THESIS .:..... 02:" HMQLJV ' -' t 4:.- * - -- ;.~~.<. a» m J 4; , .4 .f . '\ ." .V ; . _ .1 ~ I" ; .w ‘: «I 'dg‘} ‘b‘nbg'h :- 2‘ ’, ' J . C a} ‘ {3. z _ t A; (f. n x- ‘ :. 9's.» 3., »- V l 2‘ , I bwiv‘ov-kv. I “ ‘ ‘~5 \ l ‘ I;— .‘J‘. ' ‘ E 2““ i._ v, r: r ‘1;._. 09 It‘ll-"1"V V «I? “""V' (a .. £9 7‘ A; ‘~‘ ‘ .‘a'f .‘JQ'J‘ '~-“ ’ r l' ‘5 This is to certify that the thesis entitled INFLUENCE OF ENVIRONMENTAL AND HOST FACTORS ON INFECTION OF ONION BY BOTRYTIS SQUAMOSA AND BOTRYTIS ALLII presented by Stephen C. Alderman has been accepted towards fulfillment of the requirements for Ph.D. degree in Plant Pathology Wwfozim jor professor fflafiflw 0-7639 MSU is an Affirmative Action/Equal Opportunity Institution " u".- IV‘ESI_J RETURNING MATERIALS: Place in book drop to LJBRARJES remove this checkout from —:— your record. FINES will be charged if book is returned after the date stamped below. fig“! USE (jaw sis-2: . - .. ‘0’“: 5 ~)_ «1 5:1 .- 54”., _- ‘5 t f; . "~ , ~ \ r _’-3 Q. - i ' ‘21 "’ . "V l . V . - -. .‘4 . J , a = ‘v -'f Ir 7; 1'. I 1 ~ . A" ‘5' ‘ i‘ ‘4 L ' I I t :‘I :8: 0 a, a Q‘. 11.." .~ .' h '1 i." , $.4- v-‘h‘ a ‘- . .J INFLUENCE OF ENVIRONMENTAL AND HOST FACTORS ON INFECTION 0F ONION BY BOTRYTIS SQUAMOSA AND BOTRYTIS ALLII By Stephen Charles Alderman A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1984 ABSTRACT INFLUENCE OF ENVIRONMENTAL AND HOST FACTORS ON INFECTION 0F ONION BY BOTRYTIS SQUAMOSA AND BOTRYTIS ALLII By Stephen Charles Alderman A dry spore inoculation procedure was developed to study infec- tion of onion (Allium cepa L.) leaves by Botrytis squamosa Walker. Dry conidia applied to onion leaves survived for several days. Under continuous dew at 20 C, conidial germination on leaf surfaces began after 2 hours and increased through 20 hours. The first lesions were visible after 8 hours. Lesion production in onion was maximal at 20 C, slower at 15 C and greatly reduced at 25 C. The minimal leaf wetness period for infection was 6 hours, and numbers of lesions increased with increasing leaf wetness duration. Infection hyphae extended beyond lesion borders in relatively few instances. Hyphal development increased with increasing leaf age (tissue maturity), and with increasing periods of continuous leaf wetness. A dry period following inoculation and 6 gours of dew followed by resumed dew reduced lesion numbers. Lesion numbers tended to decline with decreasing humidities during a dry period following a 6 hour post-inoculation wetness period. Survival studies indicated that g, sguamosa can survive as mycelia in colonized, desiccated leaf segments. Conidial formation was observed from such segments after 48 hours under moist Stephen Charles Alderman conditions. Sporulation from colonized leaf segments was abundant at 15 or 20 C and nil at 35 C. Growth of g, Elli; or g. sguamosa on artificial media or on onion leaves at 20, 25, or 30 C increased from 0.9 through -5 to -10 bars, then declined through -90 to -100 bars. At water potentials greater than -30 bars growth was greater at 20 or 25 C than at 30 C, but at potentials below -30 bars growth was similar at all three temperatures. Conidial suspensions of g, allii applied to leaves of field onions caused latent infections and increased neck-rot incidence in storage. TO My Late Mother who helped me start on this path and To Dad who helped me finish ii ACKNOWLEDGEMENTS I would like to express sincere gratitude and appreciation to Dr. Melvyn Lacy, my Major Professor, for his patience, encouragement, advice, and very generous support throughout this course of study. I extent sincere thanks and appreciation to my committee members, Dr. John Lockwood, Dr. Karen Baker, Dr. Raymond Hammerschmidt, and Dr, Christine Stephens, for their advice and encouragement. I extent thanks also to Dr. Gene Safir for his advice and helpful suggestions and to Mr. Richard Crum, for his generous assistance in many technical aspects of this project. Special thanks are extended to Lou Anne Alderman for helping me prepare the final manuscript. iii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . LIST OF FIGURES . . . . . . . CHAPTER I INTRODUCTION AND LITERATURE BOTRYTIS LEAF BLIGHT . Occurrence . . . . . Symptoms . . . . . . Morphology . . . . . Survival and Primary Secondary Inoculum . Control . . . . . . BOTRYTIS NECK ROT . . . . Occurrence . . . . . Symptomatology . . . Morphology . . . . . Inoculum Overwintering; Primary Control . . . . . . OBJECTIVES . . . . . . . REFERENCES . . . . . . . CHAPTER II INFLUENCE OF DEW PERIOD AND TEMPERATURE ON Secondary Inoculum INFECTION OF ONION BY BOTRYTIS SQUAMOSA . . INTRODUCTION . . . . . . MATERIALS AND METHODS . . Production of E. sguamosa conidia Development of a Dry Spore Inoculation Procedure . Influence of Dew Period and Temperature on Lesion Production . Influence of Temperature on Spore Germination and Infection Influence of Dew Period on Infection Hyphal Development . . Longevity of Conidia on Leaves . RESULTS . . . . . . . . . Influence of Inoculum Density on Lesion Numbers Influence of Dew Period and Temperature on Infection . . . Influence of Temperature on Spore Germination and Infection . Influence of Dew Period on Development of Infection Hyphae Longevity of Conidia on Leaves . iv Page xi 0" \OQOmNVO‘UIU'IUI 16 16 18 18 18 20 20 21 21 22 22 22 25 25 25 Page DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . 26 REFERmCES O C O O O O O O O O O I O O O O O C C O I O O 0 30 CHAPTER III INFLUENCE OF INTERRUPTIONS OF DEw PERIOD ON NUMBERS OF LESIONS PRODUCED ON ONION BY BOTRYTIS SQUAMOSA . . . . . . . . . . . . . . . . 31 INTRODUCTION 0 O I O O O O I O O O O O O O O O O O O O O O 3 1 MATERIAIJ 8 AND METHOD 8 O O O O O O O O O I O O O O O O O O 3 2 Influence of Timing of Dew Period Interruption on Lesion Production . . . . . . . . . . . . . . . 33 Influence Of Interruption Duration on Lesion Production . . . . . . . . . . . . . . . . 33 Influence of Humidity During an Interruption . . . . 34 Spore Germination Rate on Leaf Surfaces . . . . . . . 34 RESULTS 0 O O O O O O O O O O O O O O O O O O O O O 0 O 34 Influence of Timing Of Dew Period Interruptions on Lesion Production . . . . . . . . . . . . . . . 34 Influence of Interruption Duration on Lesion Production . . . . . . . . . . . . . . . . . 36 Influence of Humidity During Interruptions . . . . . 37 Spore Germination Rate on Leaf Surfaces . . . . . . . 38 DISCUSSION 0 O O O O O O O O O O O O O O O O O O O O O O O 38 REFERWCES O O O O O O O O O O O O O O O O O O O O O O O O 42 CHAPTER IV INFLUENCE OF LEAF POSITION AND MATURITY ON DEVELOPMENT OF BOTRYTIS SQUAMOSA IN ONION LEAVES . . . . . . . . . . . . . . . . . . 44 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . 44 MATERIALS AND METHODS . . . . . . . . . . . . . . . . 45 Leaf Position vs. Lesion Numbers . . . . . . . . . . 45 Leaf Position vs. Lesion Size . . . . . . . . . . . . 45 Leaf Position vs. Infection Hyphae Development . . . 46 Lesion Size vs. Infection Hyphae Development . . . . 46 Rate of Lesion Development . . . . . . . . . . . . . 47 Influence of Tissue Maturity on Infection . Hyphae Development . . . . . . . . . . . . . . . . 47 RESULTS 0 O O O O O O O O O O O O O O O O O I O O O O O O 47 Leaf Position vs. Lesion Numbers . . . . . . . . . . 47 Leaf Position vs. Lesion Size . . . . . . . . . . . . 