moth, Emmalocera sp., which does not attaek riec, has been reported 10 8 feed on Echinoch/oa species in Japan and Malaysia (Goto. 1994). However. the potential • of this indigenous species as a biocontrol agent has not been detennined. Various fungi have been isolated frorn Echinoch/oa species and are being evaluated for their biocontrol potential as candidate bioherbicides. Coch/iobolus /unatus Nelson & Haasis was evaluated for the control of E. crus-galli seedlings in corn (Zea mays L.). but because of its weak pathogenicity. it was effective only when combined with atrazine (Scheepens, 1987). In Korea. a fungal pathogen. identified as Exserohilum monoceras. was found ta cause leaf blight of E. crus-galli but this isolate was also pathogenic to severa! important crops including rice (Chung et al.• 1990). Echinochloa species have been a primary target for recent bioherbicide research in Japan (Gohbara & Yamaguchi, 1994). Various species of Fusarium. Rhizoctonia. Phoma. Drechslera. and Leptosphaerùl naturally infect Echinochloa species and severa! strains of Fusarium and Drechslera were pathogenic to Echinochloa species but not pathogenic to rice (Gohbara & Yamaguchi. 1994). Drechslera monoceras is presently being developed as a bioherbicide for Echinochloa species control in Japan (Gohbara & Yamaguchi. 1994).
1.7. Thesis objectives Fmdings to date have shown that the classical approach and herbivore management are not adequate biocontrol methods for Echinoch/oa species in rice. Research into the biological control ofEchinochloa species through the augmentation of indigenous fungal pathogens has just recently begun. Presently. no bioherbicides are being used for the control of Echinochloa species in rice, although sorne are under investigation (Bayot et al. 1994; Gohbara & Yamaguchi. 1994). In addition. there have been no reports of phytotoxin production and the possible utility of phytotoxins produced by Echinochloa fungal pathogens. In 1991. a biological weed control research program was initiated at the International Rice Research Institute (IRRl) in collaboration with the University ofthe Philippines at Los Baiios (UPLB) and McGill University. ta evaluate the possibility of utilizing indigenous fungal pathogens for the control ofmajor weeds in rice (Bayot etal• • 1994; Watson. 1994). The research reported in this thesis focuses on the biocontrol of 9 Echinochloa species through the use of indigenous fungi and their phytotoxins. Over 76 • ~trains of pathogens were isolated from naturally-infected Echinochloa species. Six different virulent funga! isolates were obtained. The specific objectives of this research wereto: 1) select the best fungal candidate for further development as a biocontrol agent
for Echinochloa species in rice, 2) quantify the efficacy of the selected candidate, 3) deterrnine the optimum conditions and limiting factors on disease development of the selected fungus, 4) characterize the growth and conidia! production of the fungus on different substrates, 5) delimitate the host range of the fungus, and 6) isolate and partia!ly characterize the phytotoxins produced by the selected fungus.
L8. Literature cited Abbas, H.K. and Boyene, C.D. 1992. Phytotoxicity of fumonisin BIon weed and crop species. Weed Techno1. 6:548-552.
Bayot, R.G., Watson, A.K. and Moody, K. 1994. Control of paddy weeds by plant pathogens in the Philippines. Pages 139-143 in Shibayama, H., Kritani, K. and Bay Peterson, J. (eds) Integrated Management of Paddy and Aquatic Weeds in Asia. FFTC Book Series No. 45. Food and Fertilizer Technology Centre for Asian and Pacific Regions, Taipei.
Boyette, CD., Quimby, P.c. Jr., Connick, WJ., Daigle, DJ. and Fulgham, F.E. 1991. Progress in the production, formulation, and application ofmycoherbicides. Pages209-222 • in TeBeest, 0.0. (ed.) Microbial Control of Weeds. Chapman & Hall, New York. 10 Charudanan, R. 1991. The mycoherbicide approach with plant pathogens. Pages 24-57 in • TeBeest, D.O. (ed.) Microbial Control of Weeds. Chapman & Hall, New York.
Chiang, M. Y., van Dyke, C. G. and Leonard, K. J. 1989. Evaluation of endemic foliar
fungi for potential biological control ofJohnsongrass (Sorghum halepense): screening and host range tests. Pla"t Dis. 73:459-464.
Chung, Y. R., Kim, B. S., Kim, H. T. and Cho, K. Y. 1990. Identification ofExserohilum
species, a fungal pathogen causing leaf blight of barnyard grass (Echinochloa crus·gal/j). Korean Journal of Plant Pathol. 6:429-433.
Cronquist, A. 1981. An Integrated System ofClassification ofAowering Plants. Columbia Univ. Press, New York.
De Dana, S. K. 1981. Principles and Practices of Riee Production. John Wiley & Sons. New York, USA.
Duke, S.O. 1986. Naturally occurring ':::lemical compounds as herbicides. Pages 17-44 in Review ofWeed Science. Vol. 2. Weed Science Society ofAmerica. Champaign, minois, USA.
Duke, S.O. and Lydon, J. 1987. Herbicides from naturaI compounds. Weed Technol. 1:122-128.
Evans, H. C. 1991. Biological control of tropical grassy weeds. Pages 39-51 in Baker, F.W.G. and Terry, PJ. (eds) Tropical Grassy Weeds. CAB International for CASAFA. Wallingford, UK. • 11 Frick. K.E., HanIey, G.G. and King, E.G. 1983. Large scale production of Bactra • verutana (Lep.:Tortricidae) for the biological control of nutsedge. Entomophaga 28:107 115.
Gohbara, M. and Yamaguchi, K. 1994. Biological control agents for rice paddy weed management in Japan. Pages 184-194 in Shibayarna, H., Kritani, K. and Bay-Peterson, J. (cds) Integrated Management of Paddy and Aquatic Weeds in Asia. FFfC Book Series No. 45. Food and Fertilizer Technology Centre for Asian and Pa~ific Regions, Taipei.
GolO, M. 1994. The reIationship between Emmalocera sp. and barnyardgrass and its potential as a biologica1 control. Pages 113-121 in Shibayarna, H., Kritani, K. and Bay PCI'el'SOn, J. (cds) Integrated Management of Paddy and Aquatic Weeds in Asia. FFfC Book Series No. 45. Food and Fertilizer Technology Centre for Asian and P-..cific Regions, Taipei.
Gould F.W. and Shaw, R.B. 1983. Grass Systematics. Texas A&M University Press. Texas, USA.
Gupta, P. C. and O'Toole, J. C. 1986. Weed management. Pages 267-279 in Upland Rice: A Global Perspective. International Rice Research Institute, P.O. Box 933, 1099 Manila, Philippines.
Hoagland, R.E. 1990. Microbes and microbial products as herbicides - An overview. Pages 2-52 in Hoagland, R.E. (cd) ACS Symposium Series 439: Microbes and Microbial Produets as Herbicides. American Chemical Society, Washington, Oc.
Holm, L. G., Plucknett, O. L., Pancho, J. V. and Herberger, J. P. 1977. The World's Worst Weeds. Distribution and Biology. The University Press of Hawaii, Honolulu, • Hawaii, USA. 12 • Huffaker. C.B. 1957. Fundamentals of biological control of weeds. Hiigardia 27: 101- 157. IRRI. 1989. IRRI Toward 2000 and Beyond. International Rice Research Institute. P.O. Box 933. 1099 Manila, Philippines.
IRRI. 1994. Integrated Pest Management: The lRRl Perspective. IRRI Infonnation Series No.3. International Rice Research Institute. P.O. Box 933. 1099 Manila Philippines. pp:9-12.
Itoh. K. 1991. Integrated weed management of direct seeded wet rice fields in South East Asian and Pacific regions. with special references to Malaysia. Pages 77-94 in Proceedings of 13th Asian Pacific Weed Science Society, Malaysia.
Jia.'lg, R. C. 1989. Handbook of Chemical Weed Control. Shanghai Scientific and Technological Publishing House, Shanghai. China.
Jiang, R., Zhang, W., Wu, J., Lu, M. 1992. Development of one-shot herbicide used in ttansplanted riec - Daocaowei. Chinese J. Weed Sci. 3:5-8.
Julien, M. H. 1992. Biological Control ofWeeds: A Worid Catalogue of Agents and Their Target Weeds. 3rd Edition. CAB International, Wellington, in association with Austraiian Centre for International Agriculture Research (ACIAR), Canberra.
Kenfield. D., Bunkeres. G, Strobel, GA and Sugawara. 1989. Potential new herbicides phytotoxins from plant pathogens. Weed TechnoL 2:519-524.
Kennedy, A.C., Ellion, LF., Young, EL and Douglas, C.L. 1991. Rhizobacterla • suppressive to the weed downy brome. Soil Sci. Soc. Am. J. 55:722-727. 13 Krishnamunhy, K., Devendra, R., Prasad, T.V.R. and Mohan, S.L. 1989. Growth pattern • of Echinochloa species in relation to rice and bio-efficacy of 2,4-D and dicamba combinations. Pages 683-688 in Proceedings of Brighton Crop Protection Conference Weeds. Vol. 3. Brighton, England.
Maun, M.A. and Barret, S.C.H. 1986. The biology of Canadian weeds. 77. Echinochloa crus-ga/li (L.) Beauv. Can. J. Plant Sci. 66:739-759.
Makowski, R.MD. and Mortensen, K. 1992. The fust mycoherbicide in Canada: Colletotrichum gloeosporioides f.sp. malvae for round-leaved mallow control. Pages 298 300 in Proc. lst Int Weed Control Conference. Weed Science Society of Victoria, Inc., Melbourne.
Matsunaka, S. 1983. Evolution of riec weed control practices and research: world perspective. Pages 5-17 in Weed Control in Rice, International Rice Research Institute, P.O. Box 933, 1099 Manila, Philippines.
Mercado, BL and Talatala, R.L. 1977. Competitive ability of Echinochloa colona L. against direet-seeded low1and rice. Pages 161-165 in Proc. 6th Asian-Pacific Weed Sei. Soc. conference. Korea.
Moody, K. 1983. Weeds: defmitions, COSts, characteristics, classification and effeets. Pages 11-32 in Weed Management in the Philippines: Report of Serninars. Walter H. cd. PLITS.
Moody, K. 1989. Weeds reported in riec in South and Southeast Asia. International Riec Research Institute, P.O. Box 933, 1099 Manila, Philippines. • 14 Moody, K. 1991. Weed control in upland nce with emphasis on grassy weeds. Pages 164 • 178 in Baker. F.W.G. and Terry, P.J. (cds) Tropical Grassy Weeds. CAB International for CASAFA. WaIlingford, UK.
Mortensen, K. 1988. The potential ofan endemic fungus, Co//etotrichum gloeosporioities. for biological control of round-leaved maIlow (Mall'a pusilla) and velvetIeaf (Abutilon theophrasnj. Weed Sei. 36: 473-478.
Nada, K. 1977. Integrated weed control in nce. Pages 17-46 in Fryer. J.R. and Matsunaka. S. (cds) Integrated Control of Weeds. University of ToJ....yo Press, ToJ...-yo.
Pancho, J.V. 1991. Grass weeds in the Philippines. Pages 183-188 in Baker, F.W.G. and Terry, P.J. (cds) Tropical Grassy Wel:ds. CAB International for CASAFA. WaIIingford, UK.
Parker, P.E. 1986. Nematode control of silverleaf nightshade, a biological control pilot project. Weed Sei. 34 (Suppl. 1):33-34.
Rao, A.N. and Moody, K. 1992. Competition between Echinoch/oa glabrescens and nce (Oryza saliva). Tropical Pest Management 38:25-29.
Ridings. W.H. 1986. Biological control of strangle-vine in citrus - a researcher's view. Weed Sei. 34 (Suppl. 1):31-32.
Seheepens, P. C. 1987. Joint action ofCoch/iobolus lunalUS and ~ttazine on Echinoch/oa crus-gaIli (L.) Beauv. Weed Res. 27:43-47.
Schroeder, D. 1983. Biological control of weeds. Pages 41-78 in Fletcher, W.W. (ed) • Advances in Weed Reseaxch. Commonwealth AgricuIturaI Bureaux, Farnham Royal 15 • Smith, R.J., Jr. 1968. Weed competition in rice. Weed Sci. 16:252-255. Smith, R. J., Jr. 1983. Weeds of major economic imponance in rice and yield losses due
10 weed competition. Pages 19-36 in Weed Control in Rice, International Rice Research Institute, P.O. Box 933, 1099 Manila, Philippines.
Stauber, L. G., Nastasi, P., Smith, Jr., R. J., Baltazar, A. M. and Talbert, R. E. 1991. Bamyardgrass (Echinochloa crus-galli) and bearded sprangletop (LeptochloafascicuJaris) control in rice (Oryza saliva). Weed Technol. 5:337-344.
Stierle, A.c.. Cardellina, J.H. and Strobel, G.A. 1988. Maculosin, a host-specific phytotoxin for spotted knapweed..from Altemaria alternata. Proc. Natl. Acad. Sci. USA 85:8008-8011.
Strobel, G.A. 1982. Phytotoxins. Annu. Rev. Biochem. 51:309-333.
Strobel, GA, Sugawara, F. and Hershenhom, J. 1992. Pathogens and their products affecting weedy plants. Phytoparasitica 20:307-323.
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Temple1On, G. E. 1982. Biological herbicides: discovery, development, deployment. Weed Sei. 30:430-433. • 16 Wan, F.H., Wang, R., and Qiu. S.B. 1994. Biological weed control in China: Cwrent • status and prospects. Unpublished paper.
Wapshere, A. J. 1982. Biological control of weeds. Pages 47-56 in Ho1zner. W. and Numata, N. (eds) Biology and Ecology of Weeds. Dr. W. Junk Publisher. The Hague.
Wapshere, A. J., Oelfosse, E. S., Cullen. J. M. 1989. Recent developments in biological control of weeds. Crop Protection 8:227-250.
Waterhouse, O. F. 1994. Prospects for Biological control of paddy weeds in southeast Asia and sorne recent successes in the biological control of aquatic weeds. Pages 10-20 in Shibayama, 8., Kritani, K. and Bay-Peterson. J. (eds) Integrated Management ofPaddy and Aquatic Weeds in Asia. FFfC Book Series No. 45. Food and Fertilizer Technology Centre for Asian and Pacific Regions, Taipei.
Watson, A.K. 1985. Host specificity ofplant pathogens in biological weed control. Pages 577-586 in Delfosse, E.S. (ed) Proceedings of VI Int. Symp. Biol. ConO'. Weeds. Rome, Italy.
Watson, A. K. 1989. Cwrent advances in bioherbicide research. Brighton Crop Protection
Conference - Weeds. 3:987-995.
Watson, A. K. 1991. Prospects for bioherbicide development in Southeast Asia. Pages 65 73 in Swarbrick, R.K. (cd) Proceedings of 13th Asia Pacific Weed Science Society Conference. Asia-Pacific Weed Science Society and Weed Science Society of Indonesia, Jakarta. • 17 Watson, A.K. 1993. Biological Control of Weeds Handbook. Weed Science Society of • American, Champaign, ID., USA.
Watson, A. K. 1994. Current status of bioherbicide development and prospects for rice in Asia. Pages 195-201 in Shibayama, H., Kritani, K. and Bay-Peterson, J. (eds) Integrated Management of Paddy and Aquatic Weeds in Asia. FFfC Book Series No. 45. Food and Fertilizer Technology Centre for Asian and Pacific Regions, Taipei.
Watson, A.K. and Wymore, LA 1990. Identifying limiting factors in the biocontrol of weeds. Pages 305-316 in Baker, R.R. & Dunn, P.E. (eds) New Directions in Biological Control: Alternatives for Suppressing Agricultural Pests and Diseases. Alan R. Liss, Inc. New York.
Zhang, Z. 1989. Field weeds and their control in mainland China. Weed Sei. Bull. 10:41 45.
Zhang, W. 1990a. Bioassay ofthe sensitivity ofpretilachlor between rice and Echinochloa crus-gal/i. J1lIIIgsu Agricultural Science 2:35-36.
Zhang, W. 1990b. Quinclorac, a new herbicide for the control of Echinochloa crus-gal/i in rice. Chinese J. Weed Sei. 4:8-10.
Zimdahl, R.L. 1980. Weed-Crop Competition -A Review. Int. Plant. Prot. Cent., Oregon State Uni., Corvallis, OR, USA. • 18 • Connecting Text A review of the literature described in the previous chapter indicates that the augmentation of indigenous fungal plant pathogens has great potential for controlling Echinochloa species in nce. Six different fungal species were isolated from naturally infected agricultural Echinochloa populations in the Philippines. It is necessary, however, ta select the most promising fungus for further development as a biocontrol agenL In this chapter, the results of screening, identification, pathogenicity (safety ta lice), and virulence (control ofEchinochloa species) of these fungi with the view ta select the most promising isolate for further study as a biocontrol agent of weedy Echinochloa species are reported. Preliminary evidence of phytotoxin production in severa! of these fungi is also presented.
• 19 Chapter 2. Responses of Echinochloa species • and lice (Oryza saliva) to indigenous pathogenic fungi
2.1. Abstract Six pathogenic fungal species were isolated from naturally infected Echinochloa species and evaluated as biological control agents of Echinochloa species in rice. Curvularia lunata var. aeria and Exserohilum oryzae were pathogenic to both rice and Echinochloa species and were not evaluated further. Bipolaris sacchari, Curvularia geniculata, Dactylaria dimorphospora, and Exserohilum monoceras were pathogenic only to Echinochloa species and were further compared for disease severity under controlled environment conditions in the greenhouse. When provided with a 24-h dew period, E. monoceras kilIed seed1ings of all three Echinochloa species tested: E. crus-gal/i, E. colona, E. glabrescens; B. sacchari resulted in 100% monaIity ofseedlings ofE. colona and E. glabrescens; C. geniculata killed seed1ings of only E. colona; and D. dimorphospora did not cause any plant death. When given a 12-h dew period, E. monoceras still kilIed the three Echinochloa species, whereas the other fungi did not cause plantdeath. Echinochloa seedlings at the 1- and 2-leafstages were more susceptible to these fungi !han seed1ings at the 3- and 4-leaf stages. B. sacchari, E. monoceras, and
E. oryzae produced phytotoxins that caused 100% leafarea chlorosis and wilting ofintact seed1ings of the Echinochloa species placed in cell-free culture filtrates. Curvularia species also produced phytotoxins but these were less toxic.
2.2. Introduction Batnyardgrass, Echinochloa crus-galli (L.) Beauv., and junglerice, Echinochloa
colona (1..) Link., ate ranked as the world'sthird and fourth worst weed species and ate two ofthe most serious weeds in rice (Oryza saliva L.) (Holm et aI., 1977). Echinochloa gltJbrescens Munro ex Hook.F. is another important weed species in rice, especially in the tropical regions (De Datta, 1981; Moody, 1989). These species severely reduce bath yieId • and quality ofnce (Holm et aI., 1977; Smith, 1983). 20 Cultural methods, hand weeding. mechanical methods. and herbicides are available • 10 control these weeds (Matsunaka. 1983; Gupta & O'Toole, 1986). The reduced availability and higher cost of water and labor resources make the control of these weeds difficult, resulting in an increased emphasis on the use of herbicides as weil as bringing about changes in the way rice is grown (IRRI. 1994). Large production areas are shifting
from transplanting 10 direct seeding. resulting in increased weed populatioils and chemical herbicide use (IRRI. 1994). The increased use of herbicides will accelerate development of herbicide resistance in weed populations and will increase environmental and societal
concerns related 10 pesticide use. The use of biological control agents is an alternative or
complernentary taetic 10 reduce herbicide inputs but has received limited study in the major rice producing areas of Asia (Watson. 1994). In Korea. a fungal pathogen. identified as Exserohilum mD/Wceras (Drechsler) Leonard & Suggs. was found to cause
leaf blight ofE. crus-galli but this isolate was aIso pathogenic 10 several important crops including rice (Chung etal. 1990). In Japan. a fungal pathogen, identified as Drechslera mD/Wceras (Drechsler) Subram. & Jain, is being evaluated as a bioherbicide for control of Echi/Wchloa species in rice (Gohbara & Yamaguchi, 1994; GolO. 1994). Recently, six different indigenousfungal species have been isolated from naturally infected E. crus-galli, E. colona, and E. glabrescens in the Philippines (IRRI, 1993; Bayot
et al, 1994; Watson, 1994). In order 10 select the best candidate for further development
as a biocontrol agent for Echi/Wchloa species in riet, this study was designed 10 1) determine the pathogenicity of these fungi on Echi/Wchloa species and riet, 2)
characterize the responses of Echi/Wchloa species 10 these fungi, and 3) obtain
preliminary evidence as 10 whether or not these fungi produce secondary metabolites which are biologically active against Echi/Wchloa species.
2.3. M 23.2./noculwn production Sma1l picccs of mycelium from the stock culture of cach fungus were aseptically transferred to fresh PDA, plates were sealed with parafilm and incubated in the dark at 28 C for 7 days. Agar plugs (6-mm e1iam.) from the margins ofyoung colonies were used as seed inoculum (Tuite, 1969). Rice Polish Agar (RPA; 20 g rice polish, 17 g agar, and 1 L water) was used as a medium for conidia production (Tuite, 1969). Agar plugs ofseed inoculum were placed in the centre of each Petri e1ish, plates were sealed with parafilm and incubated at 28 C in the dark. Conielia were harvested 15 days after incubation by fIooeling the plateS with 10 ml distilled water and scraping the surface ofthe colonies with a glass slide. Resulting suspensions were filtered through a layer ofcheesecloth and conielial concentrations were detennined using a haemacytOmeter. 233. Pathogenicity 01 the testedfungi Cultivars Dee-Geo-Woo-Gen and IR72, Chianan and Chianung 242, and Brendol putih and Rodjolele were selected as representatives of inelica, japonica, and tropical japonica nce, respectively. A single lot of seeds of each of the Echinochloa species, i.e. E. crus-galIi. E. colona, and E. glabrescens, col1ected from the International Rice Research Institute (IRRI) farm were used in al! experiments. Seeds ofeach Echinochloa species and nce cultivar were incubated in petri dishes on moistened filter paper al reom • temperatUIe for 48 h. Five genninated seeds (coleoptile and radicle just ernerged) were 22 planted per 10 cm-diameter plastic pot filled with saturated soil (Maahas clay. Haplustic • suborder). Seeded pots were placed on a push-can in the greenhouse. Throughout the experimental period, 2-3 cm of water were maint:lined in the push-earts. Greenhouse conditions were 35125 ± 5 C day/night temperature. a 12-h photoperiod. and average light intensity of 20 MJ/m2 per day. Seedlings at the 1- to 2-leaf stage were inoculated with 1 x 10' to 1 x 106 conidia/ml to run-off with 0.05% Tween 20 as a wetting agent. using a motorized sprayer (A. H. Thomas Co. Scientific Apparatus. Philadelphia) at 100 kPa. After spraying, pots were placed in a dark dew chamber with 100% relative humidity at 25 C for 24 h. Subsequently, pots were lI'ansferred to a mist room at 24-28 C with high relative humidity (about 95%). The disease reactions of Echinochloa species and rice to each of the tested fungal species were ev:ùuated 14 days after inoculation (DAI) using a oto 3 scale (0 - no symptoms, 1 - light infection. 2 - moderate infection, 3 - severe infection to death). A randomized complete block (RCB) with four replicates (five plants/replicate) was used. The experiment was perforrned twice. The control treatrnent was sprayed with distilled water cont:lining only the wetting agent 23.4. Comparison ofdisease severiry The fungal species pathogenic to rice were excluded from further ev:ùuation, !hose fungi pathogenic ooly to Echinochloa species were further ev:ùuated to assess the effect ofdew period duration on disease severity at the 1-, 2-, 3-, and 4-leafstages. Seeding was carried out at 7, 9, 11. and 13 days before inoculation for E. crus-galli and E. glabrescens, and at 6, 8, 10, 12 days before inoculation for E. colona in order for the four leafstages to be inoculated on the same day. Five seedlings were established within each 6 pot before inoculation. Pots were sprayed until run-off at a rate of 1 x lOS to 1 X 10 conidialml containing 0.05% Tween 20 as a wetting agent. Control treatrnents were sprayed with distilled water containing ooly the wetting agent. After spraying, aIl pots were immediate1y placed in a dark dew chamber at 25 C. The response ofplants to each fungus were compared for a 12-h and a 24-h dew period. Disease severity was assessed • as the percentage leaf area damage (%LAD) and estimated visually al 2, 4, 6, 8, and 10 23 DAI. The Mean %LAD for each pot was recorded. For comparative purposes, the • standardized area under disease progress curve (SAUDPC) was calculated for cach replicate by dividing the AUDPC value by the total duration of disease development (Trapero & Kaiser, 1992). Ten days after inoculation, mortality was evaluated for cach plant, results pooled, and averaged for cach pot. Completely collapsed seedlings were considered dead. The experimental design consisted of a split-split-plot in RCB with four replications. The dew period was the main plot, the fungal species was the sub-plot, and plant growth stage was the sub-sub-plot. Data were collected individually for cach plant. Mean values of five plants were used for statistical analyses. The experiment was performed tWÎce. 235. Bioassay for phytotoxin production Agar plugs (6 mm-diameter) of cach fungus taken from the edge of 7-day-old PDA cultures were placed into 5QO-ml Erlenmeyer flasks containing 100 ml of modified Fries liquid medium (30.0 g sucrose, 5.0 g ammonium tartrate, 1.0 g NH.NOJ , 1.0 g KH2PO., 0.5 g MgSO., 0.1 g NaCl, 0.1 g CaC12, 0.5 g casein hydrolysate, and 1.0 g yeast extract in 1,000 ml distilled water) (Tuite, 1969). Flasks containing only modified Fries medium served as contrais. The f1asks were placed on rotary shakers at 150 rpm at room temperature. Mer a 2-wk incubation, cultures in cach flask were separately centrifuged at 3,000 rpm for 20 min. The supematant in the centrifuge tubes was filtered through a membrane filter (pore size 0.45 !lm) to obtain cell-free culture filtrate and adjusted to pH 6.0, 30 ml ofwhich was dispersed into sterilizecl 50 ml vials (20 mm-diameter). The roots of hea1thy EchinochIoa seedlings at the 2-leaf stage were irnrnersed into the cell-free filtrates. The experimental design was a RCB with four replications. After 48 h, data were recorded. Ch1orosis and wilting of lcaves were assumed to indicate the presence of phytotoxins in the culture filtrate. Living leaf area was measured by using a leaf area meter (U-3100 Arca Meter, LI-COR. lne., Lincoln. Nebraska, USA). This test was • performed tWÎce• 24 23.6. Data analyses • AlI percentage data were arc sine-transfonned before analysis (Gomez & Gomez, 1984). Nonparametric statistical analysis was used for the rating data. Factorial experiments were analyzed with a factorial analysis of variance considering the effeet of cach factor individually and their interaction. Results for the !wo trials ofcach experiment were pooled if homogeneity of variances was confrrmed using Bartlett's test (Gomez & Gomez, 1984). However, for experiments in which the variance of trials were not homogenous, results from one trial were, nonetheless, presented given that a similar !rend was observ-:d be!Ween them. Mean values of five plants were used for statistical analyses and treatment means were separated using the least significant difference (LSD) or Duncan's Multiple Range Tests (DMRT) at the 5% level of significance. 2.4. Results 2.4.I.lsolation and identification offungi Six different species of pathogenic fungi, !wo Exserohilum spp. (92-044 and 93 136), !wo CurvuIaria spp. (92-074 and 93-128), one Bipolaris sp. (91-097), and one Dactylaria sp. (91-106), were isolated from naturally-infected Echinochloa plants. Isolates 91-106,93-128, and 93-136 were isolated from E. crus-galli; isolates 91-097 and 92-ll44 from E. colona; and isolate 92-074 from E. glabrescens. These fungi were identified by IMI as Exserohilum monoceras (Drechsler) Leonard & Suggs (92-044), Exserohilum oryzae Sivan. (93-136), CurvuIaria lunata var. aeria (Willer) Boed. (92-074), CurvuIaria geniculata (Tracy & Earle) Boedijn (93-128), Bipolaris sacchari (E. J. Butler) Shoem. (91-097), and Dactylaria dimorphospora Veenbaas-Rijks (91-106). 2.4.2. Pathogenicity ofthe tested fungi Both Exserohilum species caused severe disease on all Echinochloa species tested (Table 2.1). Necrotic flecks appearect within 24 h and a blight-like reaction was observed 2 DAI. Symptoms were charaeterized by chlorosis and a diffuse, general water-soaking • reaction which was followed by rapid collapse and necrosis ofaffected tissues, often with 2S no or only a weak expression of typicallesions. E. oryzae was also pathogenic 10 rice and • appeared 10 be more aggressive on the japonica and tropical japonica rice cultivars than on the inelica rice cultivar. Rice, however, was immune to E. monoceras (fable 2.1). Lesions induced by both Curvu/aria species appeared on Echinochloa leaves within 24 h after inoculation and wilting of the top portion of leaves occurred 2-3 DAI. C. genicu/ata caused severe e1isease on E. crus-galli and E. colona and moderate e1isease on E. glabrescens; C. lunata var. aeria resulted in moderate disease on all Echinochloa species. However, C. genicu/ata was only pathogenic to Echinochloa species, whereas C. lunata var. aeria was pathogenic to both rice and the Echinochloa species (slightly pathogenic 10 the three types of rice) (fable 2.1). B. sacchari resulted in severe e1isease on E. colono and E. glabrescens and moderate disease on E. crus-galli. Lesions induced by B. sacchari appeared on Echinochloa species within 24 h of inoculation. This fungus also caused a blight-like symptom simi1ar 10 that caused by E. monoceras and was also nonpathogenic 10 rice (fable 2.1). D. dirnorphospoTa was only s1ightly pathogenic to Echinochloa species. Lesions induced by D. dirnorphospora appeared on Echinochloa species 2-3 DAI and infected leaves wilted 5-7 DAI. Fewer lesions, however, were observed on plants inoculated with D. dirnorphospora than those inoculated with the other fungi, despite the higher concentration of conielia. Rice was immune 10 this fungus (fable 2.1). 