49 Leaf Position vs. Infection Hyphae Development . . . 49 Lesion Size vs. Infection Hyphae Lengths . . . . . . 49 Rate of Lesion Development . . . . . . . . . . . . . 49 Influence of Tissue Maturity on Infection Hyphae Development . . . . . . . . . . . . . . . . 52 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . 52 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . 55 CHAPTER V INFLUENCE OF TEMPERATURE AND WATER POTENTIAL ON GROWTH AND SPORULATION OF BOTRYTIS SQUAMOSA . INTRODUCTION . . . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . Influence of Temperature and Water Potential on Growth Rates on PLY Agar . . . . . . . . Influence of Temperature and Water Potential on Dry Weight of PLY Broth . . . . . . . . Influence of Temperature and Water Potential on Growth in Onion Leaves . . . . . . . . . Influence of Desiccation Period and Temperature on Sporulation . . . . . . . . . . . . . . . RESULTS . . . . . . . . . . . . . . . . . . . . . Influence of Temperature and Water Potential on Growth Rate on PLY Agar . . . . . . . . -Influence of Temperature and Water Potential on Dry Weight in PLY Broth . . . . . . . . Influence of Temperature and Water Potential on Growth in Onion Leaves . . . . . . . . . Influence of Desiccation Period and Temperature on Sporulation . . . . . . . . . . . . . . . DISCUSSION . . . . . . . . . . . . . . . . REFERENCES . . . . . . . . . . . . . . . . CHAPTER VI FIELD OBSERVATIONS CONCERNING SPREAD ON BOTRYTIS LEAF BLIGHT . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . Microscopic Examination of Lesions . . . . . . . Lesion Counts . . . . . . . . . . . . . . . Development of a Leaf Blight Epidemic . . . . . RESULTS . . . . . . . . . . . . . . . . . . . . . . Microscopic Examination of Lesions . . . . . . . Lesion Counts . . . . . . . . . . . . Development of a Leaf Blight Epidemic DISCUSSION . . . . . . . . . . . . . . . . . . . . . REFERENCES . . . . . . . . . . . . . . . . . . . . . CHAPTER VII INFLUENCE OF TEMPERATURE AND WATER RELATIONS ON GROWTH OF BOTRYTIS ALLII . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . . . Influence of Temperature on Growth Of B, allii . Influence of Temperature and Water Potential on Growth Rates on PLY Agar . . . . . . . . . Influence of Temperature and Water Potential on Dry Weight in PLY Broth . . . . . . . . Influence of Temperature and Water Potential on Growth in Onion Leaves . vi Page 56 56 57 57 57 58 58 6O 6O 62 62 62 67 72 73 73 74 74 74 75 75 75 75 77 77 82 84 34 85 85 85 86 86 Page Influence of Inoculum.Concentration on Lesion Development . . . . . . . . . . . . . . . 87 Influence of Temperature on Rate of Disease Development . . . . . . . . . . . . . . . . 87 Effect of Prolonged High Temperature Exposure on Survival of B, allii . . . . . . . . . . . . . . 87 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Influence of Temperature on Growth of B, allii . . . . 88 Influence of Temperature and Water Potential on Growth Rate on PLY Agar . . . . . . . . . . . . . 88 Influence of Temperature and Water Potential on Dry Weight in PLY Broth . . . . . . . . . . . . . 91 Influence of Temperature and Water Potential on Growth in Onion Leaves . . . . . . . . . . . . . 91 Influence of Inoculum Concentration on Lesion Development . . . . . . . . . . . . . . . . . 91 Influence of Temperature on Rate of Disease Development . . . . . . . . . . . . . . . . 95 Effect of High Temperature Exposures on Survival of B. allii . . . . . . . . . . . . . . . . 95 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . 95 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . 101 CHAPTER VIII FIELD OBSERVATIONS CONCERNING BOTRYTIS ALLII . . . 102 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . 102 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . 103 Seed Treatments for Control of Neck Rot . . . . . . . 103 Effect of Inoculating Onion Leaves with ‘B, allii Conidia on Neck Rot Incidence in Storage . . . . . . . . . . . . . . . . . . . . . 104 Incidence of B. allii in Onion Seed . . . . . . . . . 105 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Seed Treatment for Control of Neck Rot . . . . . . . . 105 Effect of Foliar Applications of B, allii on Neck Rot Incidence in Storage . . . . . . . . . . 107 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . 110 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . 112 APPENDIX A SIMULATION MODELS OF BOTRYTIS LEAF BLIGHT OF ONION O O O O O O O O O O O O O O O O O O 1 1 3 PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . 113 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . 113 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . 114 Description Of the Botrytis Leaf Blight Prediction Model (BOLEB) . . . . . . . . . . . . . . 114 a. The Release of Spores . . . . . . . . . . . . . 114 b. Lesion Production . . . . . . . . . . . . . . . 117 c. Infected Leaf Area . . . . . . . . . . . . . . . 117 d. Spray Component . . . . . . . . . . . . . . . . 118 vii Page Onion Growth Model . . . . . . . . . . . . . . . . . . 119 Model Runs . . . . . . . . . . . . . . . . . . . . .'. 122 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . 122 Leaf Blight Model (BOLOB) . . . . . . . . . . . . . . 122 Onion Leaf Blight Growth Model (ONLEG) . . . . . . . . 124 Areas of Future Research . . . . . . . . . . . . . . . 130 LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . 131 APPMIX B ONION MODE 0 O O O O O. C O O O O O O O O O O O O O 133 APPENDIX C SUBROUTINE BEGBLYT . . . . . . . . . . . . . . . . . 144 APPENDIX D PROGRAM BLIGHT (BOLEB) . . . . . . . . . . . . . . . 150 viii LIST OF TABLES Table Page 2.1. Percentage of infection hyphae of Botrytis squamosa falling within ranges of lengths in lesions on onion leaves after various dew periods at 20 C . . . . . . . . . 26 3.1. Effect of interruption duration of dew period on infection on onion by Botrytis squamosa . . . . . . . . . 36 3.2. Influence of interruption after a 6 hr dew period at 20 C followed by an additional 24 hr dew period . . . . 37 3.3. Influence of a 6 hr dew period, a_20 minute dew interruption at 30, 60, or 90% RH, followed by an 18 hr dew period, on infection of onion by Botrytis squamosa . . . . . . . . . . . . . . . . . . . . 38 4.1. Relationship between leaf position and incubation period in a dew chamber on lesion size following inoculation of onion plants with g, sguamosa . . . . . . . 52 6.1. Percentage of various fungi observed within small and and large lesions collected from onions grown at the M.S.U. Muck Farm during 1981 . . . . . . . . . . . . . 76 6.2. Lesions per plant and cumulative spores trapped in an onion field plot at the M.S.U. Muck Farm during 1981 . . . 76 8.1. Effect of fungicidial seed treatments on bulb weight and neck rot incidence (1980) . . . . . . . . . . . 106 8.2. Effect of artificial infestation and fungicides on recovery of 2, allii from onion seed (Asgrow Seed Co., cv. Spartan Banner) after three months storage at 5 C (1980) . . . . . . . . . . . . . . . 107 8.3. Recovery of Botrytis fungi from dead onion leaves collected from the seed treatment experiment at the M.S.U. Muck Farm, 1980 . . . . . . . . . . . . . . . . 108 8.4. Effect of timing of inoculation with E, allii conidia of foliage of field onions on neck rot incidence in storage . . . . . . . . . . . . . . . . . 