2.4.3. Comparison ofdisease severiry Since E. oryzae and C. lunata var. aeria were pathogenic 10 riec, they were excluded from further evaluation. The other four fungal species, E. monoceras, B. sacchari, C. geniculata. and D. dimorphospora, were pathogenic only 10 EchinochJoa species and their virulence 10 the Echinochloa species was further compared. Disease progress over time was significantly different amongst the four fungal species tested (Figure 2.1). Inoculation of Echinochloa species at the 2-leaf stage with E. • monoceras resulted in 100% disease severlty of all Echinochloa species within 4 DAI 26 when provided a 24-h dew period and within 8 DAI when provided a 12-h dew period. • When inoculated with B. sacchari and given a 24 h dew period. E. colona and E. glabrescens showed 100% disease severity 2 DAI and 8 DAI, respectively. whereas E. crus-galli was not completely diseased and limited regrowth occurred. When provided a dew period of 12-h, disease severity of B. sacchari on Echinochloa species decreased dramatically (i.e. 40% LAD on E. colona and a lower value for the other two Echinochloa species). Following inoculation with C. genicuJata with 24-h of dew, only E. colona expressed 100% disease severity, whereas E. crus-galli and E. glabrescens showed 90% and 77.5% LAD, respectively, and sorne regrowth was observed. When provided a dew period of 12-h, inoculation with C. genicuJata resulted in less than a 20% LAD for the three Echinochloa species. Finally, inoculation with D. dimorphospora resulted in Iess than a 20% LAD for ail three Echinochloa species even after a 24-h dew period was provided and aImost no disease occurred when the dew period was shonened to 12-h. When provided a 24-h dew period, E. monoceras caused 100% standard area under disease progress curve (SAUDPC) for the three Echinochloa species (Figure 2.2). B. sacchari resulted in 100% SAUDPC for E. colona and E. glabrescens and 93.4% for E. crus-gaili. The SAUDPC value for B. sacchari on E. crus-galli was significantiy lower than that for E. monoceras. C. genicuJata inoculation resulted in a 83.2%, 85.0%, and 60.9% SAUDPC for E. crus-gaili, E. colona, and E. glabrescens, respectively. The C. genicuJata SAUDPC value for E. crus-galli was not significantly different from that obta.:ned for B. sacchari, however, it was significantiy lower than that produced by E. monoceras. The C. genicuJara SAUDPC value for E. colona was significantly lower than the E. monoceras and B. sacchari SAUDPC values. The same trend was observed for E. glabrescens. The SAUDPC of D. dimorphospora for Echinochloa species was below 2.5%. When provided a 12-h dew period, the highest values of SAUDPC were recorded for E. monoceras: 84.0%, 76.9%, and 97.7% on E. crus-gaili, E. colona, and E. glabrescens, respectïvely. The SAUDPC of B. sacchari subjected to a 12-h dew period • was ooly 10-40%, which was significantiy lower than the SAUDPC value produced by 27 E. monaceras. With a 12·h dew period, inoculation with C. geniculata and D. • dimorphospora produced low SAUDPC values (Figure 2.2). Echinachloa species mortality varied with fungal species, weed species, plant leaf stage, and dew period duration (Figure 2.3). When provided a 24-h dew period, E. monaceras inoculation killed seedlings of ail three Echinachloa species at aIlleaf stages; the application ofB. sacchari causc:d 100% mortality of E. colona seedlings at the 1- and 2-leaf stages and E. glabrescens seedlings at the 2-leaf stage with 10wer mortality being observed at the 3- or 4-leaf stages, and did not kill all E. crus-galli; C. geniculata killed ooly E. colona seedIings at the 1- and 2-leaf Stages, with sorne mortality observed at the 3- or 4-1eaf stages and did not kill ail E. crus-galli and E. glabrescens seedlingS; and D. dimorphospora inoculation did not cause any plant death. When given a 12-h dew period, ooly E. monaceras caused 100% mortality of the three Echinachloa species at 1-, 2-, or 3-leaf stages. The other fungi did not cause any plant death when provided a 12-h dew period. E. monaceras, therefore, required the shonest dew period duration for 100% kill of Echinochloa species, followed by B. sacchari. C. geniculata, and D. dimorphospora. Seedlings at the 1-2 leaf stage were general1y more susceptible to disease from these fungi than seedIings at the 3-4 leaf stage (Figure 2.3). 2.4.4. Bioassay for phytotoxin produr.tion ChIorosis and wilting ofEchinochloa species were observed 24 h afterplacing the roots of intact seedlings at the 2-leaf stage in the cell-free filtrates of E; monoceras, E. oryzae, and B. sacchari. Symptoms were similar to those observed in plants inoculated with conidial suspensions. Fony-eight hours after immersion, seedlings ofail Echinachloa species were dead (Table 2.2). Thus, these three fungi produced secondary rnetaboliteS which were highly active against aIl three Echinachloa species. The culture filtrate of C. Junata var. aeria significantly reduced the living leaf area of E. crus-galli and E. colona by 53% and 64%, respectively, but did not affect the living leaf area of E. glabrescens• • The cell-free culture filtrate of C. geniculata did not influence the living leaf area of E. 28 crus-galli and E. glabrescens, but ree!uced living leaf = of E. colona by nearly 80%. • The culture filtrate of D. dimorphospora significantly reducee! the living leaf area of E. crus-galli but did not influence that of other two Echinoch/oa species (Table 2.2). 2.5. Discussion The responses of Echinochloa species and rice to the six indigenous pathogenic fungi variee!. E. oryzae and C.lunata var. aeria were pathogenic to the three Echinochloa species but also to rice. The former is a pathogen of rice causing rice brown spot disease and the latter w::s reported to cause slight chlorotic and faint brown spots of bean (Phaseolus vuIgaris L.) (Bisen, 1983). D. dimorphospora, C. geniculata, B. sacchari, and E. monoceras were not pathogenic to rice, but were pathogenic to the Echinochloa species. It is often assumee! that a virulent, highly aggressive pathogen (i.e.• causing a high level ofmortality) is a preferred bioherbicide candidate. D. dimorphospora caused limiteo:1 disease on the Echinochloa species, C. geniculata causee! 100% mortality of E. c%na. B. sacchari resulted in 100% mortality of E. colona and E. glabrescens. and E. monoceras caused 100% mortality of E. crus-gal/i. E. colona. and E. glabrescens. Therefore. C. genicuIata, B. sacchari. and E. monoceras have potential to control Echinochloa species. However, further research on the delimitation of host range. determination of optimum conditions and limiting factors for disease development and host damage, mass prodü,;"Li'.>n. formulation, and quantification of field efficacy is required. C. genicuIata is reported as a pathogen causing seed rot of soybean (Glycine max (1..) Merr.) and a weak pathogen causing leaf spot disease of a turfgrass species. Kentucky bluegrass (Poo pratensis L) (Brown et aL, 1972; Tangonan & Florendo. 1992). B. sacchari is the causal fungus of eyespot disease of sugarcane (Saccharum spp.) (Martin, 1961). Development ofC. geniculata and B. sacchari as bioherbicides to control Echinochloa must consider pote.'ltial risks :0 these crop species. • E. monoceras was first reportee! in 1970 as a beneficial organism to proteet wheat 29 (Triticum aestivum L.) against powdery mildew (Erysiphe graminis D.C. ex Mérat) • (Robeson & Strobel, 1982). Since then, there have been no reports on E. monoceras unti! the 199Os. In 1990, E. monoceras was reported to cause leaf blight of E. crus-gal/i in Korea but this isolate was aIso pathogenic to severa! important crops including nce (Chung et aI., 1990). In 1992, Drechslera monoceras was reported to effectively control Echinochloa species in nce in Japan (Gohbara & Yamaguchi, 1994). Our isolate collected in the Philippines was aIso identified as Exserohilum monoceras by IMI. According to Sivanesan (1987), Drechslera monoceras and Exserohilum monoceras are the same species. However, differences among the three isolates in terms ofconidiaI characteristics, host speci."icity, and phytotoxin production are apparent. Our isolate appears to be more similar 10 the isolate found in Japan because neither infect nce. However, whether or not these three isolates can be differentiated at either forma specialis or race leve1 remains to be seen. DNA fingerprinting might provide an approach for further comparison. Phytotoxins have becn reported to be produced by E. oryzae, B. sacchari, and C. lunaJa var. aeria (Steiner & Strobel, 1971; Bisen, 1983; Vidhyasekaran et aI., 1986). Our results confinned this and have demonstrated that these phytotoxins were biologicaIly active on Echinochloa spp. Further studies are needed to determine the role of these phytotoxins in the control of Echinochloa. Monocerin was the first component isolated from E. monoceras culture which was as an antibiotic to protect wheat against powdery mildew, instead ofas a phytotoxin. Subsequently, monocerin has aIso becn isoIated from Exserohilum rurcicum (pass.) Leonard et Suggsit and found to have phytotoxic activity onjohnsongrass (Sorghum halepense (1..) Pers.) and Canada thistle (Cirsium arvense (1..) Scop.) (Robeson ,& Strobe1, 1982). However, to our knowledge, there have been no reports on the phytotoxin production by E. monoceras. Further studies can be directed to isoIate, purify and characterize the phytotoxins produced by E. monoceras. Dew period duration is a key factor in the evaIuation of weed pathogens as potential bioherbicides. Chiang et aI. (1989) proposed a relative dew requirement index (RDRI) for evaIuation. RDR! was the ratio of disease severity with a 12-h dew period • relative 10 disease severity with 24-h dew period. In most cases, RDR! reflected the dew 30 requirement for specific candidates. However. in sorne cases. especially in the case with • an equal value of RDRI. RDRI did not reflect a difference for dew period requirements. For example. the RDRI of B. sacchari for E. crus-galli. is equal to the RDRI of C. geniculata for E. crus-gal/i. but the former actually required a shorter dew period duration than the latter. The problem might be solved by multiplying RDRI with the average disease severity at two dew period durations (MORI). Using SAUDPC values might provide more accurate estimation because SAUDPC values are .:Il average of the disease severity development over time while disease severity is only a single observation in the pro::ess of disease development. 2.6. Literature cited Bayot, R.G., Watson, Al(. and Moody, K. 1994. Control of paddy weeds by plant pathogens in the Philippines. Pages 139-143 in Shibayama, H., Kritani, K. and Bay Peterson, J. (cds) Integrated Management of Paddy and Aquatic Weeds in Asia. FFTC Book Series No. 45. Food and Fertilizer Technology Centre for Asian and Pacific Regions, Taipei. Bisen, P. S. 1983. Production of toxic metabolites by Curvularia /unata var. aeria and its role in leaf spot disease of bean (Phaseo/us vulgaris). Acta Botanica Indica 11:235-237. Brown, G. E., Cole, H., Jr. and Nelson, R. R. 1972. Pathogenicity of Curvularia sp. to turfgrass. Plant Dis. Rep. 56:59-63. Chiang, M. Y., Van Dyke, C. G. and Leonard. K. J. 1989. Evaluation of endemic foliar fungi for potential biological control ofJohnsongras:; (Sorghum ha/epense): screening and host range tests. Plant Dis. 73:459-464• • 31 Chung, Y. R., Kim, B. S., Kim, H. T. and Cho, K. Y. 1990. Identification ofExserohi/um • species, a fungal pathogen causing leaf blight of bamyardgrass (Echinochloa crus-galll). Korean Journal of Plant PathoI. 6:429-433. De Dana, S. K. 1981. Principles and Practices of Rice Production. John Wiley & Sons. New York, USA. Gohbara, M. and Yamaguchi, K. 1994. Biological control agents for rice paddy weed management in Japan. Pages 184-194 j' Shibayama, H., Kritani, K. and Bay-Peterson, J. (cds) Integrated Management of Paddy and Aquatic Weeds in Asia. FFTC Book Series No. 45. Food and Fertilizer Technology Centre for Asian and Pacific Regions, Taipei. Gomez, K. A. and Gomez, A. A. 1984. Statistical Procedures for Agricultural Research. 2nd Edition. John Wùey & Sons, Inc., New York. USA. GolO, M. 1994. The relationship between Emma/ocera sp. and bamyardgrass and its potential as a biological control Pages 113-121 in Shibayama, H., Kritani, K. and Bay Peterson, J. (cds) Integrated Management of Paddy and Aquatic Weeds in Asia. FFTC Book Series No. 45. Food and Fertilizer Techno10gy Centre for Asian and Pacific Regions, Taipei. Gupta, P. C. and O'Toole, J. C. 1986. Weed ManagemenL Pages 267-297 in Up1and Rice: A Global Perspective. International Rice Research Institute, P.O. Box 933, 1099 Manila, Philippines. Holm, L. G., Plucknett, D. L., Pancho, J. V. and Herberger, J. P. 1977. The World's Worst Weeds. Distribution and Bio10gy. The University Press of Hawaii, Honolulu, • Hawaii. USA. 32 IRRI. 1993. Program Report For 1992. International Riee Research Institute. P.O. Box • 933, 1099 Manila, Philippines. pp:I77-178. IRRI. 1994. Integrated Pest Management: The IRRI Perspective. IRRI Infonnation Series No.3. International Riee Research Institute, P.O. Box 933, 1099 Manila, Philippines. pp:9-12. Martin, J. P. 1961. Eye Spot. Pages 166-202 in Sugarcane Diseases of the World. Vol. 1. Elsevier Publishing Company, New York, USA. Matsunaka, S. 1983. Evolution of riee weed control praetiees and research: world perspective. Pages 5-17 in Weed Control in Riee. International Riee Research Institute, P.O. Box 933, 1099 Manila, Philippines. Moody, K. 1989. Weeds Reported in Riee in South and Southeast Asia. Intemational Riec Research Institute, P.O. Box 933, 1099 Manila, Philippines. Robeson, D. J. and Strobel, G. A. 1982. Monocerin, a phytotoxin from Exserohilum tuTcicum (Drechslera tuTcica). Agrie. Biol. Chem. 46:2681-2683. Sivanesan, A. 1987. Graminieolous species of Bipolaris, Curvularia, Drechslera, Exserohilum and their teleomorphs. Myeologica1 Papers 158: 1-261. Smith, R. J. Jr. 1983. Weeds of major economie importance in riee and yield losses due to weed competition. Pages 19-36 in Weed Control in Riec, International Riec Research Institute, P.O. Box 933, 1099 Manila. Philippines. Steiner, G. W. and Strobc:l, G. A. 1971. Helminthosporoside, a host specifie toxin from • HelminJhosporium sacchari on sugarcane. Phytopathology 61:691-695. 33 Tangonan, N. G. and Florendo, C. Q. 1992. Host Index of Plant Disease in the • Philippines. 2nd Edition. The Deparunent of Science and Technology, Bicutan, Taguig, Manila, Philippines. Trapero-Casas, A. and Kaiser, W. J. 1992. Influence oftemperature, wetness period, plant age, and inoculum concentration on infection and development of Ascochyta blight of chickpea. Phytopathology. 85:589-596. Tuite, J. 1969. Plant Pathological Methods: Fungi and Baeteria. Burgess Publishing Co., Minneapolis. Minnesota, USA. Vidhyasekaran, P., Borromeo, E. S. and Mew, T. W. 1986. Host-specific toxin production by He/minthosporium oryzae. Phytopathology 76:261-166. Watson, A. K. 1994. Current status of bioherbicide development and prospects for rice in Asia. Pages 195-201 in Shibayama, H., Kritani, K. and Bay-Peterson, J. (eds) Integrated Management ofPaddy and Aquatic Weeds in Asia. FFTC Book Series No. 45. Food and Fertilizer Technology Centre for Asian and Pacific Regions, Taipei• • 34 • • Table 2.1. Pathogenlcity of six fungi on varlous Eehinoehloa specles and rice (Oryza sativa)' Host responsesb Fungi Rice cultivar Echinochloa speciesd IN-l IN-2 JP-l JP-2 N-l N-2 ECHCG ECHCO ECHGL Bipolaris saeeharl 0 0 0 0 0 0 2 3 3 Daetylarla dimorphospora 0 0 0 0 0 0 1 1 1 Curvularia lunata var. aeria 1 1 1 1 1 1 2 2 2 Curvularia gel/ieulata 0 0 0 0 0 0 3 3 2 Exserohiiltm monoeeras 0 0 0 0 0 0 3 3 3 Exserohiium oryzae 1 1 2 2 2 2 3 3 3 ~ , Plants at the 1- to 2-leaf stage were Inoculated wlth 105 to 106 conidia/nù, placed in a dew chamber at 25C for 24 h and subsequently malntained ln a mist room. b Host responses were mted 14 days after inoculation using a 0 to 3 grading syslem where O-no symptoms, l-Iight infection, 2-moderate Infection, and 3-severe infection to death. • IN-l =Indica rice, cv. Dee-Geo-Woo-Gen IN-2 =Indlca rice, cv. m72 JP-l =Japonica rice, cv. Chianan JP-2 =Japonica rice, cv. Chianung 242 JV-l = Tropical Japonlca rice, cv. Brondol putih=20 JV-l = Tropical Japonica rice, cv. Rodjolele d ECHCG =Eehinoehloa erlls-galti ECHCO =Eehinoehloa eolona ECHGL =Eehil/oehloa glabreseens Table 2.2. Arca of living leaf tissue of three Echinoch/oa species 48 h after roots were • placed in cell-free fùttates of six fungi' Living leaf area (cm2)b Fungi Echinochloa Echinochloa EcJûnoch!oa crus-ga//i c%na g/abrescens Dactylaria ditnorphospora 0.63 bc 0.70 a 0.83 a CurvuJaria lunata var aeria 0.53 c 0.33 b 0.47 a CurvuJaria genicuJata 0.98 ab 0.22 b 0.61 a Exserohilum monoceras o d o c o b Exserohilum oryzae o d o c o b Bipolaris sacchari o d o c o b ConlIOl 1.12 a 0.92 a 0.60 a • Healthy 2-1eaf stage seed1ings of Echinoch/oa specics were separately immersed inte vials containing 30 ml ofthe cell-free fùtratcs from 2-wk-o~d cultures on modified Frics medium. Uninoculated modified Frlcs medium served as the conlIOl Data from two triais were pooled because the variances were homogenous. Data represent means of eight replicates. b Living leaf area was measured by using a leaf area meter (1.1-3100 Arca Meler, U-COR. Ine. Lincoln, Nebraska, USA). C In a column, means having a common Ietler are not significanùy different according te DMRT al the 590 level • 36 • Figure 2.1. Disease deveIopment (expressed as the mean percentage of leaf area damage (%LAD» of severa! pathogenic fungi on Echinochloa species subjected to 12 h and 24 h dew periods following inoculation. Seedlings of E. crus-galli. E. colona. and E. glabrescens at the 2-1eaf stage were inoculated with 1 x 10' - 1 x 10· conielia/ml of Exserohilum monoceras. Bipolaris sacchari. Curvularia geniculata. and Dactylaria dimorphospora, respectively. Data from two trials were not pooled because the variances were heterogenous. but trends for the two trials were similar. Data represent four replicates of one triai. The 5% LSD between dew periods, fungi, and weeds are 8.5%, 8.9%, and 8.7%, respectively. • 37 • • 100 1- "'" -- - ~.. .' .. .;:= .. ," ...... " .. ,." ~ 801- • • flA ' • • • •. • • • .• .'• 601- •• • • • • , W .. " .' Exserohilum monoceras Bipo/aris sacchari •••• • • 401- l::·· ...... ••••• • ... .. • ...... ~:::••• • j 20 .:-;: •' • t: ...... ' .• .IM...... ' ...... • ~ " , •:: • • - .. .~ 0 ~ 100 • Echinoch/oa cros-galli ~ • Echinoch/oa CO/OIIlJ j5 80 ... Echinoch/oa g/abrescens -- 24-hdew 60 ••••• 12-h dew 40 CI/rvl//aria genicl//ata Dacty/aria dimorphospora 20 ...... • ...... •...... ::1:::: ...... -A :::::..•...... ----:::: & ,: : ••• •• .1.. •• • ••• -Â ••••••• "'" •••••• .... ;:; .... o 2 4 6 8 10 o 2 4 6 8 10 Days after inoculation • Figure 2.2. Disease development (expressed as the standardized area under disease progress curve (SAUDPC) of severa! pathogenic fungi on Echinochloa spccies subjected to 12 h and 24 h dew periods following inoculation. Data represent means of four replicates. Note DD = Dactylaria dimorphospora. CG =Curvularia geniculata. BS = Bipolaris sacchari. and EM = Exserohilum monoceras• • 38 • 100 EchinochJoa crus-ga/li 80 c::J 12-h dew 60 ~ 24-hdew 40 20 100 EchinochJoa colona 100 Echinochloa g/obrescens 80 60 40 20 • DD CG BS EM Fungal species • Figure 2.3. Mortality ofEchinoch/oa species at different leafstages caused by pathogenic fungi for either 12 h or 24 h dew period. Seedlings of E. crus-gal/i. E. corona. and E. 6 glabrescens at the 2-1eaf stage were inoculated with 1 x lOs - 1 X 10 conidialml of Exserohi/um monoceras, Bipo/aris sacchari, Curvularia geniculata, and Dacty/aria dimorphospora, respectively. Datafrom two trials were not pooled because variances were heterogenous, but trends for the two trials were similar. Data represent four replicates of one trial. The S% LSD between dew periods, fungi, weed species, and leaf stages are 16.9%, 16.9%, 17.0% and 17.6%, respectively• • 39 • • Exserohi/um monoeeras Bipolaris saeellori • 100 80 60 40 20 ~ ..... 0 .~ ~ 1 Curvularia genieulata Daetylaria dimorpllOspora ~ 100 80 ~ J-Ienf llIllll!IlI!llJ 2-lenf 60 ~ 3·lenf _ 4·lenf 40 EC Ecll;lIoehloa ems-galli CO Eellilloehloa eolOll(} 20 GB Eehilloellloa glabreseells 0 EC CO GB EC CO GB EC CO GB EC CO GB 12-h dew 24-hdew J2-h dew 24-h dew • Connecting Text Findings presented in the previous chapter demonstrate that Exserohilum monoceras is the m!lst promising candidate for the control of Echinochloa species in rice. Thus, this fungal pathogen was selected for further study. During the development phase of a bioherbicide, it is .1ecessary to determine various aspects of the potential candidate including optimal conditions and limitation for disease development and host damage, development of mass production and formulation technology, delimitation of host range, and quantification of efficacy. In this chapter, the efficacy of E. mOlloceras to suppress various weedy Echinochloa species is quantified and the performance of severa! E. monoceras formulations is evaluated. • 40 Chapl.er 3. Efficacy of Exserohilum monoceras • for the control of Echinochloa species in rice (OryzJl saliva) 3.1. Abstra<:t Efficacy of an indigenous fungus, Exserohi/um monoceras, for me control of ÙIrCC Echinoch/oa species was evaluated and compared under bom regulated greenhouse and screenhouse conditions. Under greenhouse conditions, an inoculum dose of 2.5 x 107 conidialm2 killed ail seedlings of bom E. crus-ga//i and E. g/abrescens, whereas an inoculum dose of 5.0 x 107 conidialm2 was required to obtain 100% mortality of E. c%na seedIings. The 1.5-leaf stage of all ÙIrCC Echinoch/oa species was me most susceptible groWÙI stage. At lower inoculum doses, or wim younger or older plants, weed control efficacy was lower. Increasing inoculum density increased disease intensity of younger or older Echinoch/oa seedlings. The highest level ofcontrol was observed for E. giabrescens, followed by E. crus-ga//i and me lowest level of control was found for E. c%na. Under screenhouse conditions and in me absence of an artificial dew period, E. monoceras effectively controlled Echinoch/oa species when me inoculum was formulated as an oil emulsion or when applied as a dry powder. 3.2. Introduction Echinoch/oa species are me most important weeds occurring in rice (Oryza sativa L.) (Holm et al., 1977; Michael, 1983; Smim, 1983). E. crus-ga//i (L.) Beauv and E. colona (L.) Link are considered as two of me four most troublesorne paddy weeds worldwide (Holm et al, 1977; Moody, 1991) while E. g/abrescens Munro ex Hook is an important weed in tropical regions (De Dana. 1981). Almough various management strategies are available for Echinoch/oa suppression in rice (e.g. cultural measures, hand weeding, mechanical control, and chemical herbicides) (Matsunaka, 1983), each of mese strategies bas important limitations, especially in tropical regions. Following sorne recent successes in using plant pamogens as biological agents to control weeds (Charudattan, • 1991; TeBeest et al, 1992), me possibility of using an inundative approach (Le., 41 bioherbicide) to control Echinoch/oa species in rice-based cropping systems was • investigated at the International Rice Research Institute (lRRI). Los Banos. Laguna. Philippines (Watson, 1994; Zhang et al.• 1996). Six different fungal species: Bipo/aris sacchari (E.I. Butler) Shoem., Curvu/aria genicu/ata (Tracy & Earle) Boedijn, C. /unata var. aeria (Wakker) Boed., Dacty/aria dimorphospora Veenbas-Rijks.• Exserohi/um monoceras (Drechsler) Leonard & Suggs, and Exserohi/um oryzae Sivan., were isolated from diseased Echinoch/oa species in the Philippines (Zhang et al., 1996). Of these. E. monoceras was selected for further study as a biocontrol agent of weedy Echinoch/oa species in rice because it was the most virulent fungal species, non-pathogenic to rice, and had the shortest free moisture requirement. The objectives of this study were (1) to quantify the efficacy ofE. monoceras to suppress weedy Echinoch/oa specie.~ at different inoculum densities and plant growth stages under regulated environment conditions and (2) to evaillate the performance of various formulations of E. monoceras under screcnhouse conditions in the absence of artificial dew. 3.3. MateriaJs and methods 3.3.1.1nocu/um production A single-conidium isolate of Exserohi/um monoceras growing on half-strength potato dextrose agar (1/2 PDA) (Difco, Detroit, MI) slants in small vials was maintained under minerai oil at 4 C as the stock culture (Tuite, 1969). Small pieces of mycelium from the stock culture Wci;: aseptically transferred to PDA in petri dishes. Each culture was sealed with parafilm and incubated at 28 C for 7 days, Agar plugs (6 mm diameter) containing mycelia were removed from the margins of these young colonies and were used te inoculate lima bean agar (LBA) plates. The LBA plates were prepared by grinding 15 g of dry lima beans te a very fine powder, cooking in 1 L of water for 45 min. and adding 10 g of agar. Inoc.:1ated LBA culture plates were sealed with parafilm and incubated at 28 C in the dark for 3 wk. Conidia were harvested from the culture plates by flooding With 10 mi distilled water and scraping the surface of the colonies with a • glass slide. The resulting suspension was filtered through a layer of cheesecloth, the 42 inoculum concentration determined with the aid of haemocytometer and adjusted to the • desired density by adding water. 332. Plant production A singl, batch ofseeds of cach ofthe three Echinochloa species, Le. E. erus-galli, E. colona, and E. glabrescens, collected from natural Echinochloa populations on the IRRI fann, was used in aIl experiments. Seeds of each species were incubated in petri dishes on moist filter paper at room temperature for 48 h. Five germinated seeds (coleoptile and radicle visible) were planted in 10-cm diameter plastic pots filled with saturated soil (Maahas clay, Haplustic suborner). Seeded pots were placed on a push-cart in the greenhouse and 3-cm water lev~1 was maintained in the push-cart throughout the testing period. Greenhouse conditions wer~ 35123 ± 5 C day/night temperature, a 12-h photoperiod, and an average light intensity of 20 MJ/m2 per day. 333. General inoculation procedure Seed\ings were inoculated with a conidial suspension containing 0.05% Tween 20 as Il wetting agent, using a motorized sprayer (A. H. Thomas Co. Scientific Apparatus, Philadelphia) at 100 kPa. Immediately aiter spraying, aIl pots were placed in a dark dew chamber at 100% relative humidity and 25 C for 24 h. Subsequently, pots were transferred to a corner of greenhouse having a temperature of 24-28 C with 85-95% relative humidity (Yeh & Bonman, 1986). 33.4. Assessment ofejficacy Morta1ity ofplants and dry weight of living above-ground biomass were assessed 10 days lifter inoculation (DAI). Morta1ity was evaluated for cach plant, results pooled, and averaged for each pot. Complete1y collapsed seedlings were considered dead. Dry weight was obtained by cutting aerial parts at soil leve\, drying in paper bags for 4 to 5 days at 60 C, and weighing. Dead tissue was not included in dry weight measurements. • The dry weight data were expressed as % reduction in biomass compared with biomass 43 • of non-inoculated controls. 335. Effect ofinoculum density S~gs ofE. crus-galli. E. colooo. and E. glabrescens at the 1.5-leaf stage were inoculated with O. 0.16. 0.31. 0.63. 1.25.2.50. 5.00. and 10.00 x 107 conielialm'. placed in a dew chamber for 24 h. and subsequently transferred to the greenhouse for 10 days. Mortality and dry weight of aboveground bicmass were then deternùned. The doses causing 50% and 90% reduetion in dry weight (ED,. and ED~ of the three Echinochloa species were calculated using dose-response curves with the POLQ-PC software progrnm (Robertson et al.• 1980). 33.6. Effect ofplant growth stage Seedlings ofE. crus-ga/li. E. colooo. and E. glabrescens at the 0.5-. 1.0-. 1.5-. 2.0. and 3.0-1eaf stages were inoculated with 5.0 x 107 conielialm'. placet! in a dew chamber. and subsequently transferred to the greenhouse for la days. Mortaiity and dry weight of abovegrcund biomass were deternùned. 33.7. Interaction between inoculum density and plant growth stage Seedlings ofE. crus-galli, E. colooo, and E. glabrescens at cach of0.5-, 1.0-, 1.5-, 2.0-, 3.0-1eaf stages were inoculated at inoculum densities of 0, 1.25, 2.50, 5.00. and 10.00 x 107 conielialm', placed in a dew chamber, and subsequently transferred to the greenhouse for la days. Mortality and dry weight n:easurements were then obtained. 33.8. Effect offormulalion Conidia of E. monoceras were formulated either as an oil emulsion or as a dry powder. The oil emulsion consisted of a 10% oil phase and a 90% water phase. The oil phase was composed of corn oil with 0.5% (w/v) soybean lecithin, homogenïzed at high speed in a blender for 1 minute. The water phase contained the conielia suspension, 1% • (w/v) dextrose, and 0.2% (w/v) carboxymethyl ccllulose (Couch & Ignoffo, 1981). Prior 44 lO inoculation. the two phases were nüxed and blended at high speed in a biender for 45 • seconds. The dry powder fonnulation was prepared by grinding dry Echinochloa foliage and sieving through a 4O-mesh sereen. Freshly harvested conidial suspensions were centtifuged at 3,000 rpm for 20 min. The supematant was discarded. The conidial pellet was added lO the dry leaf powder (1:3 (v/v» and nüxed at a high speed in a blender for 45 seconds. Pre-germinated seeds of the three Echinochloa species as well as rice (cultivar IR 72) were sown inlO bath 35 x 25 cm trays and 2~m diameter pots at a rate of 15 kg/ha and ISO kg/ha for Echinochloa and ricc, respectively. After seeding, the trays and pots were placed in the sèreenhouse. Seedlings in trays were treated with the oil emulsion fonnulation, whereas seedlings in pots were treated with the dry powder fonnulation. The fonnulations were applied in treatment pairs, aloce or with conielia. Seedlings within additional trays and pots were only treated with distilled water and served as contrais. Prior lO treatment, a 2-cm water levcl was established in bath trays and pots. The oil emulsion fonnulation was applied using a motorized sprayer and the dry powder fonnulation was applied by hand to the water surface where it floated. Seedlings were 2 treated at the l.5-leaf stage at an inoculurn density of 5 x 10' conidia/m • Mortality and dry weight were detennined 14 DAI for the oil emulsion application and 20 DAI for the dry p"wder application. 33.9. Data analyses AlI experiments were perfonned twice. A randomized complete black design with four replicates was used for all experiments. AlI percentage data were arc sine transfonned prior lO analysis (Gomez & Gomez, 1984). Faetorial experiments were analyzed with a factorial analysis of Vatiance considering the effect of each factor individual1y and their interaction. Regr>:ssion analysis was perfonned on all significant (P S 0.05) dependent variables. Results for the two trials ofeach experiment were pooled ifhomogeneity ofvariances was confinned using Bart1ett's test (Gomez & Gomez, 1984)• • However, for experiments in which the variance of trials were not homogenous, results 45 from one trial were presented given that a similar trend was observed between them. • Mean values of five plants for cach treatment were used for statistical analyses and treattnent means were separated using Duncan's Multiple Range Tests (DMRT) at the 5% level of significance. 3.4. Results 3.4.1. Effect ofinoculum density Increasing inoculum density significantly (P < 0.0001) increased mortality and dry weight reduction of EchinochLoa species (Figure 3.1). No significant differences in mortality and dry weight reduction were observed between E. crus-galli and E. glabrescens atcach inoculum density, but mortaIity and dry weight reduction of E. coLona were significantly (P < 0.004) lower than those of E. crus-galli and E. gLabrescens at 7 2 densities below 2.50 x 10 conidia/m • For E. crus-ga/li and E. gLabrescens, inoculum densities below 0.63 x 107 conidia/m2 did not cause any mortality, 1.25 x 107 conidia/m2 resulted in 31-36% mortality, and 2.50 x 107 conidia/m2 or above caused 100% mortaIity in bath species. For E. coLona, inoculum densities below 1.25 x 107 conidia/m2 did not cause any mortality, 2.50 x 107 conidia/m2 resulted in only 24% mortality, while inoculum densities of 5.00 x 107 conidialm2 or above caused 100% mortality of E. coLona seedIings (Figure 3.1A, B, C). Similar trends in dry weight reduction were observed among the three Echinochloa species (Figure 3.10, E, F). Even though no mortality was observed with inoculum 7 2 densities below 0.63 x 10 conidia/m , E. crus-galli and E. gLabrescens dry weights were reduced by 23-47%. Application of conidia! suspensions at a density of 1.25 x 107 conidialm2 resulted in 64-71% reduction in E. crus-galli and E. gLabrescens dry weight. For E. colona, inoculum densities below 0.63 x 107 conidialnr'reduced dry weight by 18 25%, whereas the 1.25 x 107 conidialm2 inoculation treatment resuIted in a 43% reduction "in dry weight despite the absence ofmortality. Although only 24% seedIing mortality was • observed for the 2.50 x 107 conidia/m2 density treatment, plant weight was reduced by 46 65%. 7 2 7 The ED,. for E. crus-galli (0.57 x 10 conidialm ) and E. glabrescens (0.55 x 10 • 2 7 2 conidialm ) were very similar, whereas the ED,. for E. colona (0.89 x 10 conidialm ) was almost 1.5 times greater than for E. crus-galli and E. glabrescens (Table 3.1). Simi1arly, 7 conidialm~ the ED90 Jor E. colona (4.12 x 10 was also greater than the ED90 for E. crus 7 7 galli (2.63 x 10 conidialm~ and E. glabrescens (256 x 10 ) (Table 3.1). 3.4.2. Effect ofplant growth stage The response of E. crus-galli, E. colona, and E. glabrescens seedlings to E. monoceras inoculation varied with plant growth stage (Figure 3.2). E. crus-ga/li and E. glabrescens mortality and dry weight reductions across the different growth stages showed a quadratic respcnse (Figure 3.2A, C. D, F). In contrast, E. colona mortality and dry weight reductions across the different plant growth stages were best described by a cubic response (Figure 3.2B, E). Mortality and dry weight reductions were significantly different among the three Echinochloa species (P < 0.001 and P < 0.003), however, there were no significant interactions between weed species and plant growth stage. For a11 three Echinochloa species, seedlings at the 15-leaf stage were completely controlled, but younger or older seedlings were not ail killed (Figure 3.2A, B, C). For example, 80% and 95% of the E. crus-ga/li and E. glabrescens seedlings at the 0.5-leaf stage were killed, while less than 50% of E. colona seedlings were killed. Seedlïngs of ail three Echinochloa species suffered over 90% mortality when inoculated at the l.o-leaf stage. The lowest mortality levels were observed for seedlings sprayed at the 3.0-1eaf stage. Even though not ail seedlings at the 0.5-, 1.0-, and 2.o-leaf stage were killed, over 80 % dry weight reductions were observed (Figure 3.20, E, F). 3.4.3.1nleraction between inoculum density and plant growth stage AU growth stages ofthe three EchinochIoa species responded ta inoculum density • (Figure 3.3; Table 3.2). For each inoculum density, the highest leve1 of control was 47 observed for seedlings at me 1.5-leaf stage of aH three Echinochloa species. whereas me • lowest leveI of control was observed for seedlings at me 3-leaf stage. For E. crus-gal/i. over 80% mortality was obtained wim inoculum densit:es greater man 5.0 x 10' conidialm2 at growm stages of 1. 1.5. and 2 leaves (Figure 3.3A). E. colona displayed similar mortality levels at a density of 10.0 x 10' conidialm2 (Figure 3.3B). Application of inoculum at densities greater man 2.5 x 10' conidialm2 caused over 80% mortl1ity of E. glabrescens seedlings at me 0.5. 1. 1.5. and 2 leaf stage (Figure 3.3C). Dry weight reduction increased much less drarnatically wim increasing inoculum density man did mortality (Figure 3.30, E, F; Table 3.2). For example, dry weight reduction for ail three Echinochloa seedlings at me 1-, 1.5-, and 2-leaf stages ranged between 80-100% for ail inoculum density treatment levels used. The linear regression equations adequately described the effects of inoculum density on mortality and dry weight reduction of plants at different growth stages (fable 3.2). 3.4.4. Effect offormulation E. monoceras formulated in an oil emulsion resulted in 93%, 95%, and 92% mortality of E. crus-gal/i, E. colona, and E. glabrescens seedlings, respectively, and dry weights were reduced by 92.4%, 95.7%, and 95.4%, respectively (Table 3.3). Almough me leaf tips ofme three Echinochloa species and ofrice which were inoculated wim me oil ernulsion aJone became slight1" chlorotic, no mortality ordry weight reductions in rice were observed (Table 3.3). Application of me fungal spores using a dry powder formulation resulted in 90%, 95%, and 92% mortality ofE. crus-ga/li, E. colona, and E. glabrescens, respectively, and dry weights were reduced by 92%, 87%, and 94%, respectively (Table 3.4). The dry powder formulation itself was not phytotoxic to me treated plants. 3.5. Discussion Law initial inoculum level contributes to me failure of disease epidernics to • develop and persist in weed populations (Ho1comb, 1982; Watson & Wymore, 1990). In 48 this study, increasing the inoculum density of E. monoceras increased control of • Echinochloa species, demonstrating that this potentia! bioherbicide is capable of causing artificial epidemics through manipulation of initia! inoculum dose. Under regulated greenhouse conditions, complete kill of E. crus-ga//i, E. c%na, and E. g/abrescens was 7 7 7 2 achieved using an inoculum density of 2.50 x 10 ,5.00 X 10 , and 2.50 x 10 conidialm , respectively. Since seedlings of all three Echinoch/oa species were sprayed using the same inoculum densities, various responses among these Echinoch/oa species may he due either to different host defense reactions or host morphology. Results from this study demonstrated no significant differences in mortality and dry weight reductions among the three Echinochloa species when the conidia of E. monoceras were formulated as an oil foliar application or when applied to the water surface as a dry powder. This suggests that differences in host morphology such as plant size and leaf shape, instead of differences in host defense reaction, may he largely responsible for observed differences in susceptibility among the three Echinoch/oa species. A determination of the growth stages at which the host is susceptible to disease deve10pment is an ûnportant prerequisite for any potential bioherbicide candidate (Holcomb, 1982; Watson & Wymore, 1990). The 1.5-leaf stage of all three Echinochloa spccies was the most susceptible growth stage to E. monoceras, whereas younger or older plants were less susceptible. This finding is similar to that obtained for the bioherbicide Co/Jetotrichum g/oeosporioides (penz) Sacco f.sp. ma/vae used to control ofround-leaved mallow (Ma/va pusi//a Smith) and velvetleaf (Abuti/on theophrasti Medic.) (Makowski, 1993), but contrasts with reports of other potential bioherbicides in which younger seedlings were shown to have the greatest susceptibility to disease (TeBeest et al, 1978; Boyette & Walker, 1985; Charudattan, 1989). The decreased susceptibility of 0.5- or 1 leafstage seedlings to E. monoceras may he due to Echinochloa seedlings outgrowing the disease or to the 1imited leaf area that could he covered with inoculum. E. crus-ga/li usually reaches its emergence peak at 7-10 days after the soil is • level1ed in wet-seeded rice and 10 days after rice has been transplanted in transplanted 49 rice (Jiang, 1989; Jiang et al., 1990). Seedlings at this time are usually at the 1.0- or 2.0 • leaf stages and are, therefore, at the most suitable stage for control (Jiang, 1989). Therefore, the application window for E. monoceras to control Echinochloa species in rice is similar 10 that of conventional postemergence chemical herbicides and is valid for practical use. Since increasing inoculum density increased disease severity of younger or older Echinochloa seedlings, it is likely that increasing E. monoceras inoculum density may increase the application window for effective control of Echillochloa species in rice based cropping systems. In a small field trial, a 50% dry weight reduction of Echillochloa species was obtained with a foliar application of E. mOlloceras conidial suspension containing the wetting agent, Tween 20 (data not shown). The requirement for an extended dew period may he largely responsible for the low mortality of Echinochloa species in the fust field trial. This faet could limit its potential usefulness in a biocontrol strategy as has occurred with many fungal-weed mycoherbicide efforts (Watson & Wymore, 1990; Charudattan, 1991; TeBeest, 1991). However, when E. mOlloceras conidia were formulated as an oil emulsion or a dry powder, excellent control was obtained without any supplemental dew period. The invert emuIsion has successfully been used to overcome the need for a dew period in sorne mycoherbicides (Quirnby et al., 1989; Daigle et al., 1990; Connick et al., 1991), but this system requires special air-assisted application equipment. A more sirnplified system, a low-oil-content suspension emulsion made from vegetable oils. also has the potential 10 reduce dependence on dew for a bioherbicide (Auld, 1993). In this study, the low-oil-content suspension emulsion made from corn oil emulsified by soybean lecithin plus carboxymethylcellulose and dextrose largely overcame the dew requirement of E. 17IDnoceras by effective1y controlling Echinochloa species without adversely affecting the activity of E. 17IDnoceras. However, this formulation also produced phytotoxic symptoms in all three Echinochloa species and rice, which were reduced by decIeasing the oil content. Surfactants or adjuvants in a formulation may result in • membrane solubilization, interaCtion with proteins. and alteration of epicuticular wax 50 morphology which may aid the bioagent in the penetration of the host or protect the • organism during early growth and development on the host (Falk et al.• 1994). Hence. the oil emulsion used in this study may have aided E. menoceras te penetrate Echinochloa seedlings. or may have enhanced the activity ofphytotoxins produced during the infection process. When E. menoceras was applied in the oil emulsion formulation to rice. it did not infect rice. even though the oil emulsion was slightly phytotoxic causing slight chlorosis on sorne leaves. The biocontrol of Echinochloa species in iIrigated rice-based systems. or even in rainfed systems might take advantage of the abundant water in the field to completely bypass the dew requirement throu:;h the use of the dry powder formulation which floats conidia on the water surface. Inoculum delivery systems are critical to the success of microbial biocontrol agents (Baker & Henis. 1990). and thus this dry powder formulation may be an efficient method for the delivery ofconidia te target weeds. E. menoceras aise infects culms ofEchinochloa seedlings and thus high levels ofcontrol should be achieved when using the dry powder formulation of conidia which floats on the water surface. This floatation method has becn used in the formulation of chemical herbicides in Japan and m'lY be useful in the bioherbicide approach as an easy and reliable way te deliver microbial biocontrol agents te target weeds in paddy rice. Further research is necessary te develop a basic formulation that maintains conidia viability and vinJlence during the production process as weIl as developing a produet formulation which preserves or enhances these properties before being released for commercial use. The data presented here, however. demonstrate that E. monoceras can be easiIy manipulated te provide effective control of the three Echinochloa species. 3.6. Literature cited Auld, B.R. 1993. Vegetable oil suspension emnIsions reduce dew dependence of a mycoherbicide. Crop Protection 12:477-479. • 51 Baker, C.A. and Henis. J.M.S. 1990. Commercial production and fonnulation ofmicrobial • bioconttol agents. Pages 333-344 in Baker, R.R. & Dunn. P.E. (eds) New Directions in Biological Control: Alternatives for Suppressing Agricultural Pests and Disea.o;es. Alan R. Liss Inc.. Boyette. e.D. and Walker, H.L. 1985. Factors influencing bioconttol of velvetleaf (Aburilon theophrasn) and prickly sida (Sida spinosa) with Fusarium lateritium. Wecd Sei. 33:209-211. Charudalf1>~., R. 1989. AssessmentofeffiC3cy ofmycoherbicide candidates. Pages455-464 in Delfosse, E.S. (cd) Proc. 7th Int. Symp. Biol. Control Weeds. Rome, ltaly. Charudattan, R. 1991. The mycoherbieide approach with plant pathogens. Pages 24-57 in Tebeest, 0.0. (cd) Microbial Control of Weeds. Chapman & Hall, New York. Connick, W.J. Jr., Daigle, DJ. and Quimby, P.e. Jr. 1991. An improvcd ir.ve:t emulsion wilh high water ICtention for mycoherbicide delivery. Weed Technol. 5:442-444. Couch, T.L. and Ignoffo, C.M. 1981. Fonnulation of insect pathogens. Pages 621-634 in Burges, H.D. (cd) Microbial Control of Pests and Plant Diseases. 1970-1980. Academic Press Inc. Ltd., London. Daigie, DJ. and Cony, P.J. 1990. lnver! emulsions: carrier and water source for the mycoherbicide, Alternaria cassiae. Wecd Technol. 4:327-331. De Datta, S.K. 1981. Prineiples and Practices of Riec Production. John Wiley & Sons. New York. • 52 Falk, R.H., Guggenheim, R. and SchuIke, G. 1994. Surfactant-induced phytotoxicity. • Weed Technol. 8:519-525. Gomez, K.A. and Gomez, A.A. 1984. Stalistical Procedures for Agricultura1 Research. 200 Edition. John WiJey & Sons, loc.. New York. Holcomb, G.E. 1982. Constraints on disease development Pages 61-71 in Charudattan, R. and WaIker, H.L (eds) Biological Control ofWeeds with Plant Pathogens. WiJey, New York. Hoim, LG., Plucknett, D.L, Pancho, J.V. and Herberger, J.P. 1977. The World's Worst Weeds. Distribution and Biology. The University Press of Hawaii, Honolulu, Hawaii, USA. Jiang, R. 1989. Field weeds chemical control series and systematic management Pages 731-739 in 12th Asian-Pacific Weed Science Society Conference. Soul, South Korea. Jiang, R., Sun, Y. and Zhang, W. 1990. Weed occurrence pattern and control in direct seeded riec. J. of Jiangsu Agricu1tura1 Science. 4:24-30. Matsunaka, S. 1983. Evolution of rice weed control practices and research: worid perspective. Pages 5-17 in Weed Control in Rice. International Rice Research Jnsliiute, P.O. Box 933, 1099 Manila, Philippines. Michael, P.W. 1983. Taxonomy and distribution of Echinochloa species with special reference to their occurrence as weeds of riec. Pages 291-306 in Weed Control in Rice. International Rice Research Institute. PO. Box 933, 1099 Manila, Philippines. • Moody, K. 1991. Weed control in upland rice with emphasis on grassy weeds. Pages 164 • 178 in Baker, F.W.G. and Terry, P.J. (cds) Tropical Grassy Weeds. CAB International for CASAFA. Wallingford, UK. Quimby, P.C., Jr., Fulgham, F.E., Boyette, C.D. and Connick, WJ. Jr. 1989. An invert emulsion replaces dew in biocontrol of sicklepod -A preliminary study. Pages 264-270 in Hovde, D.A. and Beestrnan, G.B. (cds) Pesticide Formulations and Application Systems. vol. 8, ASTMSTP 980. American Society for Testing Materials, Philadelphia, PA. Robertson J.L., Russell, R.M. and Savin, N.E. 1980. POLO: A user's guide to probit or logit analysis. Gen. Tech. Rep. PSW-38. USDA, Forest Service, Pacific Southwest Forest and Range Experirnent Station. Ber'-eley, CA. Smith, RJ., Jr. 1983. Weeds of major economic importance in rice and yield losses due to weed competition. Pages 19-36 in Weed Control in Riec. International Rice Research Institute, P.O. Box 933, 1099 Manila, Philippines. TeBeest, 0.0. 1991. Ecology and epidemiology of fungal plant pathogens studied as biological control agents ofweeds. Pages 97-114 in TeBeest, D.O. (ed) Microbial Control of Weeds. Chapman & Hall, New York. TeBeest, 0.0., Templeton, G.E. and Smith,RJ., Jr. 1978. Temperature and moisture tequirements for development of anthracnose on northem jointveteh. Phytopathology 68:389-383. TeBeest, 0.0., Yang, X.B. and Cisar, c.R. 1992. The status of biological control of • weeds with fungal pathogens. Annu. Rev. Phytopathol. 30:637-657• 54 Tuile, J. 1969. Plant Pathologieal Methods: Fungi and Baeteria. Burgess Publishing Co., • Minneapolis, MN. Watson, A.K. 1994. Curr....nt status of bioherbicide developme:lt and prospects for riee in Asia. Pages 195-201 in Shibayama, H., Kiritani, K. and Bay-Peterson, J. (cds) Inlegrated Management of Paddy and Aquatie Weeds in Asia. FFTC Book Series No. 45. Food and Fertilizer Technology Centre for Asian and Pacifie Regions, Taipei. Watson, A.K. and Wymore, LA 1990. Identifying limiting factors in the biocontrol of weeds. Pages 305-316 in Baker, R.R. and Dunn, P.E. (cds) New Directions in Biological Control: Alternatives for Suppressing Agrieultural Pests and Diseases. Alan R. Liss. Ine. New York. Yeh, W.H. and Borunan, J.M. 1986. Assessment of potential resistance to Pyriculan'a oryzae in six riee cultivars. Plant Pathology 35:319-323. Zhang, Wenming, Moody, K. and Watson A.K. 1996. Responses of Echinochloa speeies and riee (Oryza sativa L.) te indigenous pathogenie fungi. Plant Dis. (Submitted). • 55 Table 3.1. Exserohilum menaceras inoculum doses causing 50% and 90% dry weight • reduetion of three Echinochloa spccies". Echinachloa ED~ ED~ Species (x 107 conidia/m~ E. crus-ga/Ii 0.57 2.63 E. colona 0.89 4.12 E. glabrescens 0.55 2.56 " Seedlings of E. crus-ga/Ii, E. colona, and E. glabrescens at the 1.5-1eaf stage were inoculated with 0, 0.16, 0.31, 0.63, 1.25,2.50, 5.00, and 10.00 x 107 conidia/m', placed in the dew chamba for 24 h, and subsequenùy transferr.:d to the greenhouse mist room for 10 days. Data from two trials were pooled because variances were homogenous. The doses causing 50% and 90% dry weight reduction (ED~ and EDgo) ofEchinochloa species were calculated using dose-response curves with the POLo-PC software prog:arn (Robertson et al., 1980). • S6 • • Table 3.2. Estimated regression pararneters and associated statistics for the regression of arc sine-transformed mean percenl mortallty and dry weight reduction of Echinochloa crus-galll, E. colona, and E. glabrescells al various growth stages as a funclion of the log inoculum density 10 days aCter inoculation. MortaIity % Reduction in dry weight Echlnochloa F Estimate (SE)' F Estimate (SE) specles (P value) r bo bl (P value) r bo bl E. crus-galll 0.5-leaf" 0.0177 0.85 -4.04 (1.42) 0.64 (0.19) 0.0074 0.99 -1.44 (0.22) 0.34 (0.03) I.O-leaf 0.0152 0.90 -4.97 (1.48) 0.82 (0.20) 0.0173 0.86 -I.l6 (0.74) 0.34 (0.12) 1.5-leaf 0.0225 0.80 -0.89 (0.37) 0.31 (0.18) 0.0225 0.80 0.64 (0.51) 0.12 (0.07) 2.0-leaf 0.0163 0.97 -5.09 (0.79) 0.81 (0.11) 0.0496 0.90 -0.56 (0.45) 0.26 (0.06) !:3 3.0-leaf 0.0305 0.94 -8.77 (1.65) 1.23 (0.22) 0.0457 0.91 -2.73 (0.85) 0.51 (0.1 1) E. co/ana 0.5-leaf 0.0222 0.96 -6.85 (I.l2) 0.98 (0.15) 0.0488 0.90 -1.70 (0.63) 0.37 (0.08) 1.0-leaf 0.0135 0.97 -8.21 (1.09) 1.23 (0.14) 0.0488 0.86 -3.11 (1.25) 0.59 (0.17) 1.5-leaf 0.0455 0.90 -6.58 (1.87) 1.03 (0.25) 0.0381 0.85 -2.93 (1.30) 0.57 (0.17) 2.0-1eaf . 0.0463 0.88 -7.24 (2.08) 1.05 (0.28) 0.0451 0.72 -1.92 (1.40) 0.42 (0.19) 3.0-leaf 0.0453 0.90 -5.92 (1.49) 0.82 (0.20) 0.0437 0.75 -3.45 (1.83) 0.59 (0.24) P.. g/abrescens ., 0.5-1eaf 0.0180 0.85 -3.18 (1.31) 0.57 (0.17) 0.0342 0.84 -0.01 (0.01) 0.16 (0.13) 1.0·1eaf 0.0484 0.75 -3.58 (1.41) 0.65 (0.35) 0.0422 0.86 -1.S5 (0.23) 0.39 (0.30) 1.5-1eaf 0.0425 0.65 -1.45 (1.00) 0.39 (0.22) 0.0425 0.70 -1.36 (0.86) 0.37 (0.22) 2.0·leaf 0.0103 0.98 -8.11 (0.93) 1.21 (0.12) 0.0192 0.82 -1.85 (1.03) 0.42 (0.14) 3.0-leaf 0.0175 0.86 -7.34 (1.37) 1.08 (0.31) 0.0459 0.89 -2.23 (0.85) 0.44 (0.1 1) , SE =standard error of the estimate. " Leaf stages • • Table 3.3, Percent morta1ity (M) and dry weight (DW) of Echinocllloa species and rice seedlings 14 days aEter bcing treated with water, Exserohflum monoceras at a density of a rate of 5 x 107 conidia/m2 in oil emulsion. or with the oil emulsion alone'. Echinochioa Echinochloa Echfl/ochloa Oryza saliva crus-galfi c%lla g/abrescel/s M DW M DW M DW M DW Treatment (%) (g) (%) (g) (%) (g) (%) (g) ls:l Conidia + oil emulsion 93 a" 0,020 b 95 a 0.029 b 95 a 0.011 b oa 0.770 a Oil emulsion Ob 0.253 a Ob 0.227 a Ob 0.191 a oa 0.639 a Water Ob 0.255 a Ob 0.230 a Ob 0.190 a oa 0.780 a , Data presented are from only one of trials since variances were not homogenous, however, similar trends were observed in the two trials. oo Within a column, means having a common letter are not significant!y different at the 5% level of significance according to DMRT. • • Table 3.4. Percent mortallty (M) and dry weight (DW) of Ech/noch/oa specles and rice seedllngs 20 days after being treated \\':;i; water, a dry powder formulation of Exserohllllln monoceras at a rate of 5 x 107 conldia/m', or with a dry powder formulation not contalnlng conidia'. Ech/noch/oa Ech/noch/oa Ech/noclJloa Or)'za sol/l'a crus-galll c%na g/abrescel/s M DW M DW M DW M DW Treatment (%) (g) (%) (g) (%) (g) (%) (g) VI 'D Coniefja + dry leaf powder 90 a" 0.026 b 95 a 0.013 b 92 a 0.014 b Oa 0.750 a dry leaf powder Ob 0.342 a Ob 0.300 a Ob 0.304 a Oa 0.742 a Water Ob 0.339 a Ob 0.305 a Ob 0.310 a Oa 0.755 a , Data presented are from only one of trials since variances were not homogenous, however, similar trends were observed in the two trials. " Withln a column, means having a common leller are not slgnificantly different at the 5% level of significance according to DMRT. • Figure 3.1. Effect of inoculum density (ln the control of Echinoch/oa species by Exserohi/um monoceras, expressed as percer.! mortality and reduction in dry weight 10 days after inoculation. Sœdlïngs ofE. crus-galli, E. c%na, and E. g/abrescens at the 1.5 leaf stage wete inoculated with 0, 0.16 x 10',0.31 X 10',0.63 X 10', 1.25 X 10',2.50 X 2 10', 5.00 X 10', or 10.00 x 10' conidia/m and provided with a 24 h dew period. Observations f."om two trials Wete pooled because variances wete homogenous. Each data point represent the mean of eight replicates. The relationships of mortality and % reduction in dry weight versus log inoculum density are described by logistic equations generated from aetual data. A, B, and C: Percent mortality versus log inoculum density for E. crus-ga/li, E. c%na, and E. g/abrescens, respectively; D, E, and F: Percent dry weight reduction versus log inoculum density for E. crus-ga/li, E. c%na, and E. g/abrescens, respectively• • 60 • • • 1001- y=IOO/(1+e74.QO.lo.Ul)r A y=1 00/(1 +e69.37.9.211) y=100/(1+e7J.8S-lo.Ul) / c 2 .....,~ 80 ~ ~=o.8163 ~=0.7501 ;: r =0.8314 .~ ~ 60 )1 40 20 0 ... • .~"ffiJ 100r ~II ~II / F -S' 80 , .... 1 1 • = 60 1 / • 1 1 / • 0= '3 ::> 40 • • ~ y=100/(1+e29.08-4.391) . / y=;00/(I+e30.9S.Wl) y=100/(1 +e29.42.4.431) ~ 2 20 tJ• ·~=0.8163 • • • r =0.7350 ·1 ~=0.7742 0 6.4 6.8 7.2 7.6 ·8.0 6.4 6.8 7.2 7.6 8.0 6.4 6.8 7.2 7.6 8.0 Log inoculum density • Figure 3.2. Effect of plant growth stage on the control of Echinochloa species by Exserohilzun monoceras, expressed as percent mortality and reduction in dry weight 10 days after inoculation. Seedlings of E. crus-gaili, E. colona, and E. glabrescens at 0.5-, 1.0-, 1.5-, 2.0-, and 3.0-1eaf stages were inoculated with 5.0 x 107 conidialm' and provided with a 24-h dew period. Data from two trials were pooled because variances were homogenous. Data points represent the mean of eight replicates. The relationships of mortality and % reduction in dry weight versus leaf stage are described by polynomial equations from . :tual data. A, B, and C: Percent mortality versus leaf stage for E. c:rus gaili, E. colona, and E. glabrescens, tcSpectively; D, E, and F: Percent dry weighr reduction versus leaf stage for E. crus-gaili, E. colona, and E. glabrescens, respectivdy• • 61 • • ~ 100 t- ~ •• A B - ~ ~ C '-' .~ ~ l :r "'- ~ 40 1 y=52.l+72.2x-27.0x2 \ ~=-135.5~60.5X-277.7X~ y=79.3+27.5x.9.0x2 20 2 1 r2=0.8350 +47.13x • ~=0.8144 r =0.7924 0 ... I 1 ~ 1 1 • '€b~ 100 r- . DI 1 / E .a- 80 '" .S 1': 0 60 'Q .g40 ~' 2 2 3 2 y=64.1 +36.lx-8.7x 1 1Y2=9.9+176.9X-103.1x +16.6x y=89.5+17.2x-9.2x bI! 20 r. ~=0.7380 r =0.8765 1 1 1=0.7020 . 0 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0 Planlleaf stage • Figure 3.3. Effect of Exserohilum moooceras inoculum density and plant growth stage on the control of Echinochloa species, expressed as percent mortality and reduction in dry weight 10 days after inoculation. Seedlings of E. crus-gal/i, E. colona, and E. glabrescens 7 7 at the 0.5-, 1.0-. 1.5-.2.0-. and 3.0-leaf stages were inoculated with 1.25 x 10 • 2.5 X 10 , 7 7 2 5.0 X 10 , and 10.0 x 10 conidia/m , respectively, and provided with a 24-h dew period. Data from two trials were pooled because variances were homogenous. Data represent the mean of eight replicates. A, B, and C: Percent mortality for E. crus-gal/i, E. colona, and E. glabrescens, respectively; D, E, and F: Percent dry weight reduction for E. cl'us-galli, E. colona, and E. glabrescens, respectively. • 62 1\t~\ C,) • _ CCl ,'\. \ ...... ~ . .\ . '\ \ \. ~ - ...... \ \\. \ . \ ..... \'\ \~ ...... , ':'--" .... , '.'-...... , , .., ...... \ ". '.. . ~ Cl .... '\ Q. ~ oci \ ,\ \ \. \ . \\ '. \ . \ \'. '\...... \ ~ ~\ •...... \ . '.' \ . \ \ \. \ \ \ \ \ \ . . \ \ "".\ \'\\ \ \ ' \\ ~ '. ~ ~ , 1\ ". .'\. \ \\ 1 '. '. . \ r 1 \ ..... '\.. \ \ 1 '. '.. ~ \ ~·,.i « ~Jt , .\\ \ l'" .\ l''. '\ - 00r.: . \ l ". Jr.. '..~ .\ '.' \ . . 1 \ i \0 .... \. \ \. . \. - r.: ... \ \ . \ \' '" \ \ . \ \ .... \ \ '. f\ \ i ~ ~ ~. •.-\. '\ \\ \ \ \'\. , : \, \ : ...., \ " \ \ .... '\.. , . ~ \' ~ ~, 1.\ 1 1'. ,\. '., 1 1 , Cl Cl Cl Cl Cl Cl 5? Cl • 8- 00 N -8 00\0""",, • Connecting Text For a pathogen to be successfulIy employed as a bioherbicide. a high Ievel of weed mortality is required. However. the infection. disease development, and degree of weed control caused bv a pathogen are usually suppressed under nanrral conditions. Therefore. a better understanding of the environmental conditions under which a high level of weed control can be achieved is essential. In this chapter. the optimum environmental conditions for disease development and subsequent weed control are determined as weil as the limiting factors which shoulà be overcome or bypassed for the development of E. monoceras as a bioherbicide. • 63 Chapter 4. Effect of dew period and temperature on performance • of Exserohüum monoceras for the control of Echinoclzloa species 4.1. Abstract The effeet of dew pcrioè and temperature on disease development caused by the indigenous fungal pathogen, Exserohi/um mOlloceras. on three Echinochloa species was evaluated under controlled environment conditions. The optimum dew period temperature for disease development was between 20 and 30 C. The minimum dew period duration 10 attain 100% disease severity was 16 h fo~ E. colO/la, 12 h for E. crus-galli, and 8 h for E. glabrescens. Increased dew period duration increased the optimum dew period temperature window and supplying the optimum dew period-temperamre decrea.o:ed the dew period duration requirement. Delaying t.J,e initiation of the dew period by 24 h din not adversely affect disease development ofthis pathogen. Increased disease development occurred for inoculated seedlings treated with sequential dew periods of shorter duration than the optimal period. E. monoceras has potential as a bioherbicide for the control of Echinochloa species in riec. 4.2. Introduction Bamyardgrass, Echinochloa crus-galli (L.) Beauv., is ranked as the world's third worst weed and is important in 36 crops in 61 countries, especially in rice (Holm et al., 1977). Under intense competition from E. crus-galli, tillering in rice is reduced by up 10 50% and height, number and weight of the grains is a1so substantially reduced (Holm et al, 1977; Smith, 1983; Maun & Barrett, 1986). Riec yields were decreased by 18% and 30% when E. crus-ga/Ii was transplanted with riee seedlings at a density of 20 and 40 plantslm~, respectively (Lubigan & Vega, 1971). E. colona (L.) Link. and E. glabrescens Munro ex Hook.F. are two other important grass weed species in rice (Holm et al., 1977; De Dana, 1981; Moody, 1991). Cultural techniques, hand-weeding, mechanical control, and ehernical herbicides • are common strategies for Echinochloa suppression in rice (Matsunaka, 1983), but each 64 of these strategies has important limitations. Utilization of indigenous plant pathogens as • biological agents to control weeds has shown sorne promise (Charudattan. 