109 8.5. Effect of fungicides applied to onion foliage on neck rot incidence in storage . . . . . . . . . . . . . 109 ix Figure 2.1. 2.2. 2.3. 3.1. 3.2. 4.2. 4.3. 5.1. 5.2. LIST OF FIGURES Page Relationship between mass of Botrytis squamosa conidia and lesion numbers on l-month-old onion plants after incubation in a dew chamber for 24 hours . . . . . . . . 23 Influence of temperature and dew period on lesion development in one-month-old onion plants following inoculation in a settling tower with 2 mg conidia of Botrytis squamosa . . . . . . . . . . . . . . . . . . 24 Effect of periods of time following inoculation without dew (20 C and 60% RH) prior to exposure to 24 hours continuous dew (20 C) on numbers of lesions per plant . . . . . . . . . . . . . . . . . . . 27 Influence of leaf wetness duration prior to a 2-hour interruption (dry period), followed by rewetting plants for a total of 22 hours total wetness period, on lesions/plant . . . . . . . . . . . . 35 Percent germination of Botrytis squamosa conidia on leaf surfaces at 20 C and continuous leaf wetness . . . . . . . . . . . . . . . . . . . . . . 39 Influence of leaf position on lesion numbers on onion after inoculation with Q. sguamosa and incubation in a dew chamber at 20 C for 24 hours . . . . 48 Influence of leaf position on lesion size on onion after inoculation with E, sguamosa and incubation in a dew chamber at 20 C for 24 hours . . . . 50 Influence of leaf position on infection hyphal lengths within lesions on onion after inoculation with E. sguamosa and incubation in a dew chamber at 20 C for 24 hours . . . . . . . . . . . . . . . . . . 51 Growth rate of g. sguamosa at 20, 25, and 30 C on PLY agar adjusted to various water potentials with KCL, NaCl, sucrose, and PEG 8000 . . . . . . . . . 61 Dry weight of g, sguamosa at 20, 25, and 30 C after 4 days in PLY broth, adjusted to various water potentials with KCL, NaCl, sucrose and PEG 8000 . . . . 63 Figure 5.3. 5.4. 5.5. 5.6. 5.7. 6.1. 6.2. 7.1. 7.2. 7.3. 7.4. 7.5. 7.6. 7.7. Radial growth of 2: squamosa in onion leaves at 20, 25, and 30 C at various water potentials . . . Numbers of conidia of E3 squamosa collected from onion leaf segments after 4 days incubation on water agar at various temperatures . . . . . . . . . . . . Numbers of conidia collected after 1-6 days from 23 sguamosa colonized leaf segments incubated at 15 C O O O O O O O O O O O O O O O O O O O O O 0 Influence of interruption duration within each 24 hour period on sporulation of g. sguamosa from colonized leaf segments . . . . . . . . . . . . . . Sporulation of E. sguamosa at 20, 25, or 30 C from colonized onion leaf segments placed on water agar adjusted to various water potentials with KCl . . . Serverity of Botrytis leaf blight at 228 sites within a 50 X 200 foot field plot at the MSU Muck Farm on August 11, 1980 . . . . . . . . . . . . Severity of Botrytis leaf blight at 228 sites within a 50 X 200 foot field plot at the MSU Muck Farm on August 20, 1980 . . . . . . . . . . . . Growth of Botrytis allii on PLY agar at various temperatures 0 O O O O O O O 0 O O O O O O O O 0 O O Growth of g, allii at 20, 25, and 30 C on PLY medium adjusted to various water potentials with KCl, NaCl, sucrose, and PEG 8000 . . . . . . . Dry weight of E. allii at 20, 25, and 30 C after 4 days in PLY broth, adjusted to various water potentials with KCl, NaCl, sucrose, and PEG 8000 . . Radial growth of E, allii in onion leaves at 20, 25, and 30 C at various water potentials . . . . Lesion diameters in onion bulbs inoculated with various concentrations of E. allii conidia, after 9 days at 20 C . . . . . . . . . . . Influence of temperature and incubation period on lesion diameter in onion bubls inoculated With E. allii O O O O O O O O O O O O O O O O O 0 Survival of Botrytis allii in onion leaf segments after various incubation durations at 32 or 37 C . . xi Page 64 65 66 68 69 78 79 89 90 92 93 94 96 97 Figure 7.8. A.2. A.3. (a-d) A.3. (e-f) A.4. Survival of Botrytis allii in agar plugs at various temperatures after 24, 48, or 72 hours . Simulation of yield as percent of control after defoliation of 30, 60, or 90% of the leaf tissue on various days after germination of onion Actual cumulative conidia / m3 air sampled, and simulated conidia sampled with and without the spray option . . . . . . . . . Green leaf surface area and bulb weight simulated by ONLEG with and without the spray option . . . . Green leaf surface area and bulb weight simulated by ONLEG with and without the spray option . . . . Simulated green leaf surface area and bulb weight in response to various levels of infestation by E, sguamosa early in the season xii Page 98 121 123 125 127 129 CHAPTER I INTRODUCTION AND LITERATURE REVIEW Since ancient times, the onion (Allium cepa L.) has been valued as a food crop. Little is known regarding the origin of A, 232$, although reports of its consumption, as early as 3200-2780 B.C., have been found in Egyptian tombs (Jones and Mann, 1963). Reports of onion consumption were also documented by herbalists in ancient Greece, in India during the 6th century B.C., and during the middle ages in Europe (Jones and Mann, 1963). In modern times the onion is commonly found worldwide as a food crop (Jones and Mann, 1963). Some 43 pathogens of onion have been described (Anonymous, 1960). In the United States the major pathogens attacking A, 2222 include Botrytis squamosa Walker, which causes Botrytis leaf blight; Botrytis allii Munn, which causes Botrytis neck-rot; Peronospora destructor, Berkeley, which causes downy mildew; Alternaria porri (Ellis) Ciferri, which causes purple blotch; Pseudomonas cepacia Burkholder, which causes sour skin; Urocystis colchici (Schlecht.) Rabenh. (2g, cepulae Frost) which causes onion smut; Sclerotium cepivorum Berleley, which causes white rot; Colletotrichum circinans (Berk.) Vogl., which causes smudge on white-skinned onions; Pyrenochaeta terristis (Hansen) Gorenz, Walker, and Larson, which causes pink root; Fusarium oxysporum Schlecht. E. JR. cepa (Hans.) Snyd. and Hans., which causes basal rot; and the aster yellows mycoplasma, a mycoplasma-like organism, which causes aster yellows. The studies described in this dissertation were directed towards the epidemiology and histopathology of Botrytis sguamosa and Botrytis allii. Numerous Botrytis species have been described on onion. However, similarity between species of Botrytis and overlapping of symptoms induced in onion by the various species led to ambiguities in the early literature. Perhaps the first reports of a Botrytis disease of onion were by Sorauer in 1876 and Frank in 1880. Sorauer (1886) identified the conidial state as Botrytis cana (Pers.) Fr. and Sclerotium cepa as the sclerotial state. Frank (1896) published a similar report. The first report of a Botrytis disease of onion in the United States was by Halsted (1890) who identified the causal agent as Botrytis parasitica Cav. Smith (1900) described a Botrytis disease of onion but considered the pathogen to be an atypical Botrytis cinerea. Clinton (1903) described a neck-rot disease of onion in Connecticut occurring in 1902-1903 and suggested it was the same disease described by Halsted (1890). In 1917 Munn published a detailed description of a neck rot disease in Michigan and named the pathogen (n. sp.) Botrytis allii, He compared his isolate with literature descriptions or samples of other Botrytis species found on onion and described B, allii as a new species, claiming that it was morphologically different from the other species described on onion. In inoculation experiments, Munn (1917) demonstra- ted that B, allii was an aggressive pathogen on the bulbs, and an aqueous suspension of B, allii_conidia applied to onion leaves also induced leaf lesions. In 1925 walker described two additional pathogens of onion, Botgztis.byssoidea and Botrytis squamosa. He compared and contrasted both of these species with B, allii_Munn. In 1926 Walker published a more detailed account concerning the biological differences between the three Botgztis species. He defined B, sguamosa as the cause of small sclerotial neck rot and B, byssoidea as the cause of mycelial neck-rot. walker (1926) recognized B, bzssoidea as the most common bulb pathogen in Wisconsin. At a later date, however, Walker (1969) considered B, gliii_as the most important bulb pathogen on the world level. In 1938 Yarwood claimed to have isolated B, cinerea from onion leaves, and induced leaf lesions by applying conidial suspensions to onion leaves. Olgivae (1941) also implicated B, cinerea as the cause of leaf Spotting of onion. Thus, by the 1940's B, §Bl$i_was recognized as the major cause of bulb rot, although 5 species of Botrytis were implicated as causing a foliar disease of onion. Hickman and Ashworth (1943) reported that B, sguamosa and B, cinerea were responsible for leaf spotting and dieback of onion leaves in Great Britain, with B, sguamosa reported to be the predominant pathogen. The two species were weakly pathogenic to onion bulbs. Page (1955) and Viennot-Bourgin (1953) also identified B, sguamosa as the major cause of leaf spotting and blighting in onion. These studies established B, sguamosa as the major cause of leaf spotting and blight- ing in onion. Some confusion was created when Segall and Newhall (1960) reported that B, allii_was responsible for a leaf spotting and blighting disease of onion in New York. It is possible the Segall and Newhall (1960) either did not recognize B, sguamosa as a species of misidentified B, sguamosa as B. allii, The confusion was resolved by Hancock and Lorbeer (1963) who studied symptom expression in onion foliage induced by B, sguamosa, B, cinerea, and B, Ellii, Based on their observations they proposed that the disease caused by B. sguamosa be named Botrytis leaf blight, and that caused by B, cinerea be named Botrytis leaf fleck. They did not recognize B. allii_as a significant foliar pathogen. B, Ezssoidea also is not recognized as a significant pathogen of onion foliage (Walker, 1926) or flowers (Ellerbrock and Lorbeer, 1977a). Recent reports indicated that B, giiii_may be associated with onion in a more subtle fashion. Studies by Tichelaar (1967) and Maude and Presly (1977a, 1977b) indicated that B, allBB could colonize healthy onion leaves asymptomatically. Further, these studies suggested that B, éilli could move down the leaves asymptomatically and colonize the bulb tissues. The disease would then appear in bulbs placed in storage. Recent studies also indicated that B, sguamosa may act in associ- ation with another pathogen. Maude and Presly (1980) reported that B, cinerea and B, sguamosa, occurring jointly, were responsible for a decay of the neck region of overwintered salad onion. Thus, B, sguamosa is a foliar pathogen of onion which causes a leaf spotting and die-back disease, although it may also colonize neck tissues. 0n the other hand, B, géiii causes a storage decay of onion bulbs, although leaf flecking and symptomless colonization of the foliage may occur. Although both diseases are caused by species of Botrytis, the plant parts attacked and the symptoms they induce are sufficiently different so as to recognize them as two distinct diseases. Although B, byssoidea has been associated with bulb decay (walker, 1926), I.l.|i..l.l I r lltll.ll| |.l I _ i I did not observe it among onion bulbs during my course of study. This dissertation addresses leaf blight and neck-rot separately. Within this introduction, and among the Chapters, leaf blight, then neck-rot, will be discussed. BOTRYTIS LEAF BLIGHT Occurrence Botrytis leaf blight has been reported in Belgium (Henebert, 1964), Canada (Page, 1953), England (Maude and Presly, 1980), France Viennot-Bourgin, 1953), Italy (Crotti, 1969), Japan (Takakuw, 1974), the Netherlands (Tichelaar, 1967), New Zealand (Dingly, 1961), Poland (Lutynska, 1968), and the United States (McLean and Sleeth, 1959). The disease is economically important in the northeastern United States, where weather conditions are favorable for disease development. Symptoms Detailed descriptions of leaf blight symptoms were first published by Walker (1926), Hickman and Ashworth (1943), and Page (1955). Lesions induced by B, sguamosa on onion leaves are discrete, greyish-white, desiccated spots, 2-5 mm.in length and 1-4 mm in width. Under prolonged warm, moist weather conditions leaf blighting and collapse may occur. Leaves die from the tip downward and older leaves are more severely attacked than younger ones. Sporulation occurs on necrotic or blighted leaves, leaf tips, or, occasionally, from expanding lesions. Segall and Newhall (1960) described leaf blight as a two phased phenomenon where phase one was leaf spotting and phase two was blighting. Leaf spotting and blighting have been reported to occur most commonly when plants were near maximum foliar development (Small, 1970; Segall and Newhall, 1960; Swanton, 1977; Lacy and Pontius, 1983). However, leaf blight attributed to B, sguamosa was reported on seed- lings by Lafon (1962). Morphology Morphological characteristics of B, sguamosa were described by Walker (1925, 1926). Mycelia of B, sguamOsa are hyaline, multibranched, septate, and variable in diameter. Conidia are borne on short sterig- mata at swollen apical tips of conidiophores. Conidia are ovoid to ellipsoid, smooth, hyaline at first but darkening with age. Most conidiophores are 11-15 X 15-22 um. Following fructification, side branches of conidiOphores degenerate to form characteristic accordian— like folds. Microconidia are hyaline, globose, about 3 um in diameter. Sclerotia are roughly circular, flat, scale-like, k—4 mm in diameter, white at first but darkening with age until black. Cronshy (1946) was the first to report the perfect state of B, sguamosa. Viennot-Bourgin (1953) made a detailed study of the apothecia of B, sguamosa and named the teleomorph Botryotinia squamosa. McLean (1960) examined apothecia of B, sguamosa and felt that the teleomorph should be named Sclerotinia squamosa (Viennot-Bourgin) Dennis. However,, no subsequently published reports that I am aware of have referred to the fungus as Sclerotinia squamosa. Apothecia of B. sguamosa are 2-3 mm tall with a cup diameter of 3-5 mm (Viennot-Bourgin, 1953). Asci were observed by McLean (1960) to be 162.5-200 um X 13.8-16.5 um, each containing 8 ascospores averaging 10.0-12.5 X 15.0-17.5 um. Ascospores were measured by Viennot-Bourgin (1953) as 11.7 X 8.11 um. Survival and Primary Inoculum Walker (1926) reported that sclerotia of B, sguamosa survived on a window