1991; TeBeest et al.• 1992). and the possibility of using the inundative approach (bioherbicide) to control Echinochloa species in rice is presently being investigated (Watson, 1991; 1994). Recent work has demonstrated that an indigenous fungus. Exserohilum monoceras (Drechsler) Leonard & Suggs, which was isolated from naturally-infected Echinochloa species was able to cause blight-like symptoms on Echinochloa crus-galli. E. colona, and E. glabrescens but was non-pathogenic to rice (Zhang et al., 1996). An inoculum dose of 2.5-5.0 x 107 conidialm2 killed all three Echinochloa seedlings at the I.5-leaf stage in the greenhouse (Zhang & Watson, 1996b). Since infection, disease development, and subsequently weed control efficacy caused by a pathogen are usually suppressed under natura! conditions (Holcomb. 1982; Watson & Wymore, 1990; TeBeest, 1991; TeBeest et al., 1992), it is essential to rletermine the optimal environmental conditions for disease development as well as the limiting environmental factors which should be overcome or bypassed for effective field control of Echinochloa species by the fungus Exserohilum monoceras. Dew period and temperature have been recognized as two important environmental components which contribute to the effects on control efficacy of a bioherbicide (TeBeest, 1991; Yang & TeBeest, 1993). Various studies on the effects of dew period and temperature in many other potential bioherbicide pathosystems have been reported and provided important information on epidemiological charaeteristics with a view to reduce their environmental dependency (TeBeest & Templeton. 1978; Walker. 1981; Walker & Boyene, 1986; McRae & Auld, 1988; Makowski. 1993). Thus. the objectives of this study were to evaluate the effeet of dew period duration. frequency. and timing on infection. disease development, and control efficacy of Echinochloa species by Exserohilum monaceras. In addition. the effects ofdew period temperature as well as post-dew temperature on disease development were also investigated. • 65 4.3. Materials and methods • 43.1./noculum production A single-conidium isolate of ExserolzilunJ mOlloceras growing on half-stn:ngth potato dextrose agar (1/2 PDA) (Difco. Detroit, Ml) slants in small vials was maintained under nùneral oil at 4 C as the stock culture (Tuite, 1969). Small pieces of mycelium from the stock culture were aseptically transferred to PDA in petri dishes. Each culture was sealed with parafilm and incubated at 28 C for 7 days. Agar plugs (6-mm diameter) containing mycelia were collected from the margins of these young colonies and used to inoculate lima bean agar (LBA) plates. The LBA plates were prepared by grinding lS g ofdry lima beans te a very fine powder, cooking in 1 L of boiling water for 4S min, and adding 10 g of agar. Inoculated LBA plates were sealed with parafilm and incubated at 28 C in the dark for 3 wk. Conidia were harvested from plates by flooding with 10 ml distilled water and scraping the surface of the colonies with a glass slide. The resulting suspension was filtered through a layer of cheesecloth. The inoculum concentration was deternùned with the aid ofhaemocytometer and adjusted to the desired density by adding water. 432. Plant production A single batch ofseeds ofeach ofthe three Echinochloa species (i.e. E. crus-galli, E. colona, and E. glabrescens) collected from natura! agricultura! Echinochloa populations at the International Rice Research Institute (IRRl), Los Baiios, Philippines, was used in al! experiments. Seeds of each species were incubated in petri dishes on moistened filler paper atroom tcmperature for 48 h. Five germinated seeds (coleoptile and radicle visible) were planted in llkm diameter plastic pots fllied with saturated sail (Maahas clay, Hap1ustic suborder). Seeded pots were placed on a push-cart in the greenhouse in which a 3-cm water leve1 was maintained throughout the experimental period. Greenhouse conditions were 3S/23 ± SC day/night temperature, a 12-h photoperiod, and an average • light intensity of 20 MJ/m2 per day. 66 433. General inoculation procedure • Each treatrnent consisted of inoculating 4 pots of each of the three Echinochloa species (each pot containing 5 seedlings at the 1.5-leaf stage) with a conidial suspension at a rate of 5.0 x 10' conidia/m' containing 0.05% Tween 20 :!s a wetting agent, using a motorized sprayer at 100 kPa (A. H. Thomas Co. Scientific Apparatus. Philadelphia). Immediately after each treatment, pots were transferred to a corner of greenhouse having a temperature of 24-28 C and an 85-95% relative humidity (Yeh & Bonman. 1986). 43.4. Assessment ofdisease development Disease intensity was assessed as percentage leaf area damage (% LAD) and recorded visually every 2 days for 10 days after inoculation (DAI). Mortality of plants and dry weight ofliving aboveground biomass per pot were assessed 10 DAI. Completely coIlapsed seedlings were considered dead and dead plants were given a 100% LAD score. Dry weight was obtained by cutting aerial pans at soil level. drying in paper bags for 4 to 5 days at 60 C. and weighing. Dead tissue was not included in dry weight measurements. The dry weight data were expressed as % reduction in biomass compared with the biomass of non-inoculated controls. 435. Effect ofdew period temperature After inoculation, pots with inoculated seedlings were placed in a dark dew chamber at 10, 15, 20, 25, 30, 35, or 40 ± 1 C for 24 h and then returned to the greenhouse. 43.6. Effect ofpost-dew temperature After inoculation, pots with inoculated seedlings were placed in a dark dew chamber at 25 ± 1 C for cither 12 h or 24 h. After the dew period treattnent, pots were transferred to growth chambers al temperatUres of 10, 15, 20, 25, 30, 35, or 40 ± 1 C, 2 • with a photoperiod of 12 h and light intensity of 300 J1I1l m' S'1 for 10 days. 67 43.7. Effect ofdew period duration • After inoculation. pots with inocul:lted seedlings were placed in a dark dew chamber at 25 ± 1 C for O. 4. 8. 12. 16. 20. c: ~~ h. Except for the 24 h treatment. pOl~ were covered with cardboard for the remains of the 24 h to simulate the dari.: period in the dew chamber. After the dew period treatment, pots were returned to the greenhouse. 43.8. Interaction berween dew period temperarure and dl/ration After inoculation, pots with inoculated seedlings were placed in a dark dew chamber at 15, 20, 25, 30, or 35 ± 1 C and exposed to dew periods of 8. 12, 16. 20, or 24 h in a 5 x 5 factorial design. Except for the 24 h treatment at each temperature, pots were covered with cardboard for the rernains of the 24 h to simulate the dari.: period in the dew chamber. Following the dew period treatment, pots were returned to the greenhouse. 43.9. Effect ofdelaying initial dew period After inoculation, pots with inoculated seedlings were placed in the greenhouse for 0, 1, 2. 3, or 4 clays before being placed in a dark dew chamber for 4, 8, 12. 16 , or 20 h. The experirnent was arranged in a 5 x 5 factorial design. Except for the 16 h treattnent at each clay, pots were covered with cardboard for the remains of the 20 h to simulate the clark period in the dew chamber. After treatment, pots were retumed to the greenhouse. 43.10. Effect ofsequential dew periods Pots with inoculated seedlings were provided one of the following clark dew periods at 25 ± 1 C: (1) 6 h dew periodl18 h dry period on 1,2,3, or 4 consecutive clays; (2) 12 h dew period/12 h dry period on 1 or 2 consecutive claiS; and (3) a single 24 h dew period. AlI pots were kept in the dark for a similar length of rime by covering pots • with cardboard. After treatrnent, pots were retumed to the greenhouse. 68 43.JI. Data analyses • Ali experiments were perfonned twice. A randomized complete block design with four replicates was used for ail experiments. AlI percentage data were arc sine transfonned prior to anaiysis (Gomez & Gomez, 1984). Factoriai experiments were anaiyzed with a factoriai anaiysis of variance considering the effect of each factor individually and their interaction. Regression analysis was perfonned on ail significant (P S 0.05) dependent variables using PRoe REG (SAS, 1987). The best regressio:l equation was seleeted using the PROe REG step-wise procedure in SAS (Draper & Smith, 1981; Campbell & Madden, 1990). Results for the two triais of each experiment were pooled if homogeneity of variances was confmned by the Bartlen test (Gomez & Gomez, 1984). However, for experiments in which the variance of triais were not homogenous, ~..sults from one trial are presented given that a similar trend was observed between the triais. Mean vaiues of five plants for each treatment were used for statisticaI anaiy!"'-S, treatment means were separated by the Duncan's Multiple Range Tests (DMRT) at the 5% level of significance. 4.4. Results 4.4J. Effect ofdew period temperature Disease progress over time was greatly affected by the dew period temperature (Figure 4.1). For E. crus-gal/l, 100% LAD occurred 2 DAI when the dew period temperature was between 20 and 30 C. The % LAD was significantly reduced (P < 0.00(1) when the dew period temperature was above 30 e or below 20 C. Limited leaf damage was observed at 40 C, however, leaf damage at 35 e was much higher than at 15 e (Figure 4.1A). For E. colona, 100% LAD occurred 6 DAI at dew period tel1lperature5 between 20 and 25 e and 8 DAI when dew period temperature was between 25 and 30 e (Figure 4.1B). For E. glabrescens, the highest % LAD was observed al dew temperature5 between 15 and 35 C. In contrast, at dew period temperatures of40 e or 10 C, disease development was substantially limited (Figure 4.1C)• • . The SAUDPC (standard area under disease progress cu.-ve) provides a generai 69 assessment of the disease process and was used for comparative purposes (Figure 4.2). • In general, the highest and lowest SAUDPC values were observed on E. glabrescellS and E. colona. respectively. The SAUDPC values of E. crus-galli were not different from those of E. glabrescellS at dew period temperatures ranging from 20 to 35 C. but were significantly lower at 10, 15, and 40 C. Except for the 25 C. E. colona SAUDPC values were 10wer than those of E. glabrescellS across the temperature range tested. Moreover. E. colona SAUDPC values were also lower than E. crus-galli values at the 15, 20. 30. and 35 C temperature treatments. Percent mortality data closely panlleled SAUDPC results. That is, all E. crus gaili, E. colona. and E. glabrescellS seedlings were killed at dew period tempc:ratures between 20 and 30 C. However, at dew temperatures below 20 C or above 30 C, mortality declined sharply. For example. at 15 C dew temperature, E. crus-galli. E. colona, and E. glabrescellS mortality was 40%. 10%. and 56%. respectively. Similarly, at the 35 C dew temperature treatment, E. crus-galli, E. colona. and E. glabrescellS mortality was 26%, 13%, and 36%, respectively. No mortality was observed at a dew temperature of 10 C or 40 C for any of the three Echinochloa species. 4.42. Effect ofdew period duration Disease progress over time was also significantly affected by dew period duration and the three Echinochloa species responded differently to this treabnent (Figure 4.3). At 2 DAI, a 100% LAD was observed for E. crus-galli when provided more than 16 h dew and a 100% LAD at 4 DAI when provided a 12 h dew period. Less than 12 h of dew dramatica11y decreased the % LAD for this species (Figure 4.3A). At 6 DAI. a 100% LAD was recorded for E. colona when provided more than 20 h dew. A 100% LAD was also observed at 8 DAI whcn providcd a 16 h dew period. A dew period of less than 16 h sharply decreased the % LAD for E. colona (Figure 4.3B). E. glabrescellS seedlings showed a 100% LAD 2 DAI whcn providcd more than 12 h dew. A 100% LAD was also found al 6 DAT whcn E. glabrescellS secdIings were cxposcd an 8 h dew period. In • contrast, a 4 h dew rcsulted in very low % LAD levels (Figure 4.3C). 70 When provided a 24 Il dew period, no difference '.'las observed in SAUDPC JITIong • the three Echinochloa species (Figure 4.4). SAUDPC values for E. r:Jlona were significantly lower than for E. crus-galli and E. g/abrescens when dr-w period duration was less than 24 h. E. crus-ga//i SAUDPC values were not significantly different from those of E. g/abrescel'.s when a dew period of more than 12 h '.'las provided, but were lower for dew periods of less than 12 h. Mortality increased concomitantly with dew period duration. No plant death '.'las rerorded for any of the three Echinoch/oa species for the 0-4 h dew period treatments. However, more than 8 h, 12 h, and 16 h dew period resulted in 100% mortality of E. g/abrescens, E. crus-gal/i, and E. c%na seedlings, respectively. 4.43, Effect ofpost-dew temperature When provided a 24 h dew period, all seedlings of the three Echinochloa species were killed at ternperatures ranging from 15 C to 40 C. However, when provided a 12 h dew period, mortality of the three Echinoch/oa species varie.::' \Vith different post-dew temperatures and the three Echinoch/oa species responded differently (Figure 4.5). The highest level ofmortality ofthe three Echinoch/oa species was observed at the 30 C post dew ternperature treatment. Among the three Echinoch/oa species, E. g/abrescens appeared ta be 1east susceptible ta the influence of post-dew temperature. 4.4.4. Interaction between dew period temperature and duration Dew period duration and dew period temperature were closely linked (Figures 4.6, 4.7,4.8). Generally, increasing dew period duration widened the dew period temperature window for achieving high levels of mortality. Also, optimum dew ternperature reduced the dew period duration requirement in al] these species. Dew ternperatures below 20 C or above 30 C resulted in decreased plant mortality. With a 12 h dew period, 1ess than 15% of E. colona seedlings died at dew period temperatures of 20 and 25 C and no mortality was observed at dew temperatures of 15 C, 30 C, and 35 C. With a 16 h dew • period, there were significant differences in mortality observed among the temperature 71 treattnents. More man 90% of E. c%lla seedlings died al 20 C. 100% al 25 C. less man • 35% at 30 C. whereas no mortality was observed al 15 C and 35 C. Wim a 20 h dew period, E. c%oo mortality was 100% al 25 C. more man 80% al 20 C. 35% at 30 C. and 10% at 35 C. A 24 h dew period resulled in 100% monality of E. c%lla seedlings al 20 C and 25 C. Few E. c%lla seedlings died at dew lemperalures of 15 C or 35 C. even when prcvided a 24 h dew period (Figure 4.6). For each dew period lemperature and duration combination. higher levels of mortality were observed for E. crus-gal/i and E. g/abrescens seedlings man for E. c%lla (Figure 4.6). Wim a 12 h dew period, 100% mortality of E. crus-gal/i seedlings occurred at 25 C and 100% mortality of E. g/abrescens seedlings occurred at 20 C and 25 C. An 8 h dew period at 25 C also resulted in 100% mortality of E. g/abrescens seedlings. Among the three Echinochloa species.. E. g/abrescens appeared least susceptible to me influence of dew period temperature and duration, followed by E. crus-gal/i. and E. colooo (Figure 4.6). Regression analysis adequately described me effeet of dew period duration on seedling mortality when subjeeted to different dew period temperatures (Figure 4.7). At 10 or 35 C, increasing dew period duration had little impact on mortality of all three Echinochloa species. For E. crus-galli, mere were no significant differences in mortality in response to the various dew period durations for the 20 and 30 C treattnents. while mortality at the 25 C treattnent was significantly greater than for the 20 or 30 C temperature treattnents (Figure 4.7A). For E. c%lla, there were no significant differences in mortality for dew temperatures of 20 and 25 C, whereas the mortality for the 30 C treatment was significantly lower than for me 20 or 25 C treattnents (Figure 4.7B). For E. glabrescens, there were significant differences in mortality for dew temperatures of20, 25, and 30 C. The greatest seedling mortality was observed at 25 C while the lowest mortality occurred at 30 C (Figure 4.7C). Throughout the temperature range tested, E. crus-galli and E. glabrescens showed a sinùlar reIationship between dew period temperature and duration for attaining 100% • mortality, whereas E. colooo showed a different trend al temperatures below 20 C and 72 above 30 C (Figure 4.8). The predicted minimum dew period duration required to produce • 100% mortaIity was !2.4 h. 15.8 h. and 9.9 h for E. crus-gal/i. E. c%na. and E. g/abrescens. respective:y (Figure 4.8). 4.45. Effect ofde/aying initial dew period The gteatest seedling mortality for all three species was obtained following the shortest delay in providing a dew pcriod (Figure 4.9). When the initial dew period was delayed 1 day aft~ inoculation. the 12 h and 16 h dew period treatments still resulted in 95% and 100% mortaIity of E. crus-ga/li seedlings. These mortality rates were not significantly different from those in which the 12 h and 16 h dew period treatments were initiated immediate1y after inoculation. When the initial dew period was delayed by 2 days or longer after inoculation. E. crus-ga/li mortality decreased by more than 60% (Figur:. 4.9A). Sirnilar trends were observed for E. c%na and E. g/abrescens (Figure 4.9B. 4:9C). 4.4.6. Effect ofsequentia/ dew periods A continuous dew period of more than 12 h killed all E. crus-ga//i seedlings. In contrast, two dew periods of 6 h did not result in any plant death. whereas four dew periods of 6 h caused nearly 70% mortality (Table 4.1). The 6 h dew period on two consecutive days, three consecutive days. and four consecutive days increased E. crus ga/li dry weight reduction from 42.6% to 90.0% (Table 4.1). Sirnilar trends were observed for E. co/ana and E. g/abrescens (data not shown). 4.5. Discussion Although the three Echinochloa species responded differently to dew period temperature, the optimum dew period temperature range for achieving 100% mortality for all three Echinochloa species was sirnilar (i.e. between 20 C and 30 C). Conflicting results have been reported in different bioherbicide pathosystems in which a single • pathogen was used ta achieve the control of more than one weed species. Boyette and 73 Walker (1985) reponed that 25 C was the optimum dew period temperature for the • control of both Aburilon rheophrasri Medic. (velvetleaf) and Sida spillosa L.(prickly sida) with Fusarium lareritium Nees ex Fr.. However. the dew period temperature that provided satisfactory control of Mail'a pusilla Smith (round-leaved mallow) by Collerorrichum gloeosporioides (penz.) Sacco f.sp. mall'ae was different than that of A. rheophrasti (Makowski. 1993). Sinülar dew peri('ld tempe.-ature requirements for all three Echillochloa species should favour effective E. monoceras control on all weedy Echillochloa species simultaneously. Mor~over. the optimum dew period temperature range for development of Exserohilum monoceras on Echillochloa species is within the temperature range that OCCUIS during the rice growing season (lRRI. 1993) and is sinülar to that required for conidial germination of Exserohilum monoceras (Zhang & Watson. 1996b). Therefore. dew period temperature is not likely to be a limiting factor for E. mOlloceras as a bioherbicide. The optimum post-dew temperature was 30 C which falls within the optimum dew period and conidial germination temperature ranges. Therefore, temperature in much of Philippines is conducive to disease development of Exserohilum monoceras on Echinochloa species (IRRI, 1993). The effect of post-dew temperature appeared to be related te the degree of initial infection which itself is dependent upon the initial dew period duration. A 24 h initial dew at 25 C in the dark appeared to allow E. monoceras te complete the infection process on Echinochloa species and the degrees of infection on the three Echinochloa species were simiIar. In this case, E. monoceras disease development will not be affected by the post-dew temperature (from 15 to 40 C). However, when the initial dew period was reduced to 12 h, the degree of infection of E. monoceras on the three Echinochloa species differed and funher disease development was influenced by post-dew temperature. It appeared that the degree ofinfection was greatest for E. glabrescens because this weed species was least susceptible te the influence of post-dew temperature. These f'mdings suggest that temperature during the infection process (dew period temperature) had a greater effect on development of Exserohilum • monoceras on Echinochloa species than did post-infection temperatuJe (post-dew 74 temperature). Although no simiJar study on the relationship between dew period • temperature and post-dew temperature was reported in other potential bioherbicide pathosystems, results in this study support findings for crop diseases (Filajdié & Sunon, 1992; Trapero-Casas & Kaiser, 1992). These results also indicate that if a sufficient dew was initiated after inoculation or the dew requirement was overcome or bypassed through artificial inoculation techniques, such as more efficient formulations or application mr:thods (Zhang & Watson, 1996a), post-dew temperature wou1d no longer be a lirniting factor for the control of Echinoch/oa spccies by E. monoceras. The minimum dew period duration to achieve 100% mortality was 12 h for E. crus-gal/i, 16 h for E. c%na, and 8 h for E. g/abrescens. These dew period requirements are similar to those reponed for other bioherbicides or potential bioherbicides including CoUelOtrichum g/oeosporioides f. sp. aeschynomene (TeBeest & Templeton, 1978), 'Col/elOtrichum coccodes (WalIr.) Hughes (Anderson & Walker, 1985), Fusarium /ateritium (Boyette & Walker, 1985), A/rernaria cassiae Jurair & Khan (Walker & Boyette, 1986), or C. g/oeosporioides f.sp. ma/vae (Monensen, 1988; Makowski, 1993). TeBeest (1991) suggested that dew period requirements of 16 hours or longer could lirr.it the practical use of fungi as biological control agents for weeds. Therefore, simiJar to other bioherbicide pathosystems, dew period duration is a limiting factor for the control ofEchinochloa species by Exserohi/um menoceras, especially E. c%na. Fonunate1y, the dew period duration limitation for deve10pment of Exserohilum monoceras on Ecllinochloa species can be overcome or bypassed by formulating inoculum as an oil emulsion or through the application of a dry-po.....Jer formulation which fioats conidia on the water surface (Zhang & Watson, 1996a). The interaction between dew period temperature and duration demonstrated that the dew period requirement was substantially influenced by dew perïod temperature. The curve: of predieted minimal dew dlJration for 100% monality showed a flat region over the temperature range 20-30 C, predicting dew duration of 12.4-15.8 h for E. crus-ga/li, 15.8-21 h for E. colona, and 9.9-12.4 h for E. glabrescens, respectively. However, at dew • period temperatures below 20 C or above 30 C, the dew requirement increased 75 significantly for all three Echinochloa species. The majority of bioherbicide studies put • an emphasis on the application of biocontrol agents at times that favour the onse! of dew formation (Walker & Boyette. 1986; McRae & Auld. 1988; TeBeest. 1991). The findings in this slUdy suggest that application ofbiocontrol agents should also consider the ambient temperature at the time the biocontrol agents are to be applied. For Exserohilllm monoceras to cOhJol Echinochloa species. application of the fungus should not be carried out at night temperatures below 20 C or above 30 C. A few infections occurred on inoculated Echilloch/oa seedlings that did not receive a dew period. Similarly, dry periods of up to 24 h beginning immediately after inoculation did not adversely affect the weed control efficacy of Exserohilllm monoceras at cach minimum dew period. Sirnilar results were reported on the biocontrol of Cassia obtusifo/ia L (sick1epod) by A/ternaria cassiae, in which a delay of 2 days in the occurrence ofdew was tolerated without adversely affecting the efficacy of the pathogen (Walker & Boyene, 1986). Consequently. the same implication fits to Exserohilllm monoceras for the control of Echinoch/oa species, i.e. the inoculum of this pathogen can be applied throughout the day and application is not restricted to the late aftemoon or evening as is cornrnonly done (McRae & Auld, 1988; TeBees!, 1991). Sequential dew periods i1ave been reported to shorten the optimal dew period required for efficient Alternaria cassiae control ofCassia obtusifolia (Walker & Boyette, 1986) and C. gloeosporioides f.sp. malvae conttol of M. pusilla and A. theophrasti (Makowski, 1993). Sirnilar results were observed in our slUdy, i.e. shorter repetitive dew periods (i.e., 6 h rather than 16 or 24 h), more closely simulating field conditions than a single long dew period, enhanced the control of all three Echùwchloa species. The results were confirrned by the field observations in which foliar applications of Exserohilllm monoceras conidia suspensions containing 0.05% wetting agentTween 20 reduced the dry . weight of plants by approximately 5% 7 DAI to 50% 28 DAi Yang & TeBeest (1993) proposed a model for control efficacy of bioherbicides, in which efficacy was composed of primary infections established by the application of • inoculum and secondary or post-application infections. Exserohilllm monoceras bas been 76 observed to have poor dispersal ability (Zhang & Watson, 1996c). The preeise role of • secondary infection for Exserohi/um monoceras control of Echinochloa species is unknown. It is evident, however, that when the inoculum was formulated as an oil emulsion or when applied as a dry powder, the level of primary infection increased significantly and 90% mortality was achieved 14-20 DAI. Therefore, an adequate formulation cao greatly reduce the environmental dependency of Exserohi/um monoceras and increase its potential as a bioherbicide. 4.6. Literature cited Anderson, R.N. and WaIker, H.L. 1985. Colletotrichum coccodes: a pathogen of eastem black nightshade (Solanum ptycanthum). Weed Sci. 33:902-905. Boyene, C.D. and WaIker, H.L. 1985. Factors influencing biocontrol of velvetleaf (Aburilon theophrasn) and prickly sida (Sida spinosa) with Fusarium lateritium. Weed Sei. 33: 209-211. Campbell, CL. and Madden, LW. 1990. Introduction to Plant Disease Epidemiology. John Wiley & Sons, New York. Charudattan, R. 1991. The mycoherbieidè approach with plant pathogens. Pages 24-57 in TeBeest, 0.0. (ed) Microbial Control of Weeds. Chapman & Hall, New York, USA. De Dana, S.K. 1981. Principles and Practices of Rice Production. John W"ùey & Sons. New York, USA. Draper, N.R. and Smith, H. 1981. Applied Regression Analysis. 2nd cd. John W"ùey & Sons, New York. • 77 Fùajdié, N. and Sutton, T.B. 1992. Influence of temperature and wetness duration on • infection of app1e leaves and virulence of different isolates of Airernaria mali. Phytopathology 82:1279-1283. Gomez. K. A. and Gomez, A.A. 1984. Statistical Procedures for Agricultural Research. 2nd Edition. John Wiley & Sons, Inc., New York. Holcomb, G.E. 1982. Constraints on disease development. Pages 61-71 in Charudattan, R. and Walker, H.L. (eds) Biological Control ofWeeds with Plant Pathogens. Wiley, New Yorl,. Holm, L.G., Plucknett, D.L., Pancho, J.V. and Herberger, J.P. 1977. The World's Worst Weeds. Distribution and Biology. The University Press of Hawaii, Honolulu, Hawaii. IRR.I. 1993. Program Report For 1992. International Rice Research Institute, P.O. Box 933, 1099 Manila, Philippines. PP:177-178. Lubigan, R. and Vega, M. 1971. The effect of different densities and durations of competition ofE. crus-ga/li (L.) Beauv. and Monochoria vaginalis (Burm f.) Pers!. on the yie1d of 10wland rice. Pages 19-23 in Weed Science Report 1970-1971. Departrnent of Agricultural Botany, University of the Philippines, College of Agriculture, Los Baiios. Makowski, R.M.D. 1993. Effect of inoculum concentration, temperature, dew period, and plant growth stage on disease of round-leaved mallow and velvetleaf by Colletotrichum gloeosporioides f.sp. malvae. Phytopathology 83: 1229-1234. Matsunaka, S. 1983. Evolution of rice weed control practices ai'ld research: world pexspective. Pages 5-17 in Weed Control in Rice, International Rice Research Institute, • P.O. Box 933, 1099 Mani1a, Philippines. 