sill at Madison, WI from December-March. Ellerbrock and Lorbeer (1977b) studied sclerotial survival under field conditions and found that 7 and 65% of sclerotia survived for 21 months when buried 3 or 5 cm deep respectively in organic soil. Sclerotia placed on the surface of soil survived from September through May. Conidia survived for only about 2 months in soil under both controlled and field conditions. Sources of primary inoculum include sclerotia and infested onion debris (Ellerbrock and Lorbeer, 1977c). .McLean and Sleeth (1959) and Lafon (1961) suggested that conidia produced on sclerotia could serve as primary inoculum. Segall and Newhall (1960) observed sporulation of Botgytis species on cull onions discarded the previous year. Ellerbrock and Lorbeer (1977b) also observed sclerotia bearing conidia on onion bulbs and leaf debris in cull onions discarded the previous year. They trapped conidia from the air over cull piles and seed production fields 2-4 weeks prior to the appearance of the disease in commercial onion fields. In addition, Ellerbrock and Lorbeer (1977c) observed apothecial production from sclerotia in onion fields on one occasion, although this is extremely rare, and determined that ascospores could induce lesions on onion leaves., Conidia of B, sguamosa on seed have not been shown to infect onion seedlings (Ellerbrock and Lorbeer, 1977a). Secondary Inoculum The potential of B, sguamosa to cause epidemics has been well documented (Page, 1955; McLean and Sleeth, 1959; Swanton, 1977; Lacy and Pontius, 1983). Lorbeer (1966) and Lacy and Pontius (1983) observed a diurnal periodicity in Spores trapped. Spore trap studies by Swanton (1977) suggested that prolonged warm, wet weather conditions followed by increasing temperature and declining relative humidity favored spore release. The analysis of several years of weather and spore trap data by Lacy and Pontius (1983) indicated that warm, humid weather over a consecutive three day period is necessary to induce a large spore release. Control Berquist and Lorbeer (1971) examined varieties of Allium cepa for resistance t°.§; sguamosa but found none. However, Allium.bouddhae C 598 and B, schoenoprasum.were found to be immune to B, sguamosa. They suggested that it may be possible to introduce this resistance into Allium.cepa. Control of leaf blight based on calendar applications of fungi- cides. Several systems have been developed to reduce the number of spray applications. Shoemaker and Lorbeer (1977) described a critical disease level (CDL) of one lesion per 10 leaves to determine when spray- ing should be initiated. Swanton (1977) combined the CDL with weather conditions favorable for spore production so as to further reduce unnecessary sprays after the CDL was reached. Dzikowski (1980) devel- oped a similar system based on the CDL but suggested spraying in advance of expected rain. Lacy and Pontius (1983) developed a sporulation pre- diction model which provided a probability index for sporulation based on three day averages of temperature and vapor pressure deficits. BOTRYTIS NECK ROT Occurrence Botrytis diseases caused by Botrytis allii have been reported in England (Moore, 1948), Denmark (Hellmers, 1943), Holland (Tichelaar, 1967), New Zealand (Brain, 1939), Canada (McKeen, 1951), Norway (Ried, 1952), Israel (Netzer and Dishon, 1966), France (Henebert, 1964), Poland (Lutynska, 1968), and the United States (Munn, 1917). Symptomatologz, Botrytis allii has been found in association with onion bulbs, leaves, scapes, flowers and seeds. Symptoms on bulbs were described by Munn (1917) and Walker (1926). Onion bulb tissues at the early stage of infection appear soft, slightly sunken and water soaked, with a distinct margin. As the fungus develops on the older diseased areas the tissues become greyish in color and a smoke-grey mat of mycelia and conidio- phores bearing conidia develops on the surface of the scale. The fungus progresses within the bulb scales more rapidly than between scales. Mycelial mats between scales may fill the space between scales and appear dirty white to brown. Sclerotia develop in the older diseased areas and appear whitish at first, but later turn black. Walker (1926) described sclerotial bodies as hard, black, rounded, Spherical, oblong or irregular, varying from 1-3 mm or more in length and which may become aggregrated into crusty masses. Little odor is associated with the bulb decay. Botrytis allii was reported to cause leaf lesions and blighting of the foliage (Segall and Newhall, 1960), scape (Yarwood, 1938), flower (EllerbrockanuiLorbeer, 1977a) and seedling (MtKeen, 1951), although 10 later studies indicated that B, ailii_is not a serious pathogen of onion (Hancock and Lorbeer, 1963). Symptomless infections of B, allii in healthy onion leaves were observed by Maude and Presly (1977a, 1977b) and Tichelaar (1967). It is possible that Segall and Newhall (1960) either did not recognize B, sguamosa as a species or misidentified B, squamosa as B, allii. MoEphology Morphological characteristics of B, allii were described by Munn (1917). Mycelia of B, ailii varies from 4.5-9.0 um in width. Hyphae branch irregularly. Conidiophores are short and erect, occurring singly or in clusters. Munn (1917) observed that conidiophores were branched in culture and unbranched on the host plant. Conidia are hyaline, oblong in shape, and most are between 7-10 by 5-6 um in size. Unlike B, sguamosa, no perfect state has been reported for B, allii. Overwintering; Primary and Secondary Inoculum Munn (1917) observed sporulation of B, Bl;ii_in cull piles in Michigan in April and May and in onion leaves in New York onion fields in April. Reports by Maude and Presly (1977a, 1977b) indicated that B, .Eiiil can be seedborne and that B, giiii on seed can infect seedlings symptomlessly. They reported survival of B, géiii_on onion seed during storage. Munn (1917) suggested that aerial dissemination of B, allii may occur and that the disease is favored by cool wet weather. Munn (1917) stated that the optimal temperature for sclerotial germination was 10 C. Maude and Presly (1977a) believed that long periods of relative 11 humidities greater than 80% and frequent rains favored conidial produc- tion and spread of the disease. Control Maude and Presly (1977b) observed that seed infested with B, gill; resulted in increased neck-rot in storage. Further, they stated that seed treatments with a systemic fungicide (benomyl) effectively reduced the incidence of neck-rot in bulbs grown from these plants in storage. Most control efforts have been directed towards the harvesting and storage of onions. Proper handling, curing, and storage of onions has contributed greatly to the reduction of neck rot incidence. OBJECTIVES The overall objectives of this research were to determine the influence of temperature and moisture on infection of and development within onion tissues by B, squamosa and B, allii, and to integrate this knowledge with that derived from.previous studies so as to more clearly define the biology of these two fungi. 12 REFERENCES Anonymous. 1960. Index of Plant Diseases in the United States. U.S. Dept. Agric. Agricultural Handbook 165. 531 pp. Berquist, R. K. and J. W. Lorbeer. 1971. Reactions of Allium spp. and Allium cepa to Botryotinia (Botrytis) squamosa. Plant Dis. Rep. 55:394-398. Brien, R. M. 1939. List of plant diseases recorded in New Zealand. New Zealand Dept. Sci. Indust. Res. Bull. 67. 39 pp. Clinton, G. P. 1903 Report of the station botanist. 27th Ann. Rep. Conn. Agric. Exp. Stn. pp. 334-336. Cronshey, J. F. H. 1946. The perfect stage of Botrytis squamosa Walker. Nature (London) 158:379. Crotti, M. 1969. Botrytis squamosa Walker su Cipalla (Allium cepa). Riv. Ortoflorofruttic. Ital. 53:392-398. Dingly, J. M. 1961. New records of fungus diseases in New Zealand. New Zealand J. Agric. Res. 4:336-347. Dzikowski, P. A. 1980. The use of weather information for timing fungicide applications to control Botrytis leaf blight in onions. M. Sci. Thesis, Univ. Guelph, Guelph, Ontario. 62 pp. Ellerbrock, L. A. and J. W. Lorbeer. 1977a. Etiology and control of onion flower blight. Phytopathology 67:155-159. Ellerbrock, L. A. and J. W. Lorbeer. 1977b. Survival of sclerotia and conidia of Botrytis squamosa. Phytopathology 67:219-225. Ellerbrock, L. A. and J. W. Lorbeer. 1977c. Sources of primary inoculum of Botrytis squamosa. Phytopathology 67:363-372. Frank, A. B. 1880. Die Krankheiten Der Pflanzen. Breslau. 844 pp. Frank, A. B. 1896. Die Krankheiten Der Pflanzen. Breslau. Halsted, B. D. 1890. Fungous diseases of various crops. Rep. N. J. Agric. Exp. Stn. 3:352. Hancock, J. G. and J. W. Lorbeer. 1963. Pathogenesis of Botgytis cinerea, B. squamosa and B, allii on onion leaves. Phytopathology 53:669-673. Hellmers, E. 1943. Botrytis on Allium species in Denmark. Botgytis allii Munn and B, globosa Raabe. Medd. Plantepat. afd., Den kgl. veter. og landboh¢jsk., Khvn, 25:1-51. 13 Henebert, G. L. 1964. Botryotinia squamosa nouveau parasitica de l'eigon en Belgique. Parasitica 20:138-153. Hickman, C.J. and D. Ashworth. 1943. The occurrence of BotEytis spp. on onion leaves with special reference to B. squamosa. Trans. Br. Mycol. Soc. 26:153-157. Jones, H. and L. K. Mann. 1963. Onions and their allies. Leonard Hill Limited, New York. 286 pp. Lacy, M. L. and G. A. Pontius. 1983. Prediction of weather mediated release of conidia of Botrytis squamosa from onion leaves in the field. Phytopathology 73:670-676. Lafon, R. 1961. Botrytis on onion seedlings (Botrytis squamosa Walker) Bull. Tech. Ing. Serv. Agric. 162:1-8. Lafon, R. 1962. Le Botrytis des Feuilles de L'Oignon (Botrytis squamosa Walker) XVIth International Horticultural Congress 2:367- 371. Lorbeer, J. W. 1966. Diurnal periodicity of Botrytis squamosa conidia in air. Phytopathology 56:887 (Abstr.). Lutynska, R. 1968. Investigations on diseases of seed onion caused by the fungus Botrytis in the vegetable plantations of the Cracow Province. Acta. Mycol. 4:3-22. Maude, R. B. and A. H. Presly. 1977a. Neck rot (Botrytis allii) of bulb onions: 1. seed-borne infection and its relationship to the disease in the onion crop. Ann. Appl. Biol. 86:163-180. Maude, R. B. and A. H. Presly. 1977b. Neck rot (Botrytis allii) of bulb onions: II. seed-borne infection in relationship to the disease in store and effect on seed treatments. Ann. Appl. Biol. 86:181-188. Maude, R. B. and A. H. Presly. 1980. Studies on the biology of Botgytis species on overwintered salad onions. Ann. Appl. Biol. 94:174-184. McKeen, C. D. 1951. An occurrence of rot of Spanish onion seedlings caused by Botrytis allii. Sci. Agric. 31:541-545. McLean, D. M. and B. Sleeth. 1959. Tip and leaf blight on onions in the lower Rio Grande Valley. Journal of the Rio Grande Valley Horticultural Society 13:152-154. McLean, D. M. 1960. The apothecial stage of Botrytis squamosa, cause of tip and leaf bright of onions. Pland Dis. Rep. 44:585-586. 14 Moore, W. C. 1948. Report on fungus, bacterial and other diseases of crops in England for the years 1943-1946. Bulletin No. 139. Ministry of Agriculture and Fisheries H.M.S.0., London. Munn, M. T. 1917. Neck rot disease of onions. New York Agric. Exp. Stn Bull. 437:363—455. Netzer, D. and I. Dishon. 1966. Occurrence of Botrytis allii in onions for seed production in Isreal. Plant Dis. Rep. 50:21. Olgive, L. 1941. Diseases of Vegetables. Bulletin No. 123. Ministry of Agriculture and Fisheries H.M.S.0., London. 29 pp. Page, 0. T. 1953. Botrytis spot of Onion leaves in Ontario. Plant Dis. Rept. 37:513-514. Page, 0. T. 1955. Botrytis leaf spot on onions and its control. Can. J. Agric. Sci. 35:358-365. Rded, H. 1952. Botgytis (grey mould) on Allium cepa and Allium ascolonicum in Norway. Acta. Agric. Scand. I:20-39. Segall, R. H. and A. G. Newhall. 1960. Onion blast or leaf spotting caused by Botgytis. Phytopathology 50:76-82. Shoemaker, P. B. and J. W. Lorbeer. 1977. Timing initial fungicide application to control Botrytis leaf blight epidemics on onions. Phytopathology 67:409-414. Small, L. W. 1970. The epidemiology of leaf blight disease of onions incited by Botrytis squamosa. M. Sci. Thesis, McGill Univ., Montreal, Quebec. 249 pp. Smith, R. E. 1900. Botgytis and Sclerotinia: their relation to certain diseases and to each other. Bot Gaz.. Sorauer, P. 1876. Das Verschimmeln der Speisezqiebldeln. Oesterr. Landw. Wchnbl. 147 pp. Sorauer, P. 1886. Sclerotienkrankeiten (das verschimmeln) der Speisezqielbln. Handbuch der Pflanzenkrankheiten Aufl 2. pp 294-297. Swanton, C. J. 1977. Influence of environmental factors on develop- ment and control of Botrytis leaf blight on onions. M. Sci. Thesis, Univ. Guelph, Guelph, Ontario. 89 pp. Takakuw, M., I. Saito, A Tanii, and O. Tamura. 1974. Leaf spots in onions and leeks caused by Botrytis spp. Bulletin of Hokkaida Prefectural Agricultural Experiment Station No. 29, pp. 1-6. Tichelaar, G. M. 1967. Studies on the biology of Botrytis allii on Allium cepa. Neth. J. Plant Path. 73:157-160 15 Viennot-Bourgin, G. 1953. Un parasite nouveau de l'oignon en France: Botrytis squamosa Walker et sa forme par forte Botryotinia squamosa sp. nov. Ann. des Epiphytes 4:23-43. Walker, J. C. 1925. Control of mycelial neck rot of onion by artificial curing. J. Agric. Res. 30:365-373. Walker, J. C. 1926. Botrytis neck rots of onions. J. Agric. Res. 33:893-928. Walker, J. C. 1969. Plant Pathology. McGraw Hill, New York. 819 pp. Yarwood, G. E. 1938. Botrytis infection of onion leaves and seed stalks. Plant Dis. Rep. 22:428-429. CHAPTER II INFLUENCE OF DEW PERIOD AND TEMPERATURE ON INFECTION OF ONION BY BOTRYTIS SQUAMOSA INTRODUCTION Early investigations concerning leaf blight established that the disease was favored by prolonged moist weather conditions (Clinton, 1903; Page, 1955; McLean and Sleeth, 1959). Subsequent investigations have established that temperature and leaf wetness are important epide- miological parameters in spread of Botrytis leaf blight under field conditions (Segall and Newhall, 1960; Swanton, 1977; Lacy and Pontius, 1983). Lacy and Pontius (1983) identified temperature and humidity as the most important factors in production and release of B, squamosa conidia, and devised a disease predictive system based on these factors. Production of inoculum, however, is only part of the disease spread process. Understanding the influence of temperature and leaf wetness on germination and infection of onion by B, squamosa is also epidemio- logically important, and would allow a more complete predictive system to be synthesized. Germination of B, squamosa conidia has been examined in water