78 Maun, M.A. and Barrett, S.C.H. 1986. The biology of Canadian weeds. Echinoch/oa crus • ga//i (1...) Beauv. Can. J. Plant Sci. 66:739-759. McRae, C.F. and Auld, B.A. 1988. The influence ofenvironmental factors on anthracnose of Xanthium spinosum. Phytopathology 78: 1182-1186. Moody, K. 1991. Weed control in upland rice with emphasis on grassy weeds. Pages 164 178 in Baker, F.W.G. and Terry, P.J. (eds) Tropical Grassy Weeds. CAB International for CASAFA. Wallingford, UK. Monensen, K. 1988. The potential of an endemic fungus, Co//etotrichum g/oeosporioides, for biological control of round-leaved mallow (Ma/va pusil/a) and velvetIeaf (Aburi/on theophrasn). Weed Sci. 36:473-478. SAS Institute Inc. 1987. SAS/STAT Guide for personal computers. Version 6 edition. SAS Institute Ine., Cary, NC. Smith, R. J., Jr. 1983. Weeds of major economic importance in rice and yield losses due to weed competition. Pages 19-36 in Weed Control in Rice, International Rice Research Institute, P.O. Box 933, 1099 Manila, Philippines. TeBeest, 0.0. 1991. Eeology and epidemiology of fungal plant pathogens studied as biological control agents ofweeds. Pages 97-114 in TeBeest, 0.0. (ed) Microbial Control of Weeds. Chapman & Hall, New York. TeBeest, 0.0. and Ternpleton, G.E. 1978. Temperature and moisture requirements for development of anthracnose on northern jointveteh. Phytopathology 68:389-393• • 79 TeBeest, D. O.• Yang. X. B.• and Cisar. C.R. 1992. The status of biological control of • weeds with fungal pathogens. Annu. Rev. Phytopathol. 30:637-657. Trapero-Casas, A. and Kaiser. W. J. 1992. Influence oftemperature. wetness period, plant age, and inoculum concentraùon on infecùon and development of Ascochyta blight of chickpea. Phytopathology. 85:589-596. Tuile, J. 1969. Plant Pathological Methods: Fungi and Bacteria. Burgess Publishing Co., Minneapolis, Minnesota, USA. Walker, H.L. 1981. Fusarium lateritium: a pathogen of spurred anoda (Anoda cristata). prickly sida (Sida spinosa). and velvetleaf (Aburi/on theophrasti). Weed Sei. 29:629-631. Walker, H.L. and Boyene, C.D. 1986. Influence of sequential dew periods on biocontrol of sicklepod (Cassia obtusifolia) by Alternaria cassiae. Plant Dis. 70:962-963. Watson, A.K. 1991. Prospects for bioherbicide development in Southeast Asia. Pages 65 73 in Swarbrick, R.K. (cd) Proceedings of 13th Asia Pacific Weed Science Society Conference. Asia-Pacific Weed Science Society and Weed Science Society of Indonesia, Jakarta. Watson, A. K. 1994. Current Status of bioherbicide development and prospects for rice in Asia. Pages 195-201 in Shibayama, H., Kritani, K. and Bay-Peterson, J. (eds) Integratect Management ofPaddy and Aquaùc Weeds in Asia. FFTC Book Series No. 45. Food and Fertilizer Technology Centre for Asian and ?acific Regions, Taipei. • 80 Watson, A.K. and Wymore, L.A. 1990. Identifying limiting factors in the biocontrol of • weed:.. Pages 305-316 in Baha, R.R. and Dunn, P.E. (cds) New Directions in Bio1ogicaI Control: Alternatives for Suppressing Agriculrural Pests and Diseases. Alan R. Liss, !nc. New York. Yang, X.B. and TeBeest, 0.0. 1993. Epidemio1ogical mechanisrns of mycC'herbicide effectiveness. Phytopatho1ogy 83:891-893. Yeh, W.H. and Bonman, J.M. 1986. Assessment of potential resistance to Pyriculana oryzae in six rice cultivars. Plant Pathology 35:319-323. Zhang, W. and Watson, A.K. 1996a. Characterization of growth and conidial production of Exserohilum monoceras on different substrates. (Unpublished paper). Zhang, W. and Watson, A.K. 1996b. Efficacy of Exserohilum monoceras for the control ofEchinoch/oa species in rice (Oryza sativa L.). Weed Sci. (reviewed by IRRI and ready ta he submitted). Zhang, W. and Watson, A.K. 1996c. Host range of Exserohilum monoceras, a potential bioherbicide for the control of Echinochloa species. (Unpublished paper). Zhang, Wenming, Moody, K. and Watson, A.K. 1996. Responses ofEchinochloa species and rice (Oryza sativa L.) to indigenous pathogenic fungi. Plant Dis. (Submitted)• • 81 Table 4.1. Effect of sequential dew periods on mortality and dry weight reduction of • Echinochloa crus-gaut Dew periods Mortality reduction in dry weight (h/day) (%) (%) 0 Oc·· 10.0 d 6 Oc 23.8 c 6; 6 Oc 42.6 b 6; 6; 6 5c 59.1 b 6;6;6;6 70 b 90.0 a 12 100 a 100.0 a 12; 12 100 a 100.0 a 24 100 a 100.0 a • Seedlïngs at the 1.5-leafstage were sprayed with a conidial suspension of E. monoceras 7 2 at 5 x 10 conidialm • Data from two trials were not pooled because variances were not homogenous. Data represent means of four replicates. .. Values having the same letter are not significantly different according to DMRT at the 5% significance leveL • 82 • Figure 4.1. Effect of dew temperature on disease development caused by Exserohilum monoceras on three Echinoch/oa species, expressed as percent leaf area damage (% LAD). Seedlïngs at the 1.5-1eaf stage were sprayed with a conidial suspension of E. 7 2 monoceras at rate of 5 x 10 conidialm • Dew period duration was 24 h. Data from two trials were pooled because variances were homogenous. Data represent means of eight replicates. For comparative purposes, the disease progress curves over time were summarized by using the standardized area under the disease progress curve (SAUDPC) (Figure 4.2). • 83 Echinochloa crus-ga/li • 100 80 60 40 20 0 Echitwchloa colona ...... 100 B Cl -< ...l 80 ~....., ?;- ·C., 60 .,> ..., 40 ...,;j Q 20 0 Echitwchioa glabrescens 100 C 80 0 10C 0 15C 60 • 20C • 2SC 40 • 30C v 35C 20 <> 40C 0 0 2 4 6 8 10 • Days aiter inoculation • Figure 4.2. Effect of dew period temperature on disease developrn~nt caused by Exserohilum monoceras on three Echinochloa species, expressed 3li the standardized area under the disease progr:ess cwve (SAUDPC). Data represent rneans of eight replicates. Bars within each ternperature treatment having the same letter are not significantly different according to DMRT at the 5% significance leveL , , . • 84 • 120 Echinochloa crus-galli _ Echinochloa colona l!l!l!lIlI Echinochloa glabrescens 100 a a a a a a a ~ '-' CJ 80 p., Q ;::l -<: ~ 60 ~ c s 40 1'1) 20 0 ...... 10 lS 20 2S 30 3S Temperature (C) • • Figure 4.3. Effect ofdew period duration on disease development caused by Exserohilum monoceras on three Echinochloa species, expressed as percent leaf area damage (% LAO). Seedlings at me 1.5-leaf stage were sprayed wim a conidial suspension of E. 7 2 monoceras at a rate of 5 x 10 conidia/m • Dew temperatuIe was 25 C (in clark). Data from two trials were pooled because variances were homogenous. Data represent means ofeight replicates. For comparative purposcs, me disease progress curves over rime were summarized by me standardized area under me disease progress curve (SAUDPC) (Figure 4.4). 85 • Figure 4.4. Effect ofdew period duration un disease development caused by Exserohilum monoceras on three Echinochloa species, expressed as the standardized area under the disease progress curve (SAUDPC). Data represent means ofeight replicates. Bars within the same dew period duration having the same Ietter were not significantly different according to DMRT at the 5% significance Ievel. fi 86 • 120 ~ Echinochloa crus-}?allj _ Echinochloa calant. llIl!llI Echinochloa glabrescens 100 a a ..... ~...... ~ 80 Q ::J -< "§ 60 ~ c 5 40 20 ab 0 4 8 12 16 20 24 Dew period duration (il) • • Figure 4.5. Effect of post-dew ternperature on disease development caused by Exserohilum monoceras on three Echinochloa species, expressed as percent plant morta1ity 10 days after inoculation. Seedlings at the l.5-leaf stage were sprayed with a conidial suspension of E. monoceras at a rate of 5 x 10' conidialm2 and given a 12 h or 24 h dew period. Data from two trials were not pooled because variances were not homogenous. Data represent means of four replicates. Bars within each temperature treatment in a dew period having the same letter are Dot significantly different according ta DMRT at the 5% significance level. • 87 • Echinochloa crus-gal/i 120 == - Echinochloa c%na ...... Echinochloa g1alJrescens 100 a aaa aaa aaa aaa aaa aaa aaa 80 60 40 20 ° 15 202S 303540 15 20 2S 30 35 40 12-hdew 24-hdew Temperature (C) • • Figure 4.6. Interaction effect between dew period temperature and duration on disease development causeè by Exserohi/um monoceras on three Echinochloa species, expressed as percent plant mortality 10 days after inoculation. Data from two trials were pooled because variances were homogenous. Data represent means of eight replicates. • 88 • 100 80 . ....'. A ~ '. '. ~ ...... ~ .... ~ 40' o '. ::E ". 20 , 100 80 . . . ~ ';;' ~ .... " . ; 60 ...... 40" '. j ...... ::E 20 ...... 100 c • • Figure 4.7. Mortality response (arc sine-transformed) of (A) Echinochloa crus-gal/i, (B) Echinochloa colona, and (C) Echinochloa glabrescens 10 days after Exserohilum monoceras inoculation to increasing dew period duration at each offive temperatures. The response was calculated by fitting a linear regression to the means• • 89 2.4 _ ..- 15 C y=-0.246+O.032x. ?=OAO A • --20 C y=-0.371+O.0S2x. ?=O.S5, 2.0 _.- 2S C y=-0.122+O.0S5x. r·=O.77 ...... 30 C y=-0.270+0.075x. r2=O.SI 1.6 , .- ---35 C y=-0.075+O.011x. 1'""=0.15_._.- 1.2 _._._._. O.S _._. -~~ 0.4 .... -----_ ... --.-- ~~------0.0 2.4 -"-15 C y=-0.064+0.00Sx, ?=O.19 B --20 C y=-056S+O.096x. ?=o.S4 ,...., 2.0 .;;;c -'- 2S C y=-053SiO.09Sx, ?=o.SO ~ ··········30 C y=-0.632+O.0S6x, r2=O.S3 ...... 1.6 2 a~ ---35 C y=-0.202+O.02Sx, r =O.53 ---=:: • ]1 1.2 ---=:: • ... .~ . 0 .~ .....- :!: O.S ~ . .~...... ~ ..-.-- 0.4 -~ --_.-- ~ ..------.... - _--.::o-~ --- 0.0 L,;.;._.c==.-- .....=-::.::=:.:.:=::.:.:===:..:...;.:;:...... J._--_._- -"- 15 C y=-0.262+O.040x, ?=O.35 C 2.4 1-.__ 20 C y=O.114+0.074x. ?=O.77 2.0 ---- 2S C y=O.510+0.056x, ?=O.63 --..--.-.. 30 C y=-0.275+O.091x, ?=o.S5 1.6 - --- 35 C y=-0.137+O.029x, ?=O.24 • ~. ~~ . ---- ....-- 1.2 - _.-.- -••••••••------_o- .00.-..... - O.S -:- ....-.. -- o·· _00 0.4 ,=.. .. --_..... - -:------_.----- 0.0 ~::=:===:::il==- __.L-I .ul S n 16 ~ • Dew period (il) • Figure 4.8. Effect of dew period temperature on the predicted dew period duration required for 100% disease severity of tIuee Echinochloa species by Exserohilum molUJceras expressed as percent plant mortality 10 days after inoculation. The relationslùp was best described by the following equations: 1) Echinochloa crus-galli: ln 0 = (M+1S.1039)/(-36.1946+6.s039T-o.1323~, (P=O.OOOl, f2=0.7113); 2) Echinochloa colona: ln 0 = (M+33.4S)/(-47.14+7.44T-o.14~, (P=O.OOOl, f2=0.7828); 3)EchilUJchloa glabrescens: ln 0 =(M+3.7930)/(-3S.0961+6.S739T-O.1344~, (P=O.OOOl, r=O.9254). Where M = mortality, 0 = dew period duration, and T = dew temperature. Percent mortality data were arc sine-transfonned before analysis. • 90 • 50 r-T------..., -- Echinochloa crus-ga/li - ° Echinochloa colona • 00 000 Echinochloa glabrescens g 40 \ =o . ""i: \ -6 30 • "8 01: \ . 8.. ,. • . ~ • . "0 20 •, "'- . ] .• •, '---"'"'""-- .. - ... -- ... .' -, - '" .' 00 i -- 0 .o c.. 10 ..... -... -.- ... _--_ ..._... ----- ..- 0'------'...... -----"----...... ----..... 15 20 25 30 35 Temperature (C) • • Figure 4.9. Effect of de1aying initial dew period on disease development caused by Exserohilwn monoceras on three Echinochloa species, expressed as percent plant mortality 10 days after inoculation. Seedlïngs at the 1.5-leaf stage were sprayed with a 7 2 conidial suspension ofE. monoceras at a rate of5 x 10 conidialm • Data from two trials were not pooled because variances were not homogenous. Data represent means of four replicates. • 91 • 100 .... ~ :~ . 0. 0::: '0. S 40 . 6 ::E 100 80 B ...... 60 . 40 . 100 20 • • Connecting Text Optimization of spore production is often a critical aspect in detennining the success or failure ofa bioherbicide prospect. To date. littIe is known about the conditions required for conidial production of E. monoceras. In !bis chapter. the results of preliminary characterization of growth and sporulation of E. monoceras on standard agar media, liquid media, and agriculturally-based substrates are presented. • 92 Chapter 5. Cbaracterization of growtb and conidial • production of Exserohilum monoceras on different substrates 5.1. Abstract On agar media optimal conielial production of Exserohilum monoceras occurred on V-8 juice agar fYA) and centrifuged V-8 juice agar, whereas optimal raelial mycelial growth occurred on Czapek-Dox agar. Optimum temperatures for radial mycelial growth and sporulation were 28 to 30 C and 28 C. respectively. Sporulation elid not take place at temperatures below 15 C or above 35 C. Constant temperature was better for radial myceliaI growth and spo.ulation than altemating temperature. Linear growth was not significantly different among treatments with continuous light, continuous dark, and a1ternating 12 h light/clark. No sporulation occurred in co.~!inuous Iight and sporulation in continuous dark was 10 times greater than in a1temating 12 h Iight/clark. Near ultra violet Iight (NUV) elid not stimulate sporulation on agar media. Zonation (a1temation of zones ofmyceliaI and conielial producti('\n) elid not occur in continuous light or continuous clark but was pronounced in aItemating 12 h Iight and clark. Echinoch/oa leaf decoction significantly increased conielial production on potato dextrose agar (PDA) and VA and germ tube length on PDA,lima bean agar. and VA but elid not affect conielia germination. No conielia were produced in liquid media. On solid substrates, the most abundant sporulation (1.81 x IIf conielialg dry weight) was produced on corn leaves arter 3 to 4 wks. However, conielial production of E. monoceras on corn leaves was affecteel by incubation perioci. moisture content, and substrate quantity. There were no elifferences in germination rate, germ tube length, and virulence of conielia produced on agar media or corn leaves. 5.2. Introduction Ranked as thethird world's worst weed, bamyardgrass, Echinoch/oa crus-ga/li (L.) Beauv., has been listed as a weed in 36 different crops in 61 countries (Holm et al., • 1977). Junglerice, Echinoch/oa c%na (L.) Link., is ranked as the fourth world's worst 93 weed and has been listed as a principal or the most serious weed in 35 elifferent crops in • more than 60 countries (Holm et al., 1977). Echinochloa glabrescens Munro ex Hook.F. is also an important grass weed species, especially in tropical regions (De Dana, 1981). AlI these spccies are target weeds of a biological weed control program initiated at the International Rice Research Institute (IRRI), P.O. Box 933, 1099 Manila, Laguna, Philippines in collaboration with McGill University, Montréal, Canada (Watson, 1991; Bayot et al., 1994; Watson, 1994). Exserohilum monoceras (Drechsler) Leonard & Suggs was isolated from naturally-infected agricultural Echinochloa populations collected on the IRRI farm and was virulent on E. crus-galli, E. colona and E. glabrescens. Inoculation with this pathogen initially produces small dark-brown longitudinal necrotic streak:s (0.3 3 mm) which were often restricted to between leaf veins and exposed sheaths of infected Echinochloa plants. Three ta four days after inoculation, this pathogen produced blight- like symptams. In the greenhouse, E. crus-ga/li, E. colona and E. glabrescens seedlings at the 1.5-leaf stage were killed by the pathogen when applied at 5.0 x 107 conielialm2 over a range of dew perïod durations (Zhang & Watson, 1996; Zhang et al., 1996). In screenhouse and field trials, Echinochloa seedlings were severely eliseased (Zhang & Watson, 1996), demonstrating the potential of E. monoceras to be an effective biocontrol agent against these weeds. Ifan organism is to be considered for augmentation in the bioherbicide strategy, it is essential ta develop an efficient and economic inoculum production system (Boyette et aL, 1991). Agar mec\ia, liquid mec\ia, and solid substrates have been used to produce sufficient amounts ofinoculum ofvarious fungi used for biological weed control (Daniel et aL, 1973; Hildebrand & McCain, 1978; Walker, 1980; 1982; Walker & Riley 1982; Boyeue et aL, 1984; Wymore et al., 1988; Wmder & van Dyke, 1990). Although Sivanesan (1987) recommended sorne general media and temperature and light conelitions for anamorph production of graminicolous fungal specles in the genera Bipolaris, CurvuIoria, Drechslera, and Exserohilum. little is known about the specific culture and conidial production requirements of E. menaceras. Hence, the objectives of this study • were: 1) ta characterize mycelial growth and conielia! production on clifferent standard 94 agar media over a range of temperatures and under different light conditions. 2) to • investigate conidial production on liquid media and agriculturally-based solid subslI'ates. and 3) to study the effeet of conidial production methods on conidial production. germination, and virulence. 5.3 Materials and methods 53.1. Pathc'Jen isolation and culnue maintenance Diseased leaves of Echinochloa species were colleeted on the IRRI farm. Leaf pieees with lesions were surface sterilized with 0.5% sodium hypochlorite solution and incubated on potato dextrose agar (PDA; Difco. Detroit, MI). Single-eonidium isolates of E. monoceras were prepared and stored on half-strength potato dextrose agar (1/2 PDA) slants at 4 C in the dark as stock cultures. Small pieces of mycelium from the stock culture were aseptically transferred to the centre of fresh PDA plates. Plates were sealed with parafi1m and incubated at 28 C for 7 days. Agar plugs with mycelium (6-mm diameter) from the margin of these young colonies were used as "seed" inoculum. 53.2 Growth and conidial production on standard agar media 5.3.2.1. Effect of nutrient media on mycelial growth and conidial production Growth and conidial production of E. monoceras were observed on different agar media. Media tested were corn meal agar (CMA), centrifuged V-8 agar (CVA), Czapek Dax agar (CDA), lima bean agar (LBA), malt extract agar (MEA), oat meal agar (OMA), patata dextrose agar (PDA), half strength PDA (1/2 PDA), rice polish agar (RPA), V-8 juice agar 01A). CMA, CDA, LBA, MEA, OMA, PDA, and 1/2 PDA were prepared as recommended by the label, RPA and VA according to methocls described by Tuite (1969), and CVA by following the method of Grbavac (1981). Media were dispensed into glass petri dishes (20 ml ofmedium per 9O-mm diameter dish) and were inoculated with 6-mm agar p1ugs from 7-day-old PDA cultures of E. monoceras. Six replicate disbes of eacb medium were incubated for 7 days at 28 C in continuous darkness in an incubation • chamber, except for a brief period every 24 h when they were rernoved from the chamber 95 50 that mycelia! growth could be assessed. A wax pencil was used to out1ine the edge of • mycelia! growth at 24 h interva!s. Raelial mycelial growth during cach 24 h period was recorded as the mean of twO perpenelicular e1iameters minus the e1iameter of the inoculum plug (6 mm). The radia! mycelial growth rate was calculated in millimeters per 24 h. At the end of the growth study. plates were incubated for one more week and then assessed for production of conielia. Conielia were harvesled by flooeling the plates with 10 ml e1istilled water and scraping the surface of the colonies with a glass slide. Resulting suspensions were fùtered through a layer ofcheesecloth and conielia from six colonies per growth medium were counted inelividually in a haemocytometer. 5.3.2.2. Effeet of temperature on mycelial growth, conielial production, and conielia! germination Effect ofconstant temperatures on linear growth and sporulation were determined on VA in the dark. Radial mycelial growth and conielial production were measured for nine temperatures between 5 and 40 C at 5 C intervals following the procedure out1ined above. Effect ofconstant temperature on conielial germination were investigated on PDA. PDA was cut into 20-mm e1iameter e1isks and placed on microscopic slides that were supported on glass rads within 90-mm e1iameter petri e1ishes with moistened filter paper. Disks were inoculated separately with 15-J.Ù drops containing approximately 1 x 10" conielia/ml. Disks were incubated in the dark from 5 tO 40 C at 5-C inrerva!s. Twenty-five conidia in four replicates were observed for germination 6 h after inoculation. Prior to observation, germinating conielia were killed and stlined with lactophenol-cotlon blue (Tuile, 1969). The germination of conielia was observed using a bright-field compound microscope (X 250) and conielia were assumed to have germinated ifthe germ tube was as long as the width of a single conielium. The experiment was ammged in a completely randomized design (CRD)• • 96 5.3.2.3. Effect oflight, dark, and NUV on mycelial growth and conidial production • Effects oflight, dark. and r-.ruv on radial mycelial growth and conidial production were a!so tested on VA. Cultures were incubated on VA for 24 h at 2S C in the dark and then exposed to continuous light (SO IlE-m·~-s·l). continuous darkness. 12 h of altemating light (SO J,IE -m'~-S.l) and dark. continuous NUV (GE F 40 BL). and 12 h of altemating NUV and dark. The incubation temperature was 2S C. Radial mycelial growth and conidia! production were measured following the procedures outlined above. The effect of light on zonation (a!temation of zones of mycelial and conidial production) was observed on colonies. 5.3.2.4. Effect of Echinochloa leaf decoction on mycelial growth. conidial production and conidia! gemùnation Effects ofEchinochloa leafdecoction on conidial production. germination rate. and germ tube length were observed on PDA. LBA. and VA. The leafdecoction was prcpared by boiling 30 g of E. crus-galli green leaves in 1 L water for 15 min and straining the mixture through cheesecloth. The resulting broth was used as the "water" component in agar preparation. Corresponding media prepared with distilled water served as checks. Radia! mycelia! growth was measured following the procedures outlined above. For conidial production tests, six replicates of each medium were inoculated with 6-rnm agar plugs from 7-day-old PDA cultures ofE. monoceras and incubated for 3 weeks. Conidia! production and germination per medium were measured following the procedures outlined above. An ocular micrometer was used for germ tube measurements. As many as 30 randoltÙy selected geml tubes were measured in each replicate. 533. Conidial production on liquid media Czapek Dox solution (35 g Czapek Dox broth, 1 L ~o), Richard's solution (50 KH~ g sucrose., 10 g of KN03, 5 g of •• 2.5 g of MgSO., 0.02 g Fe03, and distilled water to 1 L), Modified Richard's (V-S) solution (50 g sucrose., 10 g of KN03, 5 g of ~., • 2.5 g of MgSO., 0.02 g FeC13• ISO ltÙ of V-S juice, and distilled water to 1 L) 97 (Daniel et al.• 1973; Wymore et al.• 1988). corn and soy floU!" (15 g soy flour. 15 g corn • rnea1. 30 g sucrose. and 3 g CaCOJ (Hildebrand & McCain, 1978). and EZ8 (EZ8 is made from V-8 juice modified with green Echinochloa leaf decoction. The decoction was prepared as described above and afler straining through cheesecloth, 0.75 g KH2PO., 1.5 g MgSO•• 3 g CaC03• and 180 lTÙ V-8 juice were added (Winder & Van Dyke. 1990» were used. One hundred ml ofeach medium were placed in 250-ml Erlenmeyer flasks and autoelaved for 15 min (100 kPa and 121 C). After cooling, each medium was inoculated with 6-mm agar plugs from 7-day-old PDA cultures of E. monoceras. Inoculated flasks were incubated on rotary shakers at 150 rpm at room ternperature (28 ± 2 C) in the dark. After 1. 2, 3. and 4 wks incubation period, conidia were harvested by filtering the contents in each flask through a layer of cheesecloth. Each treatment was replicated six tirnes. 53.4. Conidial production on agriculturally-based solid substrates 5.3.4.1. Evaluation of solid substrates The following agricultura1 products were evaluated as solid substrates for conidial production: corn (Zea mays L.) leaves. fresh corn seed. dry corn seed, cracked corn, corn grilS, corn mea1, corn cob, sorghum (Sorghum bicolor (L.) Moench) grain, rice straw, polished rice, rice grain, rice kemel, wheat (Triticum aestivum L.) seed, millet (Pennisetum glaucum (L.) R.Br.) seed, barley (Hordeum vulgare L.) seed, lima bean (Phaseolus lunatus 1-) seed, fmely ground lima bean seed, finely ground lima bean seed supplemented with 2% yeast and 0.5% dextrose, mungbean (Vigna radiata (L.) R. WÙCZek:) seed, soybean (Glycine max (L). Merr.) seed, cowpea (Vigna unguiculata (L.) Walp. subsp. unguiculata) seed, and coconut (Cocos nucifera L.) meat. The corn leaf material was prepared by collccting mature senescing leaves, cutting into 1 cm x 1 cm pieces, drying at 80 C for 4 days, and then placing 1 g of dried leaf material in 25D-ml Erlenmeyer f1asks and moisting with 10 ml of distilled water. Rice straw was cut into 1 cm lengths and processed in the same way as corn leaves. For other substrates, 10 g of • each substrate was placed into a 250-ml Erlenmeyer flask and moistened with IS ml of 98 distilled water. AIl preparations were aUloclaved twice for 15 min (100 kPa and 121 C). • After coo1ing. flasks were inoculated with 6-mm agar plugs from 7-day-old PDA cultures of E. monoceras and incubated at 23 C in the dark. After 3 wks, conidia were harvested by adding 50 où of distilled water to each flask, shaking the flasks on a rotary shaker at 250 rpm for 5-10 min, and then filtering through a layer of cheesecloth. Six replicates were used for cach substrate. 5.3.4.2 Effect of incubation period on conidial production on corn leaves Corn 1eaf material was prepared as described above. Conidial production was assessed after 1. 2. 3. 4, 5. and 6 weeks of incubation following the procedures outIined above. Each tteatment was replicated six times. 5.3.4.3. Effect of moisture content and substrate quantity on conidial production on corn 1eaves One and two grams of corn leaf material were moistened with 5. 10. or 15 où of distilled water and tested for conidial production in a factorial design. Percent moisture content of corn 1eaves after autoclaving was evaluated on a wet weight basis using the following formula: (g ~O/g wet corn leaf) x 100. Conidia were harvested aiter 3 wks. Each treatment was replicated six times. 535. Effect of conicüal production metJwds on conidial production, germination, and virulence EZ8A (consisted of 163 où V-S juice. 0.75 g KH2PO•• 1.5 g MgSO•• 3 g CaCO), 20 g agar. and 1 L Echinochloa 1eaf decoction) and corn leaves were selected as representatives ofstandard agar medium sporulation and solid fermentation. respectively. Each medium was seeded with agar p1ugs. using the procedure described previously for each method. The conidia were harvested after 1. 2, 3. and 4 weeks. The germination rate and germ tube length ofconidia produced at cach harvest were determined following the • procedure outlined above. 99 For the virulence test, E. crus-gal/i seecls were incubatecl in petri dishes on • moistenecl filter paper at room temperature for 48 h. Five genninatecl seeds (coleoptile and radicle just emergecl) were planted per 10-cm diameter plastic pot fillecl with saturatecl soil (Maahas clay, Haplustic suborder). Seedecl pots were placecl on a push-cart in the greenhouse and water level was maintainecl in the push-eart at a depth of 2-3 cm throughout the experimental period. Greenhouse conditions were 35/25 ± 5 C day/night temperature, a 12-h photoperiod, and an average light intensity of 20 MJ/m2 per day. Seedlîngs were inoculated with 2.5 x lOs conidia/m2 with 0.05% Tween 20 as a wetting agent, using a motorizecl sprayer (A. H. Thomas Co. Scientific Apparatus, Philadelphia) at 100 kPa. In Experiment l, inoculation was carried out on seedlings at the 3-leaf stage and in Experiment 2 on seedlings at the 1.5-leaf stage. After spraying, pots were placed in a dark dew chamber with 100% relative humidity at 25 C for 6, 12, and 16 h and then returned 10 a corner of greenhouse having a temperature of 24-28 C and an 85-95% humidity (Yeh & Bonman, 1986). The control treatrnent was sprayed with distilled water containing only the wetting agent. Virulence ofconidia was assessed as percent reduction in dry weight 10 days after inoculation (DAI) where % reduction in dry weight = (dry weight of check plant - dry weight of treated plant)/ dry weight of check plant. The dry weight was obtained by cutting aerial parts at soilleve! and drying living foliage in paper bags for 4 to 5 days at 60 C, and weighing. Dead leaves were Dot included in the dry weight measmements. Each treatrnent for the virulence test was replicated four times. 5.3.6. Data analyses AlI experiments were performed twice. A completely randomized design (CRD) was used for all experiments, except for virulence tests which were atranged in a randomized complete black design (RCB). Analysis ofvariance (ANOVA) was performed on the pooled data from the two experiments, and treatrnent means were separated using Duncan's Multiple Range Tests (DMRT). Data from conidial counts were subjected 10 10g(1+x) ttansformation for zero values and log(x) for non-zero values; percentage data • were arc sine-ttansformed (Gomez & Gomez, 1984). Regression analysis was used to 100 model the effect of temperature on radial mycelial growth. conidial production. and • gemûnation rate (Analytis. 1977). Results for the two trials of each experiment were pooled if there was homogeneity of variances as determined by Bartlett's test (Gomez & Gomez, 1984). Simïlar trends were observed between trials of experiments where variances were not homogeneous and results from one trial or both trials were presented. AlI analyses were conducted using SAS/STAT™ software for IBM-PC (SAS. 1987). 5.4. Results 5.4.1. Growth and conidial production on standard agar media 5.4.1.1. Effect of nutrient media on mycelial growth and conidial production Radial mycelial growth and conidial production of E. monoceras varied with the different nutrient media (Table 5.1). Maximum radial mycelial growth occurred on Czapek-Dox agar. however. this growth was not significantly different from growth rates on rice polish agar. lima bean agar. V-8 juice agar. and centrifuged V-8 juice agar. The greatest number of conidia was produced on V-8 juice agar and centrifuged V-8 juice agar. 5.4.1.2. Effect of temperature on mycelial growth. conidial production. and conidial gemünation The optimum temperature range for radial mycelial growth of this fungus on V-8 juice agar in the dark was 28 to 30 C (Figure S.lA). Temperatures higher than 30 C or lower than 28 C significantly reduced the growth rate. The optimum temperature for conidial production was 28 C (Figure S.lB). E. monoceras did not sporulate at temperatures below 15 C or above 35 C. High genni:'ation rates occurred across the 20 35 C range (Figure S.le). The predicted optimum temperature for radial mycelial growth. conidial production. and conidial germination were 28.5 C. 28 C. and 25 C. respectively. 5.4.1.3. Effect oflight, darI<, and NUV on mycelial growth and conidial production • The mycelial growth ofE. monoceras on V-8 juice agar was significantly reduced 101 when exposed to continuous NUV or alternating NUV/dark (Table 5.2). There was no • difference arnongst dark, light, and alternating darkllight on mycelial growth. Light had a drarnatic effect on conidial production as so conidia were not produced when the culture was exposed to continuous light and conidia production was greatly reduced when exposed to alternating darkllight, continuous NUV, and alternating NUV/dark (Table 5.2). Conidia production in continuous dark was 10 tirnes greater than that in alternating dark/light. With alternating darkllight, zonation occurred in the cultures with sporulating and non-sporulating sectors. Zonation did not take place in continuous light or continuous dark. 5.4.1.4. Effeet of Echinoch/oa leaf decoction on mycelial growth, conidial production, and conidial germination The E. nwnoceras mycelial growth was not affected by Echinochloa leafdeeoction (data not shown). However, The addition of Echinoch/oa leaf deeoctior. significantly influenced E. nwnoceras conidial production and its effeet varied with base medium (Figure 5.2A). In general, Echinoch/oa leaf deeoction increased conidial production on PDA and VA but inhibited sporulation on LBA. Echinoch/oa leaf deeoction did not affect conidial germination rate (Figure 5.2B). However, germ tube length was significantly increased with the addition of leaf deeoction on all three media tested (Figure 5.2C). 5.4.2. Conidia/ production on /iquid media Mycelial growth in liquid media was observed 2 days after incubation. AlI the cultures produced dark pigmentation 7 days after incubation. None of the liquid media supported E. nwnoceras sporulation under submerged culture conditions. 5.4.3. Conidia/ production on agriculturally-based so/id substrates. 5.4.3.1. Evaluation of saUd substtates Twcnty-two agricultural produCt5 were evaluated as saUd substrates for mass • production of E. nwnoceras. The most abundant sporulation occurred on corn leaf 102 material at 1.81 x 106 conielialg dry corn le:lf (Table 5.3). Mungbean. soybean. and • cowpea elid not support rnycelial growth or conidial production of E. monoceras. 80th dry corn seed and barley grains allowed mycelial growth but elid not support sporulation. The remaining substrates produced less than 1 x 103 conielia per g substrate up to more than 1 x Hf conielia per g of substrate. Consequently. the corn leaves were seleeted as solid substrate for further evaluation. 5.4.3.2. Effeet of incubation pe..;od on conielial production on corn leaves Conidial production increased with increasing incubation period but deelined aiter 4 wks (Figure 5.3). At three weeks of incubation, more than 1 x la· conidialg were produced on corn leaf material. 5.4.3.3. Effect of moisture content and substrate quantity on conidial production on corn leaves There was an interaction between moisture content and substrate quantity on conielial production on corn leaf material (Table 5.4). Different substrate quantities and moisture content deterrnined the thickness ofthe corn leaf material. After autoclaving. the dry corn leaf material absorbed the water and expanded in the flasks. One gram of dry corn leafmaterial with la ml elistilled water (93.8%) fully occupied the space in the flask and produced the highest number of conielia. Other weight and volume combinations of dry corn leaf material and water elid not optirnize space utilization in the flasks. 5.4.4. Effect of conidial production methods on conidial production, germination. and virulence Conidia produced on EZ8A and corn leaf material were morphologically sirnilar and showed no significant difference in conielial size (fable 5.5). Ateach incubation time, germination, germ tube length. and virulence (% dry weight reduction of Echinochloa seedlings) of conielia produced on both EZ8A and corn leaves were not significantly • different (fable 5.5). 103 For the various incubation periods, germination was not significantly different, but • germ tube length and virulence of conidia were significantly different. The longest germ tube length was observed for conidia produced at 1 week incubation while the shortest germ tube length for conidia harvested at 4 weeks incubation. No clear relationship was observed between virulence and germ tube length. The virulence at cach incubation period depended upon the dew period duration provided. When provided a sufficient dew period (12 h or 16 h), a 100% reduction in dry weight was observed for an incubation periods. When provided an 8 h dew period, virulence differed arnong incubation periods with the highest dry weight reduction occurring at 3 weeks incubation. In Experiment l, less infection occurred because plants were inoculated at the 3-leafstage which is not the most susceptible growth stage to E. monoceras (Zhang & Watson, 1996). 5.5. Discussion Under laboratory conditions, optimal radial mycelial growth and conidial production of E. monoceras were dependent on the culture media, temperature, and light conditions. Sporulation was best on V-8 juice-based agar media (VA, CVA, and EZ8A). Temperature and light had profound effects on Illdial mycelial growth and conidial production on VA. Temperature may have affected sporulation directly by influencing conidial production or indirecdy by influencing colony development which later provided a large colony area for further production of conidia. As a result, both production of conielia and radial mycelial growth had sirnilar response curves. The optimum temperature range for growth and sporulation was usually 28-30 C. At 35 C. radial mycelial growth decreased gready and conidial production ceased. The fungus produced more spores when exposed ta continuous dark. Continuous light complete1y inhibited sporulation. Near UV did Dot stïmulate sporulation of this pathogen on agar media. This contrasts with other findings (Leach, 1967; Sivanesan, 1987). Zonation was observed when culture plates were exposed ta alternating dark and light periods at 28 C and supports the results of other workers (Kaiser et al, 1994). This is the first time ta report findings on the growth and • sporulation characteristics of E. monoceras on standard agar media (Sivanesan, 1987). 104 Abundant conidia wc:":: obtained when Echillochloa leaf decoction was added to • the media. The magnitude of the increment, however, was influenced by the basaI medium used. When Echillochloa leaf decoction was added into VA, more conidia were produced (EZ8A) (Table 5.5). However, when Echillochloa leaf decoction was added into LBA, conidia! production was decreased. Media not containing Echillochloa leaf decoction produced the same shape of conidia as media containing Echinochloa leaf decoction. The different responses in conidial production on different agar media supplemented with Echinochloa leaf decoction may be related to the different nuttitional composition and C:N ratio of the various media (Hildebrand & McCain 1978; Churchill 1982). These fmdings suggest that Echinochloa leaf decoction rnight be used to enhance conidia! production on certain types of media. The conidia of E. monoceras did not germinate and grow in distilled or deionized water (data not shown). On PDA, conidia began to germinate 1 h after incubation. For optimal germination and germ tube development. the conidium requires an exogenous nuttient source (Gottlieb. 1978). It is thus apparent that PDA provided adequate nuttients for good germination and germ tube development. Nuttitional factors have been known to play an important role in sporulation when the nuttient requirement is higher than that needed for growth (Cochrane, 1958). These agriculturally-based solid substrates tested contain different carbohydrate-protein ratios (Purseglove, 1968; 1972). There seems to be no clear relationship between nuttient content and sporulation on solid substrate. However. a lower protein content appears ta encourage greater sporulation as indicated by the corn leaf materia!. rice straw, fresh corn, and sorghum grains media. Particle size, perhaps expressed as surface area, also played an important role in conidial production since finely ground lima bean seed produced more conidia that did whole seed. Stawell et al. (1989) categorized mycoherbicides into two groups based upon differences in their physiology, pathology, and sporulation in culture. Group 1 consists of fungi which attaek plants and sporulate within the plant tissues or sporulate under the • plant cpidcrmis in pycnidia or acervu1i. Fungi within this group are able ta sporulate in 105 liquid culture. Group II consists of fungi which push conidiophores through the plant • surface and fonn conidia in the gaseous environment outside the plant. Fungi within this group do not sporulate weil in liquid culture. E. monoceras belongs to group II as it did not sporulate in submerged cultures instead requiring a solid surface for sporulation. However, recent work has revea1ed that Group II fungi can be induced to sporulate by manipulating C~ concentration (Cotty, 1987). Further research into the way in which E. monoceras sporulation is affected by COz regulation in liquid media rnight prove vaIuable. Submerged production techniques are preferred because of the availability of teehnology and the scaIe-up process is relatively easy (Churchill, 1982; Hall & Papierok, 1982), but solid substrate fermentation aIso provides sufficient conidia for sorne of the biocontrol agents which do not sporulate in Iiquid culture such as Cercospora rodmanii Conway (Kenney et ai., 1979) and Alremaria spp. (Walker, 1981, 1982). For E. monoceras, corn leaf materiaI provided abundant conidiaI production. The germination and virulence of conidia produced on corn leaves are sirnilar to those on agar medium. Corn leaves are readily available after corn harvest and often considered a non-useable portion of the plant. SmaI1 scaIe mass production of E. monoceras using corn leaves as a solid substrate may be feasible. 5.6. Literature cited AnaIytis, S. 1977. Über die relation zwischen biologischer Enkwick1ung un Temperatur bei phytopathogenen Pï1zen. PhytopathoI. Z. 90:64-76. Bayot, R.G., Watson, A.K. and Moody, K. 1994. Control of paddy weeds by plant pathogens in the Philippines. Pages 139-143 in Shibayama. H., Kiritani, K. and Bay Peterson, J. (cds) Integrated Management of Paddy and Aquatie Weeds in Asia. FFTC Book Series No. 45. Food & Fertilizer Technology Centre for the Asian and Pacifie Region. Taipei. • 106 Boyette. C.D., Templeton, G.E. and Olivier. LR. 1984. Texas gourd (Cucurbira rexana) • control with Fusarium so/ani f.sp. cucurbirae. Weed Sci. 32:649-655. Boyette, C.D., Quimby, P.C. Jr., Connick. WJ.• Daigle, D.J. and Fulgham. F.E.. 1991. Progress in the production. formulation. and application of mycoherbicide. Pages 209-222 in TeBeest, 0.0. (ed) Microbial Control of Weeds. Chapman & Hall. New York. Churchill, B.W. 1982. Mass production of microorganisms for biological control. Pages 139-156 in Charudattan, R. and Walker, H.L. (eds) Biological Control of Weeds with Plant Pathogens. John Wiley & Sons. New York. Cochrane, V. 1958. Physiology of Fungi. John Wiley & Sons. London. Couy, P.J. 1987. Modulation of sporulation of A/remaria ragetica by carbon dioxide. Mycologia 79:508-513. Daniel, J.T., Templeton, G.E., Smith, RJ. Jr. and Fox, T.W. 1973. Biological control of northem jointvetch in rice with an endemic fungal disease. Weed Sci. 21:303-307. De Dana, S.K. 1981. Principles and Practices of Rice Production. John Wiley & Sons. New York. Gomez, K.A. and Gomez, A.A. 1994. Statistical Procedures for Agricultural Research. 2nd Edition. John Wiley &.5ons, !nc.. New York. Gottlieb, D. 1978. The Germination of Fungus Spores. Meadowfield Press Ltd. UK. Grbavac, N. 1981. A simple technique for inducing sporulation in Drechslera graminea • in culture. Trans. Br. MycoL Soc. 77:218. 107 Hall. R.A. and Papierok. B. 1982. Fungi as biological control agents of arthropods of • agricultural and medical importance. Parasitology 84:205-240. Hildebrand, D.C. and McCain. A.H. 1978. The use of various substrates for large-scale production of Fusarium oxysporum f.sp. cannabis inoculum. Phytopathology 68:1099 1101. Holm, L.G., P1ucknett, D.L., Pancho, J.V. and Herberger, J.P. 1977. The World's Worst Weeds. D~bution and Biology. The University Press of Hawaii, Honolulu, Hawaii, USA. Kaiser, WJ., Hannan, R.M. and Rogers, J.O. 1994. Factors affecting growth and sporulation of Ascochytafabae f.sp. lentis. Plant Dis. 78:374-379. Kenney, O.S., Conway, K.E., and Ridings, W.H. 1979. Mycoherbicides-potential for commercialization. Dev. Ind. Microbiol. 20:123-130. Leach, CM. 1967. Interaction of near-ultraviolet light and temperature on sporulation of the fungiAlternaria, Cercosporella, Fusarium. Helminthosporium, and Stemphyllium. Can. J. Bot. 45:1999-2016. Purseglove, J.W. 1968. Tropical Crops - Dicotyledons. John WI1ey and Sons. Ine., New York. Purseglove, J.W. 1972. Tropical Crops - Monoeotyledons. John WI1ey and Sons, Ine., New York. SAS Institute Ine. 1987. SAS/STAT Guide for persona! computerS. Version 6. SAS • Institute Ine., Cary, NC. 108 Sivanesan, A. 1987. Gr:mùnicolous species of Bipolaris. Cun'ularia. Drechslera. • Exserohilum and their teleomorphs. Mycological Papers 158: 1-261. Stowell, LJ., Nene, K., Heath. B. and Shuner, R. 1989. Fermentation alternatives ior commercial production of a mycoherbicide. Pages 219-227 in Demain. A.L.. Somkuti. G.A.. Hunter-Cevera, J.e. and Rossmoore, H.W. (eds) Novel Microbial Products for Medicine and Agriculture. Society for Industrial Microbiology. Tuite, J. 1969. Plant Pathological Methods: Fungi and Bacteria. Burgess Publishing Co.• Minneapolis, MN. Walker, aL 1980. Alternaria macrospora as a potential biocontrol agent for spurred anoda: production of spores for field studies. V.S. Dep. Agric. Adv. Agric. Tech. South. Ser. 12 (ISSN 0193-3728). Walker, H.L 1981. Granular formation of Alternaria macrospora for control of spurred anoda (Anocia cristata). Weed Sci. 29:342-345. Walker, aL 1982. Seeding blight of sicklepod caused by Alternaria cassiae. Plant Sei. 66:426-428. Walker. aL and Riley, W.R. 1982. Evaluation of Alternaria cassiae for the biocontrol of sicklepod (Cassia obtusifolia). Weed Sci. 30:651-654. Watson, A.K. 1991. Prospects for bioherbicide development in Southeast Asia. Pages 65 73 in Swarbrick, R.K. (cd) Proceedings of 13th Asia Pacific Weed Science Society Conference. Asia-Pacific Weed Science Society and Weed Science Society of Indonesia, • Jakarta. 109 Watson, A. K. 1994. Cunent status of bioherbieide development and prospects for riee • in Asia. Pages 195-201 in Shibayama, H., Kritani, K. and Bay-Peœrson, J. (eds) Integrated Management of Paddy and Aquatic Weeds in Asia. FFTC Book Series No. 45. Food and Fertilizer Technology Centre for Asian and Pacific Regions, Taipei. Wmder, R.S. and van Dyke, c.G. 1990. The pathogenicity, virulence, and biological potential of two Bipolaris species on Johnsongrass (Sorghum halepense). Weed Sei. 38:89-94. Wymore, L.A., Poirier, c., Watson, A.K., and Gotlieb, A.R. 1988. Colletotrichum coccodes, a potential bioherbieide for control of velvetleaf (ADutilon theophrasn). Plant Dis. 72:534-533. Zhang, W. and Watson, A.K. 1996. Efficacy of Exserohilum monoceras for the control ofEchinochloa species in rice (Oryza saliva L.). Weed Sci. (Reviewed by IRRI and ready to he submitted). Zhang, W., Moody, K. and Watson, A.K. 1996. Responses of Echinochloa species and riec (Oryza saliva L.) ta indigenous pathogenic fungi. Plant Dis. (Submitted). • 110 Table 5.1. Effect of nutrient media on Exserohilum monoceras mycelial growth and • conidial production· Mean growth rate Number of conidia per plate Media (mmI24 h) (1 corn meal agar 4.3 d·· 2.03 bc centrifuged V-8 juice agar 11.4 ab 13.40 a Czapek-Dox Agar 11.6 a 0.07 c lima bean agar 10.7 ab 6.33 b malt extraet agar 6.9 c 0.77 c oatmeal agar 9.9 b 0.13 c PDA 10.0 b 0.13 c ln. PDA 8.9 b 0.08 c rice polish agar 10.5 ab 5.50 b V-8 juice agar 11.2 ab 14.90 a • Six replieate dishes on each medium were incubated at 28 C in the dark. Number of conidia per plate was counted at 2 weeks after incubation. Results are from pooled expaiments. - Values in each column sharing the same letter are not significanùy different according to Duncan's Multiple Range Test (P S 0.05) • 111 Table 5.2. Effect of light, dark, and near ultraviolet light (NUV) on mycelial growth and • conielial production of Exserohilum monoceras grown on V-8 juice agarO Mean growth rate Number of conielia per plate Treatment"° (mm/24 h) (l05) O Dark 11.2 a - 14.78 a Light 11.8 a 0.00 c Dark/light 11.4 a 1.07 b NUV 8.2 b 0.20 c NUV/dark 8.9 b 0.03 c ° The incubation temperature was 28 C. Number of conielia per plate was counted at 2 weeks after incubation. Results are from pooled experiments. 2 00 Dark = continuous darkness, Light = continuous light (80 J,1E'ffi' 'S'1), Dark/light = 2 12 h of altemating light (80 J,1E'ffi' 'S'1) and dark, NUV = continuous NUV, NUV/dark =12 h of altemating NUV and dark. - Values in each column sharing the same letter are not significantly different according 10 Duncan's Multiple Range Test (P ~ 0.05) • 112 Table 5.3. Effeet of various agriculturally-based produclS as salid substrates on conidial • production of Exserohilum monoceras' Solid substrate Conidialg substrate (x Ht) corn leaves 18.1 a" fresh corn seed 1.52 b dry corn seed Od cracked corn 0.25 c corn grilS 0.03 c corn rneal 0.12 c corn cob 0.005 d ricestraw 1.84 b polished rice 0.42 c riec grain 0.36 c rice kemel 021 c sorghurn grain 1.75 b wheat seed 0.12 c millet seed 0.02 c barley seed Od finely ground lima bean seed 2.18 b finely ground lima bean seed supplemented with 2% yeast and 0.5% dextrose 029c lima bean seed 0.009 d mungbean seed Od soybean seed Od cowpea seed Od coconut meat 0.1 c • AlI substrates were incubated at 28 C in the dark for three weeks. ResullS were from pooled experiments. •• Number in each column followed by the same letter are not significantly different • according 10 Duncan's Multiple Range Test (p S 0.05) 113 Table 5.4. Effect of substtate quantity and moisture content on conidial production of • Exserohi/um Tn01UJCeras on corn leaves· Quantity volume of water (ml) of dry corn leaves (g) 5 10 15 Conidialflask 1 1.1 x lOS c- (83.3)" 16.3 x lOS a (93.8) 1ù.0 x lOs b (95.2) 2 2.8 x lOS b (71.4) 10.6 x lOS a (88.2) 9.3 x lOS a (90.9) • 250 ml Erlenmeyer flasks were used as the fermentation vessel. Flasks were incubated at 28 C in the dark. Conidia were harvested 3 weeks after incubation. Results are from pooled experiments. - Values in a row followed by the same letter are not significantly different according to Duncan's Multiple Range Test cP S 0.05). - Values in parentheses were % moistllre content of corn leaves. • 114 • • Table 5.5. Effeet of conidial production methods on Exserohifum mOlloceras conidial production. germination. and virulence Incubation Conidia Germination Germ %Dry weight reduction• per plate tube Exp 1 Exp 2 Medium period or mie f1ask length Dew period (h) Dew period (h) (weeks) (x 10~ (%) Û1m) 8h 12 h 16 h 8h 12 h 16 h EZ8A" 1 2.8c"· 99.6a 299.0a 30.6b 19.8c 52.5b 50.3b lO0.0a 100.0a 2 15.8b 97.0a 187.2b 22.4b 42.0a 51.8b 45.5b l00.0a 100.0a 3 17.6ab 97.0a 159.6b 38.2a 47.6a 73.1a 80.9a 100.0a 100.0a .... 4 22.5a 95.7a 28.2c 16.4c 34.1 b 36.3c 40.7b 100.0a 100.0a v..... corn leaves 1 3.2c 99.0a 301.0a 31.7a 20.1c 40.1b 60.3b lO0.0a l00.0a 2 16.2b 97.3a 183.2b 25.3b 38.5b 58.9b 42.5c 100.0a lO0.0a 3 18.3ab 97.7a 163.7b 30.2a 50.5a 80.0a 79.9a 100.0a lO0.0a 4 20.8a 95.5a 35.0c 17.5c 38.2b 40.8b 43.5b 100.0a 100.0a • % dry weight reduction as an indication of conidial virulence. CaCO~, " EZ8A consisted of 163 ml V-8 juice. 0.75 g KH1P04• 1.5 g MgS04, 3 g 20 g agar. and 1 L Echillochloa leaf decoction• ... Values in a row for each medium followed by the same leller are nol. significantly different according to Duncan's Mulliple Range Test (P ~ 0.05). • Figure 5.1. Effect oftemperature on radial mycelial growth (A). conidial production (B). and conidial germination (C) ofExserohi/um monoceras grown on V-8 juice agar plates. The predicted equations are: (A) GROWTIi = exp(7.28 + 3.00lnT' + 1.451n(1 - T'». R2 = 0.93; (B) PRODUcnON = exp(14.83 + 8.49InT' + 8.671n(1 - T'». R2 = 0.99 (ternperature range from 20 to 35 C); and (C) GERMINATION =-92.41 + 16.43T 0.3~. R2 =0.82, where T =temperature and T' =(T - T..J/(T_ - T..J. • 116 • 80 • ~ E .§. 60 -5 e~ CIl 40 Ol :aou ~ 20 0 ~ B sou !1 ~ lS 1- ~ 0 -0 ou :a 10 1- "S 0 ...'" ..0 .8 S 1- E z= ) 0 • \ ~ c o "::1ou C "~ o S 10 lS 20 2S 30 3S 40 • Temperature (C) • Figure 5.2. Effeet of Echinochloa leaf deeoction on conidia! production CA), germination rate (B), and germ tube length Cc) of Exserohi/um menoceras grown on V-S juice agar CVA), lima bean agar (LBA), and potato dextrose agar (PDA). • 117 25 f-A a ~ ~ c:::J Without leaf decoction • 0 ~O<': 20 f- ~ With leaf decoction --!$. B os 15 - a ~ b '3 °ë a 0 10 - CJ b 5 f- o B 100 - ~ a a " 'â' ~ 80 - c: 0 0:: ~ os 60 c: 1~ ., 40 l- 0 20 I- 0 120 C ,... 100 l- b E a h ...... =- l- oS 80 co c:., - 60 I- b ~ .,§ 40 f- 0 20 I- o PDA LBA VA • Mediwn • Figure 5.3. Effect of incubation time on Exserohilum monoceras conielia production on corn leaves. One gram dry corn leaves wen: placcd in 25o-ml Erlenmeyer flasks, moistened with la ml elistilled water, and autoclaved two times for 15 min (100 kPa and 121 C). After cooling, flasks wen: inoculated and incubated at 28 C in the clark. • 118 • • • 20 • • • 1 ... • --0..... • ~ 15 .c -QI) .~ ~ ~ 10 :a 'ë 8 5 OL- .L.- -'-- ..L...- -'- ~ 1 2 3 4 5 6 Incubation lime (week) • • Connecting Text The delimitation of host range is an important component of biological weed control programs. especiaIly when using the classical or inoculative approach. (iost specificity of the candidate agent must assure the safety of desirable plants if the biocontrol agent is to be released. Since indigenous plant pathogens are being evaluated as bioherbicides, strict host specificity is not required but must be demonstrated to be "sufficiently safe" (Wapshere, 1982; Watson, 1985). In this chapter, the findings of host range experiments for the fungal pathogen, E. menaceras are reported. • 119 • Chapter 6. Host range of ExserohiJum monoceras 6.1. Abstract Fifty-four plant species in 43 genera and 19 families. selected by using the centrifugal phylogenetic method. were screened against Exserohilllm monoceras. an indigenous fungus of Echinoch/oa species. Trials were performed under both optimum greenhouse conditions with supplemental 24 h dew and sub-optimal conditions where no dew supplement was provided. The compatibility of host-pathogen interactions wa.~ characterized by the time and level of sporulation observed on detached inoculated leaves. Ali species of the ge:lus Echinoch/oa were proved to be highly susceptible to this fungus under optimum greenhouse conditions as well as in the absence of a dew supplement Rottboellia cochinchinensis was also highly susceptible to this pathogen in either the presence or absence of a supplemental dew period. Arnong those crops tested. only corn seedlings were slightly infected under supplemental 24 h dew conditions, but no disease symptoms were observed on corn in the absence of a dew supplement Sorghum and sugarcane were hypersensitive to the pathogen under supplemental 24 h dew conditions, but no infection was detected in the absence ofa dew supplement Ali other species tested were immune to E. monoceras. Data on sporulation on detached leaves from inoculated plants indicated that E. monoceras was weakly compatible with corn. No disease symptoms and no conidia were detected on leaves of trap plants. In the field, E. monoceras did not produce any symptoms on corn plants. 6.2. Introduction The genus Echinoch/oa (poaceae) contains several important weed species. including Echinochloa crus-ga//i (L.) Beauv. (bamyardgrass), Echinoch:oa c%na (L.) Link. Gunglerice), and E. g/abrescens Munro ex Hook.F.. E. crus-ga//i is ranked as the world's third worst weed and has been listed as a weed in 36 different crops in 61 countries (Holm et al, 1977). E. c%na is ranked as the world's fourth worst weed and • has been listed as a principal or most serious weed in 35 different crops in more than 60 120 countries (Holm et al.. 1977). Yields of a number of important crops in many countries • are substantially reduced by these IWO Echinoch/oa species. These include banana (Musa sapientum L). cassava (Manihot escu/enta Crantz.). cotton (Gossypium hirsutum L.). corn (Zea mays L.). millet (Pennisetum g/aucum (L.) R.Br.). potato (So/anum tubervsum L.). sorghum (Sorghum bic%r (L.) Moench). and taro (C%casia escu/enta Schon.) (Holm et al.. 1977). E. crus-ga//i. E. c%na. and E. g/abrescens are the three most serious weeds in rice (Oryza sativa L.) (Holm et al.• 1977: De Dana. 1981). Intense competition from E. crus-gal/i can reduce tillering in rice by up to 50% (Holm et al.• 1977). Similarly. Smith (1968) reported that season-Iong competition from E. crus-gal/i reduced rice grain yields by 70%. Rice yield reductions of 25% were observed at E. c%na densities of 80 plants/m' during the initial 40 days of growth with this crop (Mercado & Talatala, 1977). Although E. c%na is a less vigorous competitor tha., E. crus-gal/i. E. c%na can pose a serious problem in rice systems because typical populations of this weed can greatly exceed the 80 plants/m' critical density. Yield losses caused by E. g/abrescens are si:nilar to those of E. crus-gal/i (Krishnamurthy et al., 1989). These Echinoch/oa species can be controlled through various management strategies such as hand weeding, cultural and mechanical methods as weil as chernical herbicides (Matsunaka, 1983). However, each of these strategies has important limitations. Recently, the possibilities of utilizing biological control agents against these Echinoch/oa species are being evaluated (Scheepens. 1987; Chung et al., 1990; Bayot et al.. 1994; Gohbara & Yamaguchi, 1994; Wapshere, 1994; Watson, 1994; Zhang et al., 1996). A funga! pathogen, Exserohi/um monoceras (Drechsler) Leonard & Suggs, was isolated from diseased E. c%na leaves collected in nce fields at the International Rice Research Institute (IRRI), P.O. Box 933, 1099 Manila, Philippines (Zhang et al., 1996). Inoculation with this pathogen initially results in dark-brown longitudinal necrotic streaks (0.3-3 mm long) which are often restricted to area between leaf veins and ~osed leaf sheaths of infected Echinoch/oa plants. Within 3-4 days of inoculation, a blight-like syrnptorn is observed on plants. Under greenhouse conditions. all E. crus-gal/i, E. colona, • and E. glabrescens seedlings at the 1.5-leaf stage were kil1ed by the pathogen when 121 applied at densities of 2.5-5.0 x 107 conidia/m' over a range of dew period durations • (Zhang et al.. 1996). Field trials using this pathogen resuIted in severely diseased Echinochloa seedlings (personal observation). Therefore. there is potential to use E. monoceras as a bioherbicide for the control of Echinochloa species. Host specificity of a biocontrol agent is one of the most important factors to consider in a biological weed control program. It is critical that the safety of desirable plants is not threatened by the release of a potential biocontrol agent (Hasan. 1983: Schroeder. 1983: Watson. 1985). To date. the host range of the fungal pathogen E. monoceras has not yet been studied. ThUs. the objective of this study was to d..,termine the host range of Exserohilum monoceras and estimate the potential risks involved in its use as a biological control agent of Echinochloa species in rice. 6.3. The organisms 63.1. The rarger weeds: Echinochloa species Echinochloa belongs to the tribe Paniceae. subfamily Panicoideae. Poaceae Family. Cypera!es OrcIer (Cronquist, 1981; Gould & Shaw. 1983). 6.3.1.1. Echinochloa crus-galli (L.) Beauv. E. crus-galli is believed native to Europe or India (Holm et al.• 1977; Maun & Barrett, 1986). It is now widely cosmopolitan and occurs throughout the tropical and temperate regions of the world from latitude 50 N to 40 S. E. crus-galli is an erect, clumped, C. annual grass growing up to 1.5 m high. with branching stems near the base and rooting when decumbent. Leaves are fiat, tapering to a point, hairless. or with a few hairs on the margins near the broad base. Inflorescences typically consist of 15 greenish (often tinged with purple) spikelets. Aowering occurs year-round in the Philippines, with each plant producing at least 200 seeds. Reproduction is exclusively via seed. Sorne seeds genninate immediately, although others remain viable in soil for severa! years. E. crus galli is a very morphologically variable species with numerous ecorypes present around • the world. 