on glass slides and on leaf surfaces with conflicting results. On glass slides, Shoemaker and Lorbeer (1977) reported optimal germination at 15 C with no germination above 27 C. Swanton (1977) reported optimal germination at 20 or 28 C with reduced germination at 33 C. McDonald 16 17 (1981) reported optimal germination at 24 C. On leaf surfaces, histo- pathology of conidial germination and subsequent infection was studied by Clark and Lorbeer (1976) at 21 C, but temperature influences on germination were not determined. Several studies have addressed the effect of temperature and/or leaf wetness on infection of onion by B, squamosa. The optimal temper- ature for leaf blight development was reported to be 18 C (McDonald, 1981; Tanner and Sutton, 1981), and 20 C (Shoemaker and Lorbeer, 1977; Swanton, 1977). Lesion development was reduced at temperatures above 24 C (McDonald, 1981; Shoemaker and Lorbeer, 1977; Tanner and Sutton, 1981). Lesion numbers increased with increasing leaf wetness duration at 9-25 C (McDonald, 1981; Shoemaker and Lorbeer, 1977; Swanton, 1977; Tanner and Sutton, 1981). Conflicting reports have been published on optimum leaf wetness periods for maximum infection. Using aqueous conidial suspensions, minimal leaf wetness durations for subsequent development of lesions at 18-20 C have reported to be 12 (Tanner and Sutton, 1981), 9 (McDonald, 1981), 6 (Shoemaker and Lorbeer, 1977), or 5 hours (Swanton, 1977) while maximum.numbers of lesions were reported to occur after 24 (McDonald, 1981), 48 (Swanton, 1977) or 60 hours (Shoemaker and Lorbeer, 1977) of continuous leaf wetness. At 24 C McDonald (1981) reported maximum lesion production after 12 hours of postinoculation leaf wetness. Preliminary experiments gave highly variable numbers of lesions when plants were inoculated with aqueous conidial suspensions. Much more consistent results were obtained by applying the conidia in a dry state in a settling tower, so we used the dry conidia inoculation technique throughout these experiments. 18 The objectives of this study were to determine the effect of . length of dew period at various temperatures on conidial germination and subsequent infection and lesion production in onion leaves exposed to a standardized number of conidia under controlled environmental conditions, with the conidia applied in a dry state similar to that occurring in nature; and to determine the effect of extended dew dura- tion on postinfection hyphal development within lesions. MATERIALS AND METHODS Production of B. squamosa Conidia Isolate BSS-4 of B, squamosa, originally isolated from Michigan- grown onions, was used in all experiments because it was highly virulent, and it sporulated somewhat more prolifically on artificial media than other isolates. The fungus was grown on potato dextrose agar (PDA) for 7-12 days, then transferred to sterilized muck soil in test tubes, incu- bated 7-12 days at room.temperature, and stored at 5 C. For conidial production, infested muck soil particles from the soil tubes were placed on autoclaved (85 minutes at 20 p.s.i.) onion leaves placed aseptically on the surface of water agar in 9-cm diameter petri plates. The plates were incubated at 20 C under a 16 hour fluorescent light photoperiod for 5-7 days. Conidia were produced abundantly using this system, but not on other agar media. Cultures survived greater than two years in muck soil tubes and did not lose virulence. Development of a Dry Spore Inoculation Procedure Preliminary inoculation experiments using aqueous conidial snapen- sions of B, squamosa gave highly variable lesion numbers using standar- dized spore suspensions. Difficulties were encountered in suspending 19 the conidia in water and in applying a uniform mist of the suSpension to leaves due to the hydrophobic nature of onion leaves and of B, squamosa conidia. Aggregations of fine droplets easily ran off the leaves. A wetting agent (Tween 20) aided in suspending conidia in water, but difficulties persisted in the application of suspensions to leaf surfaces. A dry spore inoculation technique was devised to over- come the difficulties associated with aqueous conidial suSpensions and their application to leaves. A galvanized sheet metal cylinder (61 cm diameter by 77 cm deep) mounted on a wooden base was used as a settling tower. Conidia were collected from sporulating cultures using a pasteur pipet connected to a water aspirator. At very low suction conidia were collected in the pipet without being sucked into the aspirator. Conidia were easily tapped out of the pipet onto weighing paper. The relationship between conidial weight and numbers was deter- mined by suspending known weights of conidia in 70% ethanol and esti- mating spore numbers using a hemacytometer. Ethanol solution was used since conidia readily entered into suspension in this medium. Amounts of conidia weighing 0.5, 1.0, 1.5, 2.0, and 2.5 mg were determined to contain ca. 2.5-, 4.9-, 7.4-, 10.0-, and 12.5 X 105 conidia respectively. These numbers represented the actual conidia, measured by weight, fall- ing on the floor area in the settling tower. For inoculation, plants were positioned within the settling tower and dry conidia were dispersed near the top of the chamber by directing a low velocity stream of air from a pipet tip over the conidia on a piece of weighing paper. A wooden cover was positioned over the tap of the cylinder to reduce air currents and allow the conidia to settle on the plants. 20 In experiments requiring dew formation on plants, the plants were positioned within a commercial dew chamber (Percival Mft. Co., Boone, IA 50036) for the appropriate dew period. Dew formation was evident on plants within 1 hour of their placement in the dew chamber. To determine the relationship between inoculum density and lesion numbers, groups of six replicate onion plants were each inoculated with 0.5, 1.5, 2.0, or 2.5 mg dry conidia, then placed in a dew chamber at 20 C for 24 hours. Lesion numbers were counted at the end of the incu- .bation period. Influence of Dew Period and Temperature on Lesion Production Groups of 18 onion plants were inoculated with dry conidia and placed in the dew chamber at 15, 20 or 25 C. Six randomly preselected plants were removed from the dew chamber after each period of 4, 8, 12, 16, 24, or 32 hours of continuous dew, respectively, and were then moved to a growth chamber set at the same temperature as the dew chamber set at the same temperature as the dew chamber, with 60 i 10% relative humidity (RH) and a 16 hour photOperiod. ‘Dew dried from the leaves within 5 minutes. Lesions were counted on each plant after the variable times in the dew chamber and growth chamber totaled 48 hours. The experiment was repeated three times. Influence of Temperature on Spore Germination and Infection Four onion plants were inoculated in the settling tower with 2 mg dry conidia and placed in a dew chamber for 24 hours at 20 or 25 C. The third or fourth youngest leaf on each plant was used since lesion size distribution was more uniform on these leaves. Four 1-cm2 leaf tissue pieces were removed from each of four replicate leaves on four separate 21 plants, fixed in formalin:acetic acid:50% ethanol (1:18:1 v/v) (FAA), Stained in cotton blue, and examined using light microscopy. Conidia