122 6.3.1.2. Echinoch/oa co/ana (L.) Link. • E. co/ana is native to India but its present range extends from latitude 45 N to 40 S (Holm et al., 1977). This annual, C. grass has prostrate seedlings which may attain 70 75 cm in height at maturity. E. co/ana closely resembles E. crus-ga//i, however, E. co/ana individuals do not possess a ligule, have red-putple tinged leaf sheaths and blades, typically possess awnless spikelets, and have smaller seed (caryopsis). E. co/ana can be best distinguished from E. crus-ga//i by the absence of awns on its spikelets. 6.3.1.3. Echinoch/oa g/abrescens Munro ex Hook.F. E. g/abrescens is wide::pread from the India subcontinent through mainland Southeast Asia, China to Korea and Southem Japan (pancho, 1991). It is also been reported in Togo, West Africa (Pancho, 1991). E. g/abrescens is very similar to E. crus ga//i but only grows 0.5-1 m in heighl The leaf blade is acuminate WiL't leaf sheaths almost closed and often flanened. Awns. ifpresent, are shorter than those of E. crus-ga//i (about 1 cm long). The seeds (caryopsis) of E. g/abrescens are larger than those of E. crus-ga//i. 6.32. The biocontro/ agent: Exserohilum monoceras (Drechs/er) Leonard & Suggs Exserohilum monoceras is a species of a dematiaceous hyphomycetes (Fungi Imperfecti (Ellis, 1976). It has been associated with Setosphaeria monoceras Alcom teleomotph, which is included in the Family Pleosporaceae, the Ortler Pleosporales, Ascomycotina (Alcom, 1978; Sivanesan, 1987). The motphological and growth aspects of E. monoceras isolated from the Philippines are simi1ar to the descriptions by AIcom (1983) and Sivanesan (1987) (personal observation). The Conidia were fusoid in shape, brown in color, 15.0-21.5 x 51.3-139.9 J.lm in size with 3-8-distosepte, and had a srnall protruding basal hilum. The conidia germinated from both polar cells in semiaxial direction. The frrst septum was formed close to hilum (submedian), the second supramedian, and the third median. A black pigment was usually observed on agar media • 7 days after culture. 123 E.r:serohilum Leonard & Suggs. Bipo/aris Shoem. and Drechs/era Ito are the • genera to describe the graminicolous species ofHe/minrhosporium Link and are associated with Serosphaeria. CochUobo/us. and Pyrenophora teleomorph. respectively (Alcom. 1983; Sivanesan. 1987). Bipo/aris and Cun'u/aria Boedijn anamorphs have been associated with the same teleomorph CochUobo/us Drechsler (Sivanesan. 1987). Therefore. Bipo/aris. Cun'u/aria. Drechs/era. and He/minrhosporium are considered to be related to Exserohi/um. There are now 20 formally described species in Exserohi/um. 52 in Bipo/aris. 23 in Drechs/era. and 32 in Curvu/aria (Sivanesan. 1987). Sorne of these are important species causing economic losses in several important grass crops. Important diseases caused by Exserohi/um species include: E. rosrrarum (Drechsler) Leonard & Suggs with teleomorph Serosphaeria rosrrara Leonard. which causes leaf spots and foot rot of wheat (Triticum aestivum L). seedling blight of bermuda grass (Cynodon dacty/on (L.) Pers.). leaf blight of E/eusine. damping off of sugarcane (Saccharum officînarum L.) seedlings. stalk rot and ear rot of corn (Zea mays L.); E. rurcicum (pass.) Leonard & Suggs with teleomorph S. turcica (Luttr.) Leonard & Suggs (Anahosur. 1977). causing northern leaf blight of corn and sorghum (Sorghum bie%r Pers.). Reported hosts of E. monoceras are Echinoch/oa. Oryza. Panicum. and Seraria (Sivanesan. 1987; Farr et al.• 1989). Its distribution has been reponed in nine countries: Australia. Cuba. India. Israel. Japan. Korea. Turkey. Russia. U.S.A.. and Yugoslavia (Sivanesan. 1987; Chung et al.• 1990; Gohbara & Yamaguchi. 1994). This is the first repon of this pathogen in the Philippines. 6.4. Materials and methods 6.4.1. 1noculum production A single-conidium isolate of Exserohi/um monoceras growing on half-strength potato dextrose agar (1/2 PDA) (Difco. Detriot, MI) slants in small vials was maintained under minerai oil al4 C as a stock culture (Tuite. 1969). Small pieces ofmycelium from • the stock culture were aseptically transferred to PDA in petri ùishes. Each cul:ure was 124 sealed with parafilm and incubated at 28 C for 7 days. Agar plugs (6-mm diameter) • containing mycelia were removed from the margin ofthese young colonies and were used to inoculate V·8 juice agar (VA) plates (fuite. 1969). Inoculated VA culture plates were s~ed with parafilm and incubated at 28 C in the dark for 3 wk. Conidia were harvested from the culture plates by f100ding with 10 ml distilled water and scraping the surface of the colonies with a glass slide. The resulting suspc:nsion was filtered through a layer of cheesecloth. Inoculum concentration was determined with the aid of haemocytometer and adjusted to the desired density with water. 6.42. Plant Production Using the modified centrifugaI phylogenetic and varietaI strategy (Wapshere. 1974), 54 plant species were selected for the host range trial (fable 6.1). Smce E. monoceras was isolated in the Philippines, most test plants used were common to this region of the world. Tested plants belonged to one of three groups: (A) taxonomically related plants, (B) economically important plants known to be anacked by species of Exserohilum and associated species of Bipolaris, Curvularia, Drecheslera, Helminthosporium, and their teleomorphs, and (C) economically important plants commonly found in close proximity to irrigated rice fields. The phylogenetically related plants of group A were based on the systematic classification of Cronquist (1981). The order Cyperales has two families: the Cyperaceae and the Poaceae. The Cyper.lceae are of Iittle economic importance, but the Poaceae (grasses) are a most important family. Triticwn, Ory:a, Zea, Saccharum, Sorghum, and Hordeum are among the most important genera within the Poaceae (Cronquist, 1981). Plants in group B are those plants reponed to be attacked by the candidate age.'lt, E. monoceras, as weIl as host plants of fungal species closely related 10 the candidate agent. Bipolaris, Curvularia, Drecheslera, and Helminthosporium are considered genera closely related to Exserohilum (Alcom, 1983; Sivanesan, 1987). Designations for group B follow Tangonan & Quebral (1992) while Group C plants are designated according 10 Harwood (1977), Carandang et al. (1977), • Aycardo (1977), and Gomez & Gomez (1983). 125 6.43. General inoculation procedure • For each species, four sets of test plants (each set containing 10 plant.~) were prepared. Two sets were inoculated with E. monoceras and the other {Wo sets served as uninoculated controls. For the inoculation treatment, seedlings at the 1- to 3-leaf stage were inoculated with a conidial suspension of E. monoceras at a rate of 5.0 x 107 conidialm2 with 0.05% Tween 20 as a werting agent, using a motor sprayer at a pressure of 100 kPa (A. H. Thomas Co. Scientific Apparatus, Philadelphia). For the uninoculated control. seedlings at the 1- to 3-leaf stage were sprayed with water containing 0.05% Tween 20. One inoculated set and one uninoculated set of test plants were exposed to a 24 h dew at 25 C in the dark and then \Vere transferred to the greenhouse. The other inoculated and uninoculated set of test plants were placed outdoors immediately after inoculation and exposed to prevailing natural conditions (in the absence of a dew supplement). The experiment was conducted as a factorial experiment with species tested, inoculation level (0 and 5 x 107 conidialml. and dew supplements (0 and 24 h) as factors. Each treatrnent was replicated four rimes and pots were placed randomly in the greenhouse and outdoors. Plant-pathogen interactions were evaluated using {Wo pararneters: disease severity and sporulation. Disease severity was visually assessed daily for 2 weeks following inoculation by using a disease rating scale of NS (no symptoms), HS (hypersensitive response, pinpoint lesions which did not increase in size), L (light infection, symptorns present, secondary spread Iimited), S (severe infection, systemic spread ofpathogen, some plants killed). After the rating of disease severity, one or {wo inoculated leaves were excised from each replicate for both the tr<:ated and untreated plants. Excised leaves were surface-sterilized, rinsed, and then incubated on moistened filter paper in petri dishes at 28 C in the dark. After 24, 48, and 72 h of incubation, sporulation on leaves was exarnined with a stereo microscope. Sporulation \Vas rated at three levels: - = no sporulation; + =light sporulation; ++ =moderate to heavy sporulation. Time and the level of sporulation were used to characterize the compatibility of host-pathogen interactions. • Ifno sporulation occurred within 72 h on excised leaves, then the fungus was designated 126 • as being incompatible with the host plant 6.4.4. Disease de\'elopment on trap plants Since corn was infected by E. monoceras under optimum greenhousc conditions. spore dispersal and transport were examined by using the trap plant technique (Pinnschmidt et al., 1993). Trap plants were corn and E. crus-galli grown from seed in a disease-free environment in the greenhouse. Seed of each species was incubated in petri dishes on moistened filter paper for 48 h at room temperature. Pive genninated seeds (coleoptile and radicle just emerged) were planted per lO-cm diameter plastic pot fil1ed with saturated soil (Maahas clay. Haplustic suborder). Seeded pots were placed on a push cart in the greenhouse. Greenhouse conditions were 35!25 ± 5 C day/night temperature, a 12-h photoperiod. and an average light intensity of 20 MJ/m2 per day. Fifty Wagner's pots with E. crus-galli at the 1.5-leaf stage were inoculated with 7 2 E. monoceras at a rate of 5 x 10 conidialm • After inoculation, ail pots were placed outside the greenhouse and covered with plastic bags for 24 h to maintain high humidity leveIs. Severe disease was observed for plants placed in Wagner's pots in 2-3 days. Pive days after inoculation. IWO sets of trap plants (i.e. corn plants at the 2-leaf stage and E. crus-galli plants at the 1.5-leaf stage) were placed around the inoculated pots in four directions at a distance of 0.5 ln, 1.0 m, 20 m, 5.0 ln, and 10 m. Trap plants were replaced weekly around inoculated pots so that trap plants werc present up to 3 weeks fol1owing inoculation. For each week of exposure, one set of trap plants was returned to a dark dew chamber for 24 h at 25 C and then transferred to the greenhouse to enable any conidia produced and deposited during the exposure week to germinate and possibly cause host plant lesions. The number oflesions on trap plants was counted. The other set oftrap plants was taken to the laboratory in order to obtain leafprints (Pinnschmidt et al, 1993). lmmediately after leaf prints were made, leaves were placed in petri dishes lined with mOÎStened filter paper and incubated for 5 days at 28 C in the clark. The number of lesions on each leaf blade was counted. • The experirnent was conducted twice during the dry season and twice during the 127 • wet season of 1994. 6.45. Field inoculation ofcorn Various corn cultivars were evaluated for their response to E. monoceras under the field conditions. Pirsabak. IPB varl. and super sweet were the corn cultivars used in the experiment. Seed of cultivars was directly sown into the field. Cultivars (three replicates per cultivar) were arranged in a completely randomized design (CRD) and were spaced 0.3-m apart in four 2-m rows. E. monoceras was inoculated onto corn seedlings 7 2 at the 2-leaf stage at a rate of 5 x 10 conidia/m • Infection response (size and type of lesion) and disease severity (percentage of lea! area infected) were assessed weekly for 2 months. Leaf samples from cultivars exhibiting foliar disease symptoms were collected. surface sterilized. and isolated. Cultural and conidial characteristics of isolated organisms were compared with those of E. monoceras. 6.5. Results 65.1. Host range screening No lesions appeared on any check plants. Both Echinochloa species and Rottboel/ia cochinchinensis (Lour.) W.O. Clayton. were severely infected in both the supp1emented 24 h dew treatment and the non-supplement dew treatment (Table 6.2). Ali seedlings ofthe three Echinochloa species as weil as those ofRottboe//ia cochinchinensis were killed when subjected to a 24 h dew. Oisease intensity on Echinochloa species and Rottboel/ia cochinchinensis seed1ings was found to be 60-80% in the non-supplemented dew treatmenL Corn showed light syrnptoms with limited expansion oflesions under the 24 h dew period treatmenL Lesie-n expansion covered less than 5% of infected leaves 2 weeks after inoculation and no stern lesions were observed. Lesions did not spread 10 emerging uninocuIated leaves. In contrast. no symp10ms were observed on corn in the absence of a supplemental dew period (Table 6.2). • Sorghum and sugarcane exhibited a hypersensitive response 10 E. monoceras for 128 the 24 h dew period tteatmenl SmaIl «1-2 mm) brown flecks were observed on leaves. • but these did not expand in size during the 2 week observation period. No symptoms were detected for the non-supplemental dew treatment (Table 6.2). No other monocotyledonous plants were infected by E. monoceras for either dew treatmenl Ali dicotyledonous plants were immune tO E. monoceras. Sporulation on detached incubated leaves showed a different pattern of interaction between E. monoceras and its plant hosts (Table 6.3). Heavy sporulation occurred on detached inoculated Echinochloa leaves following a 24 h incubation period in the presence or absence of supplemental dew. Heavy sporulation was aIso observed for Rottboellia cochinchinensis inoculated leaves following 24 h incubation for the 24 h dew treatment and following 72 h incubation in the absence of supplemental dew. Light sporulation was observed on detached inoculated corn leaves following 72 h incubation for the 24 h dew treatment but no sporulation occurred on corn leaves in the absence of supplemental dew. No sporulation occum:d on other plant species. 6.52. Disease development on trap plants During both the dry and wet seasons. no lesions were detected on trap plants and no conidia were counted on leaf prints. 6.5.3. Field inoculation ofcorn A few lesions were observed on corn at the heading stage, however, the fungus differed from E. monoceras in terms of cultural and conidial characteristics. 6.6. Discussion Host range testing has evolved from an early emphasis on testing important crops in the area of proposed introduction of the biocontrol agent te the present emphasis on a rational biological basis for host range testing (Harris & Zwlilfer, 1968; Wapshere, 1974). Wapshere (1974) proposed a testing strategy for c1assical biocontrol agents based • on the pbylogenetic relationships of the target weed. This approacb bas received wide 129 acceptance in biological weed control research. In this study. the phylogenetic rnethod of • selecting test plants was also used. The results demonstrated that the host range of E. monoceras is restricted to the genera: Echinochloa. Zea. and Rottboellia in the subfamily Panicoideae of the Poaceae family (Gould & Shaw. 1983). More detailed research is. however. required to determine whether other tribes within the Poaceae family may be susceptible to E. monoceras. Host range tests for 54 plant species in 43 genera and 19 families showed that corn was the only economically important crop to be infected. However. infection was light, disease did not spread to emerging uninoculated leaves. and no stem lesions were observed when the fungus was provided optimum growth conditions (i.e. 24 h dew at 25 C) (Zhang & Watson. 1996). In the absence of supplemental dew. no disease symptoms were detected on corn while a 60-80% disease intensity was observed on Echinochloa seedlings. Light sporulation was observed on derached inoculated corn leaves 72 h after incubation when plants were provided with a 24 h dew. Alternatively. no sporulation occurred on corn leaves when plants did not receive supplemental dew. These findings suggest that corn was slightly compatible with this pathogen under optimal growing conditions. However. trap plants exposed to severely diseased Echinochloa species for one week did not develop any symptoms following a 24 h incubation in a dark dew chamber. Similarly. no conidia were found on leaf prints. These results indicate !hat E. monoceras has a poor disserninating ability. It is also apparent after a number of field trips that E. monoceras-infected Echinochloa plants are rather rare under natura! conditions. Field inoculations demonstrated that corn was not infected by this pathogen. This is consistent with reports showing that corn is not a host of the fungus in all nine countries where E. monoceras is found, including the Philippines (Sivanesan. 1987; Farr et al. 1989; Tangonan & Quebral. 1992). This is the fust report of E. monoceras in the Philippines. Host specificity is an important component in biological weed control The safety of non-target econornic and wild plants must be assured before a pathogen is widely used in the field. But in the bioherbicide strategy. wherein augmentation of indigenous weed • pathogens is being considered, host specificity is a less rigorous requirement since the 130 biocontrol agent is already present in the environment and non-target plants should have • already becn exposed to the pathogen (Watson. 1985). For example, COLLEGO"'. a commercialized bioherbicide consisting of the fungus Colletotrichum gloeosporioides (Penz.) Sace. f. sp. aeschynomene used for the control of northem jointvetch (Aeschynomene virginica (L.) B.S.P.), is restricted to the subfamily Papilionoideae which has severa) economically important crops that are susceptible to this fungus. However. there have becn no reports of damage on non-target plants after commercial application of COLLEGO'" (Weidemann & TeBeest, 1990). Likewise, E. monoceras should be safe to use in fields where no corn is present. Risk analysis using modelling procedures proposed by Jong et al. (1990) might provide more insight into the risks involved in releasing E. monoceras. E. monoceras produced severe disease on bath Echinochloa andR. cochinchinensis under bath optimum greenhouse conditions and in the absence of supplemental dew. Therefore, E. monoceras also has the potential to control R. cochinchinensis. R. cochinchinensis, thought to be native to India, is an important weed that has already becn targeted within a bioherbicide program (Evans, 1987). However. there are no reports of E. monoceras occurring on R. cochinchinensis. Thus, further studies are needed to determine the efficacy of E. monoceras in controlling R. cochinchinensis. Differences in hosts ofE. monoceras are obvious. The results in this srody indicate that rice is not the host of E. monoceras, which is in agreement with the reports by Farr et al. (1989) and Gohbara & Yamaguchi (1994), but contrasts to the reports liy Sivanesan (1987) and Chung et al. (1990). Chung et al. (1990) reported that corn was moderately susceptible to E. monoceras, our results also find that E. monoceras infects corn leaves. However, corn was not reported as hosts of E. monoceras elsewhere. Setaria viridis was not infected by E. monoceras in this study, but was listed as host of E. monoceras in the United States (Farr et al., 1989). This study is the first report ofRottboellia as a host of E. monoceras. The differences in host susceptibility may suggest that these E. monoceras • isolates cao be differentiated at either forma specialis or race leveL 131 6.6. Literature cited • Alcom. J.L. 1978. Setosphaeria monoceras sp. nov.• ascigerous state of Exserohilum monoceras. Mycotaxon 7:411-414. Alcom, J.L. 1983. Generic concepts in Drechs/era. Bipolaris and Exserohilllm. Mycotaxon 17:1-86. Anahosur, K.H. 1977. CM! Descriptions of Pathogenic Fungi and Bacteria No. 304. Commonwealth Mycological Institute. Kew, UK. Aycardo. H.B. 1977. Multiple cropping with vegetables. Pages 15-25 in Multiple Cropping Sourcebook. University of Philippines at Los Baiios, College of Agriculture. Bayot, R.G., Watson. A.K. and Moody, K. 1994. Control of paddy weeds by plant pathogens in the Philippines. Pages 139-143 in Shibayama, H. Kiritani, K and Bay Petersen, J. (eds) Integrated Management of Paddy and Aquatic Weeds in Asia. FFTC Book Series No. 45. Food & Fertilizer Technology Centre for the Asian and Pacific Region. Taipei. Carandang D.A., Harwood, R.R. and Barile, C. 1977. Multiple cropping systems based on rice. Pages 11-14 in Multiple Cropping Sourcebook. University of Philippines at Los Baiios, College of Agriculture. Chung, Y.R., Kim, B.S., Kim, H.T. and Cho, K.Y. 1990. Identification of Exserohi/um spedes. a fungal pathogen causing leaf blight of bamyardgrass (Echinoch/oa crus-gal/j). Korean Journal of Plant Pathol. 6:429-433. Cronquist, A. 1981. An Integrated System ofOassification ofFlowerlng Plants. Columbia • Univ. Press, New York. 132 De Dana. S.K 1981. Principles and Practices of Rice Production. John Wiley & Sons. • New York. Ellis. M.B. 1976. More Dernatiaceous Hyphomycetes. Commonwealth Mycological Institute. Kew. UK Evans. H.C. 1987. Fungal pathogens of some subtropical and tropical weeds and the possibilities for biological control. Biocontrol News and Information 8:7-30. Farr. D.F.• Bills. G.F.• Chamuris. G.P. and Rossman. A.Y. 1989. Fungi of Plants and Plant Products in the United States. APS Press. St. Paul. MN. Gohbara, M. and Yamaguchi. K. 1994. Biologica1 control agents for rice paddy weed management in Japan. Pages 184-194 in Shibayama, H. Kiritani. K and Bay-Petersen. J. (eds) Integrated Management of Paddy and Aquatic Weeds in Asia. FFfC Book Series No. 45. Food & Fertilizer Techno1ogy Centre for the Asian and Pacific Region. Taipei. Gomez A.A. and Gomez, KA. 1983. Multiple Cropping in the Humid Tropics of Asia. The International Development Research Centre. Ottawa, Ont.. IDRC. Gould F.W. and Shaw. R.B. 1983. Grass Systematics. Texas A&M University Press. Harris. P. and Zwô1fer. H. 1968. Screening ofphytophagous insects for biological control of weeds. Can. Ent. 100:295-303. Harwood. R.R. 1977. Intensification of cropping: principles and methods. Pages 1-8 in Multiple Cropping Sourcebook. University of Philippines at Los Baiios. College of • Agriculture. 133 Hasan. S. 1983. Biological control of weeds with plant pathogens - status and prospecL~. • Pages 759-776 in Proc. 10Ùl InL Congr. Plant ProL. Australia (Vol. II.). Holm. L.G.• Plucknett D.L. Pancho. J.V. and Herberger. J.P. 1977. The World's Worst Weeds. Distribution and Biology. The University Press of Hawaii. Honolulu. Jong. M.D. de. Scheepens. P.C. and Zadok. J.C. 1990. Risk analysis for biological control: a Dutch case study in biocontrol of Prunus serotina by the fungus Chondrostereum purpureum. Plant Dis. 74:189-194. KrishnamurÙly. K.. Devendra. R.. Prasad. T.V.R. and Mohan. S.L 1989. GrowÙl pattern of Echinochloa species in relation to rice and bio-efficacy of 2.4-0 and dicamba combinations. Pages 683-688 in Proceedings of Brighton Crop Protection Conference Weeds. Vol. 3. Brighton. England. Maun. M.A. and Barret, S.C.H. 1986. The biology of Canadian weeds. 77. Echinochloa crus-galli (L.) Beauv. Can. J. Plant Sci. 66:739-759. Mercado. B.L and Talata:.... R.L 1977. Competitive ability of Echinochloa colona L. against direct-seeded lowland rice. Pages 161-165 in Proc. 6Ùl Asian-Pacific Weed Sci. Soc. conference. Korea. Moody. K. 1989. Weeds Reported in Rice in SOUÙl and SouÙleast Asia. International Rice Research Institute. P.O. Box 933. 1099 Manila. Philippines. Pancho. J.V. 1991. Grass weeds in the Philippines. Pages 183-188 in Baker. F.W.G. and Terry. PJ. (eds) Tropical Grassy Weeds. CAB International for CASAFA. Wallingf~rd. UK. 134 Pinnschmidt, H.O.• Klein-Gebbinck. H.W.. Bonman. J.M. and Kranzet, J. 1993. • Comparison of aerial concentration. deposition. and infectiousness of conidia of Pyricularia grisea by spore-sampling techniques. Phytopathology 83:1182-1189. Rao. A.N. and Moody. K. 1992. Competition between Echinochloa glabrescens and rice (Oryza saliva). Tropical Pest Management 38:25-29. Scheepens. P.C. 1987. Joint action of Cochliobolus lunarus and atrazine on Echinochloa crus-galli (L.) Beauv. Weed Res. 27:43-47. Schroeder. D. 1983. Biological control of weeds. Pages 41-78 in Fletcher. W.W. (ed.). Advances in Weed Research. Commonwealth Agricultural Bureaux. Farnharn Royal. Sivanesan. A. 1987. Graminicolous speci~ of Bipolaris. Curvularia. Drechslera, Exserohilum and their teleomorphs. Mycol. Pap. 158:1-249. Smith, R.J., Jr. 1968. Weed competition in rice. Weed Sci. 16:252-255. Tangonan. N.G. and Quebral, F.C. 1992. Host index of plant disease in the Philippines. 2nd Edition. The Deparo-nent ofScience and Technology, Bicutan, Taguig, Metro ManiIa. Tuite, J. 1969. Plant Pathological Methods: Fungi a~ld Baeteria. Burgess Publishing Co., Minneapolis, MN. Wapsherc. AJ. 1974. A strategy for evaluating the safety of organisms for biological weed control. Annu. Appl. Biol. 77:201-211. 135 Watemouse, D.F. 1994. Biological control of weeds: Southeast Asian Prospects. • Austtalian Centre for International Agricultural Research Monograph No. 26. Can~"l<:rra, Austtalia. Watson, A.K. 1985. Host speeifieity ofplant pathogens in biological weed control. Pagcs 99-104 in Delfosse, E.S. (ed) Proceedings of VI Int Symp. Biol. Contr. Weeds. British Columbia: Agriculture Canada. Watson, A. K. 1994. Current status of bioherbieide development and prospects for rice in Asia. Pages 195-201 in Shibayama, H., Kritani, K. and Bay-Peterson, I. (eds) Integrated Management of Paddy and Aquatic Weeds in Asia. FFfC Book Series No. 45. Food and Fertilizer Technology Centre for Asian and Pacific Regions, Taipei. Weidemann, G.I. and TeBeest, 0.0. 1990. Biology of host range testing for biocontrol of weeds. Weed Technology:465-470. Zhang, W. and Watson, A.K. 1996. Efficacy of Exserohilum monoceras for the control ofEchinochloa species in rice (Oryza sativa L.). Weed Sei. (Reviewed by IRRI and rcady to he submitted). , Zhang, W., Moody, K. and Watson, A.K. 1996. Responses of Echinochloa speeies and rice (Oryza sativa L.) to indigenous pathogenic fungi. Plant Dis. (Submitted). • 136 • • Table 6.1. List of test plant species used for host-specifieity sereening of Exserolrl/lln1 nlOlloeeras against EehilloelJ/oa speeies A. Economieally important plants and taxonomieally related to EehilloelJ/oa speeies Poaeeae 1. E. erllS-galli var. allstro·japonensls Ohwi. 2. E. erlls-galli var. hiSlidll 3. E. eolona (Linn.) Link. 4. E. glabreseells Munro ex Hook 5. Pellniselllnl glallellm (L.) R. Br. 6. TrlliCl/nI aeslil'lIn1 L 7. Oryza saliva L. Indiea: Dee·Geo-Woo-Gen IR66 IR68 IR70 IRn IR74 laponiea: Chianung 242 Pela Chianan 8 KaoshiulIg -~ lavaniea: Brondol putih Rodjolele 8. Bordellnl vlIlgare L. 9.Saeehartlnl officfllartlnl 1.. 10. Zea nlays L. Pirsabak !PB var 1 !PB var 2 Los Banos lagkitan Super sweet 11. Sorghllm ble%r (L.) Moeneh ICSV 217 UPLSG 5 12. Leploeh/oa ehlnellsls (L.) Nees. Cyperaeeae 13. Cyperlls difformls L. 14. Cyperlls Irla L. 15. Cyperus rOllll/dllS L. 16. Flnlbrlstylfs nlifiaeea (L.) Vahl. Liliaeeae 17. Allillnl sativllnI L. 18. Aflfllnl eepa L. • • Table 6.1. (Continued) B. Plant species reported to he infected by species of B/polar/s, Curvular/a, Drechslera, Exserohllum, Heimilllhospor/um, and their teleomorphs in Philippines Amceae 19. Colocas/a esculellla Scholl. C8rlcaeae 20. Car/ca papaya L. Convolvulaceae 21. lpomoea balala (L.) Lam. CUcurbitaceae 22. Cllrul/us lallalus l,Thunb.) M&N. 23. Cucurblta maxima Duch. Dioscoreae 24. D/oscorea alala L. -~ Euphorbiaceae 25. Mall/hol esculellla Crnntz. Fabaceae 26. Glycine max (L.) Merr. 27. Viglla rad/ota (L.) R. Wilczek 28. Viglla ullgu/culala (L.) Walp. var. ullgu/culala 29. Arachls hypogaea L. 30. Leucaella leucocephllia (Lam.) DeWit. Malvaceae 31. Gossyp/um h/rsulum L. Musaceae 32. Musa texlllis Nee. 33. Musa sap/ell/II/II L. Palmae 34. Cocos lIl/ci/era L. Poaceae 35. Roltboel1la coclrlllch/llells/s (Lour.) W.D. Clayton. 35. Eleus/lle /Ild/ca (L.) Oaertn. Solanaceae 36. Lycopers/coll esculelllum L. • • Table 6.1. (Continue) C. Crops. commonly found in close proximity 10 rice fields in Philippines Apiaceae 37. Daucus carola L. Asleraceae 38. Lacluca saliva L. Brassicaceae 39. Brassica o/eracea L. var. capilala 40. Brassica pekil/esis (Lour.) Rupr. ,1 41. Brass/ca juncea (L.) Coss. 42. Raphal/us salivus L. Cucurbitaceae 43. Cucumis me/o L. 44. M01llordica charal/lia L. ... 45. Cucumis salivus L. 46. Lagel/aria /eucanlha (Lam.) Rusby. ~ Fabaceae 47. Phaseo/us /ul/a/us L. 48. Psophocarpus lelragol/%bus (L.) OC. 49. Phaseo/IIS vu/garis L. Malvaceae 50. Abe/moschus escu/enlUs (L.) Moench. Solanaceae 51. Caps/cum al/I/I111111 L. 52. CapsicuIII chil/ese L. 53. So/a1/l11ll 1IIe/ol/gel/a L. Zingiberaceae 54. Zil/giber ojJicil/a/e Rose. Table 6.2. Results of host-spe<:ificity screening for Exserohilum monoceras - disease • severity Disease severity" ôTest plant specie's 24 h dew No dew Eehinoehloa species E. erus-galli var. austro-japonensis S S E. erus-galli var. histidu S S E. eolona S --S E. glabreseens S S lteh grass Rottboellia eoehinehinensis S S Corn (Zea mays) Pirsabak L NS !PB var 1 L NS !PB var 2 L NS Los Baiios lag\'..itan L NS Super sweet L NS Sorghum (Sorghum bieolor) ICSV 217 HS NS UPLSG 5 HS NS Sugarcane (Saeeharum ojJieinarum) HS NS • AlI other species retnained uninfecteci. b Disease severity was visually assessed each day until the 14th day after inoculation of lOS conidialm2 by using a disease rating scaIe of NS (no symptoms). HS (hypersensitive response, pinpoint lesions which did not increase in size). L (light infection. symptoms present, secondary spread limited). S (severe infection. systemic spread {Jf pathogen. sorne plants killed). 140 Table 6.3. Results of host-specificity screening for Exserohi/um monoceras - sporulation • b Sporulation Test plant species' 24 h dew Nodew Echinoch/oa species E. crus-galli var. austro-japonensis ++ ++ ++ ++ ++ ++ E. crus-gaIli var. histidu ++ ++ ++ ++ ++ ++ E. c%na ++ ++ ++ ++ ++ ++ E. g/abrescens ++ ++ ++ ++ ++ ++ Itch grass Ronboe/lia cochinchinensis ++ ++ ++ + + ++ Corn (Zea mays) Pirsabak + !PB var 1 + !PB var 2 + Los Baiios lagkitan + Super sweet + • No sporulation occurred on other species tested. b After disease severity was rated, one or 1\;'0 inoculated leaves were excised from each replication of treated and untreated plants. Excised leaves v'ere surface-sterilized, rinsed, and then incubated on moistened filter paper in peni dishes at 28 C in the dark. After 24, 48, and 72 h ofincubation, sporulation on leaves were examined with a stereo microscope. Sporulation was rated at three levels: - =no sporulation; + =light sporulation; ++ = moderate 10 heavy sporulation. 141 • Connecting Text Natural compounds produced by nùcroorganisms may be directly used as herbicides or can be utilized as building blacks for novel herbicides. although few have become commercial realities. Preliminary evidence of phytotoxin production by Exserohilum monoceras was presented in Chapter 2. In this chapter. the isolation. purification, and partial characterization ofthe phytotoxins produced by E. monoceras are reported. The possibility of using phytotoxins produced by E. monoceras to control EchinochIoa species in rice is aIso investigated. • 142 Cbapter 7. Isolation and partial cbaracterization • of phytotoxins produced by Exserohilum monoceras 7.1. Abstract Phytotoxin production has not previously been reporred for the funga! pathogen Exserohilum monoceras. Two biologieally active compounds, designated toxin 1and toxin II were isolated from E. menoceras culture filtrate and inoculated plant leaves by means of extraction and thin layer ehromatography (ILC). On 'ILC plates, Rr values of toxin 1 and toxin II were 0.59 and 0.72, respectively. In the absence of the pathogen, eaeh of the purified toxins incited syrnptorns similar to those caused by E. menoceras conidia. Toxin 1 was shown to be most patent and host-specifie. Toxin II had a broader spectrum of activity, but played a less important role in produeing disease on Echinochloa species. A root inhibition experiment indicated that toxin 1was selective for Echinochloa species and did not damage riee. 7.2. Introduction Exserohilum menoceras (Drechsler) Leonard & Suggs causes leaf blight in Echinochloa species and is presently being evaluated as a potential bioherbieide for the control of Echinochloa species (Zha..'lg et al, 1996). Inoculation with E. menoceras resulted in a blight-like reaction char 7.3. Materials and methods 73.1 Culturing The E. monoceras culture used in this study was originally isolated from naturaily infected Echinochloa species leaves coIIected in the Philippines. The organism was maintained on half-strength potato dextrose agar (1/2 PDA; Difco, Detroit, Mn slants in mla1I vials under mineraI oil at4 C (Zhang et al., 1996). For toxin production, the fungus was grown in 1-L Roux bottles containing 200 ml of Modified Fries medium (lOO g sucrase. 2 g casein hydrolysate, 15 g NaNO" 1 g ~HPO., 0.5 g Ka, 0.5 g MgSO., 0.01 g FeSO., and distiIIed water to 1 L) (Tuite, 1969). Cultures were incubated at laboratory temperature (25 ± 2 C) on a rotary shaker operating at 150 rpm. 73.2. Isolation and purification oftoxins After 21 days ofgrowth, the culture fluid was obtained by filtrating through three layers of cheesecloth and concentrating culture filtrates 10 10% of their original volume by using a flash evaporator al 50 C (Steiner & Strobel, 1971; Stierle et al., 1992). The concentrated broth was extraeted with chlorofonn (CHa,> (3 x 113 vol). The chlorofonn • extraet was then evaporated using a flash evaporator, the residue was weighed, and 144 collected in vials using chlorofonn. The chlorofonn extract was subjected to TLC. • Analytical silica gel plates, 0.25 mm thick. were used and developed in chlorofonn: methanol 9:1. Bands on TLC plates were rnarked under ultra violet (UV) light and then each band was carefully cut off. Compounds from each band (with silica gel) were re extracted in chlorofonn, dried by flash evaporator, and weighed. In order to deteet biological activity, each component was 1" :pared aud subjected to a leaf bioassay by placing in 2% aqt:.:DUS ethanol solution containing 0.05% Tween 20 as a wetting agent 733. Leafbioassay The most recently expanded leaf of each of the three Echinochloa species (i.e. E. crus-galli, E. colona, and E. glabrescens) was detaehed from plants. Sets of glass slides were prepared by inserting bath ends of the slide into filter paper (Whatman No. 3) so that a 4 cm mid-portion was exposed. The tip and basal portion of a detaehed leaf were fixed by inserting them berween the slide and filter paper and by placing a small cotton ball on the basal end of the detached leaf (tO keep the leaf from drying). The prepared leafset was placed into a petri dish. The filter paper and cotton ball were then moistened with distilled water. The detaehed leaf was wounded with a glass capillary tube and a droplet of test solution containing 50 !1g/ml of the toxin in 2% aqueous ethanol with 0.05% Tween 20 was placed on the wound (Karr etal, 1974). The plates were incubated at 28 C in the darle. Mter48 h incubation, symptoms similar to those prodnced by conidia were observed. 73.4./solation oftoxins produced in vivo E. crus-galli seedlings at the 4-leaf stage were inoculated with E. monoceras at 2 a rate of 1 x lOS conidia/m , placed in a dew ch;;.nber for 24 h, and then transferred to a mist room (Yeh & Bonman, 1986). The control treatment consisted ofplants that were inoculated with cfstilled water but otl:erwise were subjected to the same conditions as inoculatedplants. Afu.l 1 week, 30 g of severely infected leaf material was collected, • chopped, and treated overnight with 350 ml of chloroform and methanol at room 145 temperature (Vidhyaseka.-an et al., 1986). Extracts were filtered through four layers of • cheesecloth. Residues of methanol were added to 350 ml of chlorofonn for further extraction for 4 h and once again filtered through four layers of cheesecloth as weil. Ali the chlorofonn, methanol, methanol + chlorofonn filtrates were further filtered through two layers of Whatman No. 1 fIiter paper and 100 ml of water was added to them. Solvents were removed by using a flash evaporator and water fractions were partitioned with chlorofonn (3 x 1/3 vol). The water fraction was discarded and the chlorofonn was evaporated to dIyness in vacuum. Residues were then collected in small vials and then subjected to TLC. Analytical plates, 025 cm thick, were run in chlorofonn: methanol 9:1. 735. Host specificity oftoxins N'me plant species including three Echinochloa species and cultivars ofthree types ofrice were selected for host specificity testing, using leaf bioassays. Leaf sections of test species were inoculated with E. rnonoceras by preparing a spore suspension containing approximate1y 5 x 107 conidialml in 2% aqueous ethanol with o.O~~.Jw~ 20 and p1acing 50 J.Ù ofthe suspension on a leaf wound as described in section 7.3.3. The toxins were also diluted to 50 J.lg/ml in 2% aqueous ethanol containing 0.05% Tween 20 and tested on leaves of these same hosts. 73.6. Root growth inhibition A single bateh of seeds ofeach of the three Edùnochloa species, E. crus-galli, E. colona. and E. glabrescens collected from natura! agricultura! Echinochloa populations on the International Rice Research Institute (IRRI) fann was used in this experiment. The rice cultivars used were Dee-Geo-Woo-Gen ~.!ld Chianan, representing the indica and japonica rice types, respectively. Seed ofeach Echinochloa species and rice cultivars was :~cubated in petri dishes on moistened filter paper at room temperature (25 ± 2 C) for 48 h. Seedlings having primary roots 5 mm long were selected and placed in 5 cm diarneter petri dishes (5 germinated seeds/dish) aIong with 2 ml of the toxin preparation diluted • with 2% aqueous ethanol solution at concentrations of0, 0.2, 0.4, 2, 4, 20, 40, 0, 80, and 146 100 l1g1ml. After 48 h at room temperature (25 ± 2 C), the root length of seedlings was • measured. Percent root growth inhibition was obtained by comparing the root length of seedlings in the presence of toxins to that ofcontrols ('foder et al., 1977). There were 25 measurements of seedling root length for each treatment or control. . 73.7. Comparison oftoxitis with standards ofbipolaroxin and exserohilone The standards of two phytotoxins, bipoIaroxin and exserohilone were provided by Dr. G.A. Strobe1. Toxin 1 and toxin n together with these two standards were subjected to :he same TLC. Analytical silica gel plate, 0.25 mm thick, and developed in chloroform:methanol 9: 1. The Rr value was recorded for each compound. Compounds with the same Rr value were considered to be the same compound (Stierle et al., 1992). 7,4. Results 7.4.1. Isolation aroll purification oftoxins No toxin activity was detected in t.~e water fraction after partitioning with three volumes of chloroform. However, toxin activity was deteeted in the chloroform fraction. The chloroform extraet contained six different compounds, including the twO toxin fractions, detected as short UV-quenching bands. The two toxin fractions migrated with Rr values of0.59 (toxin 1) and 0.72 (toxin II) (chloroform:methanol =9:1 solvent) on the TLC plate. When these twO bands wére eluted separately and rechromatographed, no other spots were detected. In different solvent systems, the toxin fractions migrated with different he values. In chlorofonn:methanol 25:1, toxin 1 and toxin n migrated as short UV-quenching bands, with Rr values of0.42 and 0.65, respectively. In pentane:ethyl ether: acetic acid 20:80:1, toxin l and toxin n migrated at Rr values of 0.48 and 029, respectively. Visually, toxin 1 appears as a yellow powder!Uld toxin n as an orange powder. They are highly soluble in chloroform, methanol, ethanol and acetone, and sparingly soluble in water. Bath toxins are fairly stable to heal Neither autoelaving at 121 C for 15 l1lin. nor storing atroom temperature, changed the Rr value in the chlorofonn:methanol 147 • system while retaining full toxicity to Echinoch/oa. 7.42. Phytotoxiciry oftoxins Toxin 1 appears to be a more powerful phytotoxin than toxin II. In the leaf bioassay using toxin l, first symptorns appeared within 24 h as a weak chlorotic marbling which subsequenùy developed into well-defined chlorotic spOts surrounding brown necrotic lesions. Symptorns on detaeho:d Echinoch/oa leaves produced by toxin 1 were similar to those produced by the conielia of the pathogen after 48 h (Figure 7.1). Toxin II qualitatively induces the same symptorns as toxin L However, the minimum toxin concentration required to cause chlorosis was 7 to 8 rimes greater than that of toxin 1. 7.43. Specificity oftoxins E. monoceras severely infected Echinoch/oa species and slighùy infected corn (Zea mays L.), but did not infect other hosts tested (Table 7.1). Toxin 1 induced typical symptorns on leaves of plants susceptible to the fungus but did not produce any effecl~ on non-hosts. Symptom expression on Echinochloa species with toxin II was much weaker and toxin II had a broader spectrum of activity, including grass and broad-leaved plant species (Table 7.1). 7.4.4. Root growth inhibition Root growth of Echinoch/oa species was much more susceptible to toxin 1 than was rice (Figure 7.2). Concentrations of 0.2 Jlg/ml of toxin 1 inhibited Echinoch/oa roo~ growth by 46-60%, but did not inhibit rice root growth. Similarly, the 20 Jlg/ml toxin 1 tteatment inhibited Echinochloa root growth by approximately 80%, whereas rice root growth was inhibited by less than 10% by this tteatment. The root growth suppression response to toxin 1 by the three Echinoch/oa species was similar. 7.5. Discussion • Toxin 1 and toxin II are not bipolaroxin orexserohilone based on comparisQn with 148 the standards provided by Or. G.A. Strobel. However. toxin II is most likely monocerin • since it has the same R, value when run in the same solvent system and similar conditions reported elsewhere (Robeson & Strobel. 1982). !f so. monocerin also has phytotoxic properties towards Echinoch/oa species besides those reported for Canada thistle. johnsongrass, tomato, and cucumber (Robeson & Strobel. 1982). Toxin II migl.: be a novel phytotoxin which is highly active on Echinoch/oa species. However. further research is required to be able to proper1y identify these toxins. Pringle & Scheffer (1964) defined a host-specific toxin as a metabolic product of a pathogen which is toxic on1y to the host of the pathogen. Toxin 1 produced by E. monoceras has severa! charaeteristics in common with other host-specific toxins (Steiner & Byther, 1971). The host range of the pathogen and that of toxin 1were simiIar. Toxin II, if it is monocerin, is known to have a very broad spectrum of biologicai activity inc1uding antibiotic, insecticidal, and phytotoxic properties (Robeson & Strobel, 1982). These IWO toxins were aIso isolated from Echinoch/oa leaves inoculated with E. monoceras. Therefore, it cao be concluded that the IWO toxins isolated from cultures are aIso produced during the infection of Echinoch/oa species by E. monoceras. It appears that toxin 1 contributes more than toxin n to disease expression in Echinochloa species because toxin 1is more potent than toxin li There has been considerable research interest in phytotoxins produced by plant pathogens of crop plants. In severa! instances, these phytotoxins have proven useful as 100ls for screening plants for toxin insensitivity (resistance) and as probes of nonnai physio10gical plant function (Strobe1, 1982). Phytotoxins produced by weed pathogens have received less attention. However, phytotoxins produced by weed pathogens have the potentiai to be used direcùy on the target weed species or utilized as building blocks for novel herbicides (Duke, 1986; Hoagland, 1990; Strobel et al, 1992). The selectivity of toxin 1 toward rice and Echinochloa plants indicates that toxin 1 has the potentiai to be used as one of these novel herbicides or building blocks. • 149 7.6. LiteTature cited • Duke, S.O. 1986. Naturally occUIring chemical compounds as herbicides. Pages 17-44 in Review ofWeed Science. Vol. 2. Weed Science Society ofAmerica. Champaign, minois. Hoagland, RE. 1990. Microbes and microbiai products as herbicides - An overview. Pages 2-52 in HoagIand, RE. (cd) ACS Symposium Series 439: Microbes and Microbial Produets as Herbicides. American Chemical Society, Washington, DC. Karr, AL Jr., Karr, D.B. and Strobel, G.A. 1974. Isolation and partial characterization offour host-specific toxins ofHelminthosporium maydis (Race 1). Plant Physiol. 53:250 257. Pringle, R.B. and Scheffer, RP. 1964. Host-specific plant toxins. Ann. Rev. Phytopathol. 2:133-156. Robeson, D. J. and Strobel, G. A. 1982. Monocerin, a phytotoxin from Exserohilum rurcicum (Drechslera turcica). Agric. Biol. Chem. 46:2681-2683. Steiner, G. W. and Byther, R. S. 1971. Partial eharacterization and use of a host-specifie toxin from Helminthosporium sacchari on sugarcane. Phytopatltology 61:691-695. Steiner, G. W. and Strobel, G. A. 1971. He1minthosporoside, a host specifie toxin from Helminthosporium sacchari. J. Biological Chemistry 246: 43504357. Stierle, A., Strobel, G., Stierle, D~ and Sugawara, F. 1992. Analytical methods for phytotoxins. Pages 1-32 in Linskens, H.F. and Jackson, J.F. (cds) Modem Methods of Plant Analysis New Series, Vol. 13, Plant Toxin Analysis. Springer-Verlag Berlin • Heidelberg. 150 • Strobel, G.A. 1982. PhytOtoxins. Annu. Rev. Biochem. 51 :309-333. Strobel, G.A., Sugawara, F. and Hershenhorn, J. 1992. Pathogens and their produets affecting weedy plants. Phytoparasitica 20:307-323. Tuile, J. 1969. Plant Pathological Methods: Fungi and Baeteria. Burgess Publishing Co., Minneapolis, MN. Vidhyasekaran, P., BotI'Omeo, E.S., and Mew, T.W. 1986. Host-specifie toxin production by Helminthosporium oryzae. PhytOpathology 76:261-266. Yeh, W.H. and Bonman, J.M. 1986. Assessment of potential resistanee ta Pyricularia oryzae in six rice cultivars. Plant Pathology 35:319-323. Yoder, O.c., Payne, GA., and Gracen, V.E. 1977. Bioassays for detection and quantification of Helminthosporium maydis race T-toxin: a comparison. Physiological Plant Pathology 10:237-245. Zhang, W. and Watson, A.K. 1996. Efficacy of Exserohilum monoceras for the control ofEchinochloa species in rice (Oryza sativa L.). Weed Sei. (reviewed by IRRI and ready ta be submitted). Zhang, Wenming, K. Moody and A.K. Watson. 1996. Response of Echinoch!oa species and rice (Oryza sativa L) ta indigenous pathogenic fungi. Plant Dis. (Submitted). • 151 • Table 7.1. Host range of Exserohilum monoceras and its associated toxins' Pathogenicity' Plant species Conidia Toxin 1 Toxin Il Echinochloa crus-galli +++ +++ + Echinochloa c%na +++ +++ + Echinochloa g/abrescens +++ +++ + Rice (Oryza saliva L.) Indica type Japonica type Tropical Japonica type Corn (Zea mays L) + ++ Tomate (Lycopersicon escu/entum L.) ++ Banana (Musa sapientum L) + Mungbean (Vigna Tamara (L) R. Wilcz.) + Cowpea (Vigna unguicu/ata (L) Walp.) + • Toxin 1 with Rr 0.59 and toxin Il with Rr 0.72. b Symbols: +++ =severe, ++ =rnoderate, and - =no symptoms. • 152 • Figure 7.1. Effect ofExserohi/wn monoceras spores and toxin Ion. Echinochloa crus-galli leaves, in situ, 48 h after treatment Detached leaves were wounded with a glass capil1ary tube. Spore application was carried out using 50 !d ofE. monoceras sta rate of5 x 10 7conidialml in 2% aqueous EtOH and 0.05% Tween 20. Toxin 1was applied using 50 III of toxin 1solution in 2% aqueous EtOH and 0.05% Tween 20. The toxin solution was composed of50 Ilg ofpure toxin I1ml. The control treatment consisted ofapplying only 50 III of2% aqueous EtOH and 0.05% Tween 20 solution. • 153 • Figure 7.2. Inhibition of seedling root growth of riec and Echinochloa species by toxin 1 produced by Exserohilum monoceras. 154 • EchinochIoa crus-galli 100 • • Echinochloa colona ... Echinochloa glabrescens • Indica ricc ~ Japonica ricc ~ 80 • '-' -5 :t 0 6h ~ 60 ...e0 0 c .2 ~ 40 :E ~ 20 Log1o lOxin 1concentration (pg/ml) • • Chapter 8. General Conclusions The research reponed in this thesis investigated the possibility of utilizing indigenous fungi and their phytetoxins to control Echinochloa species in rice 50 as to reduce herbicide dependency for weed control in this cropping system. especially in the major rice growing counmes of Asia. This study c1early showed that three fungal species Exserohilum m01l0ceras. Bipolaris sacchari. and Cllrvularia geniculata have potential to control Echinochloa species in rice hecause of the high monaIity each inflicted against the three Echinochloa species. In contrast, these fungi were not pathogenic to rice. Among the three fungi. E. mOlloceras was shown to he the most promising candidate as a bioherbicide since it showed the mos: virulece as weB as having the shonest free moisture requirement. The experiment on the biological constraints for E. monoceras development as a bioherbicide showed that the inoculum density required to obtain 100% monality of Echinochloa species was similar to that of other bioherbicides with the most s.-.sceptible plant growth stage (1.5) weB within the application time of convential post-emergence chemical herbicides. Moreover. the optimal dew period temperature was within the temperature range occurring during t.'1e rice growing season, however, the dew period requirement of 8-16 h is rarely met under field conditions. These findings demonstrate that inoculum density, plant growth stage, and dew period temperature do not constitute biologica1 constraints for E. monoceras as a bioherbicide, but dew period duration is mos: likely to he a limitation. Nevenheless, this constraint can he largely overcome or bypassed through the use of oil emulsion or dry powder formulations. In this study, a dry powder formulation not only completely bypassed the dew requirement by floating conidia on the water surface, but aise formed an efficient method for the delivery of conidia to target Echinochloa species. Optimization of spore production is often a critica1 aspects in determing the success orfailure ofa bioherbicide prospect. E. monoceras can he easily grown and made • to sporulate on standard agar media. Although liquid fermentation may present a 155 technologicallimitation for mass production of E. monoceras. sol id fermentation on corn • leaves was demonstrated to be an alternative method for mass producing E. monoet'ras. Mass production of E. monoceras on corn leaves can be easily "repared as a dry powder formulation and can be an easily and efficiently used by farmers. Host range tests showed :hat E. monoceras was restricted to two tribcs: the Paniceae and the Andropogoneae. in the subfarnily Panicoideae of the Poaceae family. Although corn was lightly infected under optimum gr,:enhouse conditions. E. monoceras is safe to use in rice fields because no disease symptoms were detected on corn plant~ in the absence of a dew supplement, no symptoms and no conidia were detected on leaves of trap corn plants. and field inoculation of corn did not produce any symptoms. E. monoceras. B. sacchari. and E. oryzae produce phytotoxins which are biologically active against Echinochloa species. One phytotoxin produced by E. monoceras is selective for Echinochloa species and did not damage rice. The pOlential of using this phytotoxin against these weeds species warrants further research. • 156 • Chapter 9. Contribution to Knowledge The following are considered to he key contributions to knowledge arising from the research described in this thesis: 1. This research discovered three fungal pathogens, Exserohilum monoceras, Bipolaris sacchari, and Curvularia geniculata, having potential to control Echinochloa species in rice. 2. Findings in this slUdy demonstrated that Exserohilum monoceras conidial applications produced high levels ofEchinochloa mortality, were safe to rice, and could he formulated to achieve a higher degree of weed control. Moreover, this fungus was capable of producing conidia on standard agar media and crude agriculturaI products. E. monoceras can he viewed as a potential bioherbicide product 3. This is the flfSt smdy to describe phytotoxin production by Exserohilum monoceras. One phytotoxin was demonstrated to he particularly selective against Echinochloa species in rice. This phytotoxin has the potential to be used to control Echinochloa species in riec. 4. The development of a special formulation for floating funga! spores on the water surface in this slUdy was a new and innovative method to deliver funga! spores to target weeds and/or to overcome or bypass dew requirements. This novel application technique is Iikely to contribute greatly to the successful development of future bioherbicide products in similar cropping systems. 5. This is the first report to characterize on the culture and conidial production of E. monoceras. 6. This is the fust atternpt to delimitate the host range on the funga! species E. • monoceras, using phylogenetic methods. 157 • • Appendlx 1. A comparison of Exserohilllm mOl/oceras characteristics reported in different countries' Characters Philippine isolate Japanese isolate Korean isolate Conidium Shape straight or curved bent fusoid. straight or curved Size(J.lm) 15-21.5 x 51.3-139.9 15-17.5x87.5-127.5 15-19.6x53.3-138.8 septum 3-8 5-8 3-8 Hilum smal1 protruding Scar Smal1 protruding Germination Bipolar Bipolar Germ tube semiaxia1 or axial semiaxial or axial close to hilum close to hilum Septum ontogeny first submedian submedian lia- second supramedian supramedian third median median Host specificity Tomato NS NS cucumber NS NS wheat NS HS barley NS HS rice NS NS L corn L M goosegrass NS L bamyard grass S S S • - = data not available. L = light infection, NS = no symptoms. M = moderate infection. HS = hypersensitive reaction. S = severe infection. Appendix 2. Effect of Exserohilum monoceras conidial fiè'ld applications on severa! • Echinochloa crus-galli growth parameters 28 days after treatrnent" % Reduction Leaf Stage Dry Growth Tiller Leaf Weight Rate (g) (cm/24 h) No. No. l-leaf 54.8 32.3 18.3 48.1 2-leaf 49.1 41.2 13.1 38.9 3-leaf 40.1 24.0 11.1 40.8 4-leaf 24.9 20.1 16.8 19.8 • Echinochloa crus-galli seedlings were inoculated with Exserohilum monocl'~as at a rate of 2.5 x 107 conidia/m2 with 0.02% Tween 20 at the 1-, 2-, 3-, and 4-1eaf stages. • 159 Appendix 3. Effect of al! oil emulsion fonnulation having a diffen:nt r.ltio of oil: watcr • and Exserohilllm monoceras conidia on mortality of Echinoclzloa erlls-galli and rie<: (Oryza sarÏ\'a) under greenhouse conditions· Mortality ('70) Oil: Water E. crl/s-ga/li Rice 1:1 100 0 1:2 100 0 1:3 100 0 1:4 100 0 1:5 100 0 1:6 100 0 1:7 100 0 1:9 100 0 1:19 0 0 1:49 0 0 CONTROL 0 0 • Seedlings at the 1.5-1eaf stage were inoculated with Exserohilum monoceras at a rate 7 2 of 5 x 10 conidialm • • 160 Appendix 4. Effect of an oil emulsion fonnulation having a different ratio of oil: water • and Ezserohi/um monoceras conidia on the plant height of Echillochloa crus-galli and rice (Oryza sativa) under greenhouse conditions· Height (cm) Oil: Water E. crus-gal/i Rice 1:1 0.42 3.88 1:2 0.55 6.20 1:3 1.40 7.18 1:4 4.34 9.03 1:5 4.16 8.24 1:6 5.84 12.58 1:7 7.32 12.20 1:9 5.62 16.30 1:19 12.30 19.88 1:49 15.80 22.75 CONTROL 18.07 23.04 • Seedlings at the 1.5-leaf stage were inoculated with Exserohilum monoceras at a rate 7 2 of 5 x 10 conidialm • • 161 Appendix 5. Effect of wetting agents and chemical herbicides on disease severity caused • by Exsi!rohi/um monoceras under greenhouse conditions· Wetting agents Disease severity 0.02% Silwet L-77 + 0.02% Morwet M + 0.02% Citewett + 0.02% Tween 20 ++ 0.01% Oil emulsion +++ NC-31lC» +++ LondaxC» +++ • Seedlings at the 1.5-leaf stage were inoculated with Exserohi/um monoceras at a rate 7 2 of 5 x 10 conidialm • • 162 Appendix 6. Effect of suspension media on Exserohi/um monoceras conidial viability in • freeze-dried treatment Suspending media Germination(%) Water 0 10% Skimmed milk 0 10% Skimmed milk + 5% inositol 50 10% Gelatin 59 10% Giycerin 5 10% Lactose 20 10% Sucrose 20 10% Dextrose 20 • 163 • • Appendl" 7. Percent mortality and reduction in weight and dry weight between paired fiais of Eclu'lIoch/oa crlls-gall; seedlings treated either with Exserohilum mOlloceras plus an adjuvanl. or wilh the adjuvant alone. using different sprayers under greenhouse conditions• Hand spray Motor-sprayer Mortality Fresh Weight Dry Weight Mortality Fresh Weight Dry Weight (%) (%) (%) (%) (%) (%) 1% Tween 20 50.0 a 84.4 a 78.4 ab 100.0 a 100.0 a 100.0 a 1% Vegetable oil 0.0 b 37.2 ab 32.7 bcd 30.0 b 86.0 b 74.0 b 1% Vegetable oil ... 88.4 a 100.0 a 100.0 a 100.0 a ~ + 0.2% Tween 20 50.0 a 79.5 a 1% Vegetable oil + 1% G1ycerol 0.0 b 47.9 a 46.5 abcd 50.0 b 88.9 b 63.6 b 0.2% Tween 20 + 1% G1ycerol 30.0 ab 60.3 ab 57.5 abc 100.0 a 100.0 a 100.0 a 1% gelatin 0.0 b 34.6 ab 25.9 cd 20.0 c 62.5 c 50.9 c Water 0.0 b 0.0 b 0.0 d 0.0 c 31.0 c 18.2 c 7 2 • Seedlings at the 1.5-leaf stage were inoculated with ExserohilwlI II/olloceras al a raie of 10 conidia/m • .. Within a column. values with the same leller are not significantly different according 10 Duncan's Multiple Range Test (P S 0.05). • Appendix 8. Shelf life Exserohilum monoceras conidia harvested from corn leaves Survivability (%) 1 month 2 month 6 month 98 95 85 • 165 Appendix 9. Disease severity between paired flats ofEchillochloa seediings treated either • with Exserohilum monoceras plus an adjuvant. or with the adjuvant alone under greenhouse conditions' E. crus-galli E. glabrescens E. colona Ricc Formulation" T C T C T C 0-1 15 0 40 0 15 0 0 0 0-2 25 15 85 5 55 15 0 0 0-3 55 0 35 5 75 0 2.5 0 0-4 40 0 60 0 40 0 0 0 0-5 95 15 100 20 95 40 8.8 3.8 0-6 40 15 60 20 93 60 0 1.3 • Seedlings at the 1.5-leaf stage were inoculated with E. monoceras 2 at a rate of 10' conielia/m • •• 0-1 1:26.67 orr emulsion 0-2 1:26.67 oil emulsion + 3.125% (w/v) Dextrose 0-3 1:26.67 oil emulsion + 02% Carboxymethyl cellulose 0-4 1:26.67 oil emulsion + 02% Carboxymethyl cellulose + 3.125% (w/v) Dextrose 0-5 1:10 oil emulsion + 0.2% Carboxymethyl cellulose + 3.125% (w/v) Dextrose 0-6 Invert emulsion (O:W =1:3, oil phase =15.7% (w/v) paraffin wax, 15.7% (v/v) soybean lecithin, and 68.6% mineraI oil) ... T =Formulation + Conielia; C =Formulation alone • 166 Appendix 10. Conidial production of Bipolaris sacchari on different substrates' • Conidia No. Media 7 days 15 days Standard agar media (Spores/ml) lima bean agar 6.13 x 10' ab 16.77 x 10' a V-8 juice agar 5.73 x 10' ab 12.30 x 10' ab centrifuged V-8 juice agar 8.95 x 10' a 12.70 x 10' ab oatrneal agar 5.Q7 x 10' ab 0.23 x 10' b corn meal agar 0.18 x 10' b 0.83 X 10' b malt extraet agar 6.35 x 10' ab 5.96 x 10' ab rice polish agar 0.31 x 10' b 0.77 y. 10' b potato dextrose agar 3.35 x 10' ab 6.53 x 10' ab czapek-Dox agar 0.70 x 10' b 0.23 X 10' b Crude agricultural products (spores/g) milled barley 6.75 x 10' milled oat 4.00 x 10' chopped E. crus-galli 0.95 x 10' Liquid Media (Spores/ml) Modified Richard's solution 0 0 • Treatrnent rneans having a common letter were not significantly different at the 5% level of significance according to Duncan's Multiple Range Test (p S 0.05)• • 167