which washed off leaves during fixation were collected on a 13-mm- diameter filter membrane (Millipore Filter Corp., Bedford, MA 01730) with 0.33 an pore size, stained with cotton blue, and counted using light microscopy. Numbers of conidia on leaves which had formed appressoria were counted, as well as spores which had formed both appressoria and infec- tion hyphae. Appressorial counts were based on swollen germ tube tips and infection hyphal counts were based on the observation of infection hyphae within lesions. The experiment was repeated two additional times. Influence of Dew Period on Infection Hyphal Development Sixteen onion plants were inoculated and placed in the dew chamber for 2, 4, or 6 days of continuous dew at 20 C with a 12 hour photoperiod. In addition, four replicate plants were given a 2 day dew period at 20 C, followed by incubation in a growth chamber maintained at 20 C, 60 t 10% RH and a 12 hour photoperiod for 4 days. Leaf sections containing lesions were randomly removed from plants, fixed in FAA, cleared in boiling 70% ethanol, and stained in 1% aqueous trypan blue. Lengths of infection hyphae in each lesion were measured with an ocular micrometer for a minimum of 50 lesions. The experiment was repeated three times. Longevity of Conidia on Leaves To determine the longevity of spores on onion leaves following inoculation, two groups of 21 onion plants were inoculated with 4 mg conidia each and placed in a growth chamber at 20 C. after 0, 1, 2, 3, 22 4, 6, or 8 days, 6 replicate plants were removed from the growth chamber and incubated with continuous dew for 36 hours, then returned to the growth chamber. Numbers of lesions per plant were counted 24 hours after removal from the dew chamber. RESULTS Influence of Inoculum Density on Lesion Numbers Lesion numbers increased linearly up to a maximum of about 280/ plant when 0.5 to 2.5 mg conidia per 0.3 m2 area of settling tower floor were applied to onion plants subsequently incubated for 24 hours in a dew chamber at 20 C (Figure 2.1). Influence of Dew Period and Temperature on Infection Lesions were produced after 8 but not after 4 hours continuous dew at 20 C. The number of lesions per plant increased in apparent sigmoi- dal fashion with increasing dew duration, with little increase in lesion numbers after 24 hours of dew (Figure 2.2). Total lesions produced at 20 C or 15 C were approximately equal on plants incubated with constant dew for 32 hours, although the rate of increase in lesion production at incremental dew periods was faster at 20 than at 15 C. Lesion produc- tion was severely curtailed at 25 C and did not increase further after 12 hours continuous dew. To determine the minimum dew period required for infection, 6 replicate onion plants were inoculated with dry conidia and placed in the dew chamber at 20 C. After 4, 5, 6, or 7 hours plants were trans- ferred to a growth chamber set at 20 C and 60 t 10% RH. Lesions were counted after an additional 24 hour in the growth chamber. No lesions 23 300% 200- 100- LESIONS / PLANT 0 o 01.5 130 135 2.TO 215 3.0 SPORE MASS (MG) / 0.3 M2 SEITLING TOWER FLOOR AREA Figure 2.1. Relationship between mass of Botgytis squamosa conidia and lesion numbers of 1-month-old onion plants after incubation in a dew chamber for 24 hours. ‘ 24 .H<.mm3 meson mm cam m amm3umn Ano.OLMV nmA “mucoafiumaxo OumOHHmmu manna aoum vo>auom mama mmaam> emu: .mmoEmnvm mauhwuom mo mavficoo we N nuwz umsou wnHHuumm a ca aoaumasooafi wefisoaaow madman coaco paelcuaoEIweo we unuseoao>ou eoamoa co poauma Bow new unnumHOQEOu mo moemaamaH .~.N madman EIV 005mm Bun «m. 0N ON ON m. N. m V 00 d u q - u 0 mm 0m 0 m. 00. Om. LESIONS / PLANT 1._ O 0 CH 0 o~ 1 0mm Oon 25 were observed on plants given 4 or 5 hour dew periods. After 6 or 7 hours dew, 4.2 i 4 and 29 i 18 lesions per plant, respectively, were observed. Thus, 6 hours was the minimum dew period required for lesion formation. Influence of Temperature on Spore Germination and Infection At 20 C, 78 i: 10% of conidia on leaves germinated, while 62 :1: 8% induced lesions. At 25 C, 55 i 5% of the spores germinated while only 27 t 10% induced lesions. At 20 C, 61 t 8% of the applied spores formed appressoria and 36 i 6% formed infection hyphae, compared with 37 t 1% forming appressoria and 11 i 6% forming infection hyphae at 25 C. Influence of Dew Period on Development of Infection Hyphae Length of dew period following inoculation had a dramatic effect on infection hyphae development within lesions. Lengths of infection hyphae within lesions, after 2 days in the dew chamber were less than 125 um (Table 2.1). After 4 days ca. 30% of the hyphae extended beyond 125 um.with the greatest number falling into the 125-150 um catagory. After 6 days of continuous dew ca. 35% of the hyphae extended beyond 125 um, again with the greatest number in the 125-150 um.catagory. Infection hyphae in lesions on plants treated with 2 days dew followed by 4 days without dew in the growth chamber remained restricted, with only 4% reaching the 125-150 um catagory, and none falling into the longer catagories. Longevity of Conidia on Leaves Numbers of lesions remained relatively constant on leaves held in the growth chamber (60 i 10% RH and 20 C) for up to 2 days after inoc- ulation, then placed in the dew chamber for a 36 hour dew period 26 (Figure 2.3). There were ca. 425-450 lesions per plant with a 0-2 day interval between inoculation and exposure to dew. With a 3-4 day interval this declined to ca. 245 lesions per plant, and with a 6 or 8 day interval this further declined to 130 lesions per plant, reflecting a 71% loss in infectivity of conidia after 6 days in the growth chamber prior to dew period. Viability of conidia evidently declined rapidly after the first two days without dew. Table 2.1. Percentage of infection hyphae of Botrytis squamosa falling within ranges of lengths in lesions on onion leaves after various dew periods at 20 C. Infection hyphae Lenggh of dew period (days) 2 days dew + 4 lengths (um) 2a 4a 6a days no dew 1-125 100b 72 :1: 15 65 a: 14 96 :l: 6 125-150 0 13 t 8 17 i 9 4 i 6 250-375 0 7 t 4 5 t 5 0 375-500 0 3 i 3 4 i 1 0 500 0 5 i 1 9 t 3 0 aLesions were examined immediately after the end of the indicated dew period. bBased on the mean of three replicate experiments with each experimental run including a minimum of 50 lesion observations. DISCUSSION Previous dew period studies employed aqueous conidial suspensions as inoculum. However, under field conditions B, squamosa conidia are disseminated by wind and deposited on leaves as dry spores (Lacy and Pontius, 1983). Most conidia of B, squamosa are released between 0800 27 600 (n C) C) l S LESIONS / PLANT ‘ ho (A C) C) S” ‘3 A C) C? C)or1rr2 3141516'715 DAYS BETWEEN INOCULATION AND DEw PERIOD Figure 2.3. Effect of periods of time following inoculation without dew (20 C and 60% RH) prior to exposure to 24 hours continuous dew (20 C) on numbers of lesions per plant. Plants were inoc- ulated in a settling tower using 4 mg dry Botgytis squamosa conidia. 28 and 1300 hours (Lacy and Pontius, 1983; Sutton et al., 1978) where they must survive in a dry state until a period of leaf wetness occurs. Shoemaker and Lorbeer (1977) reported that dry conidia brushed onto leaf surfaces of plants in growth chambers survived 2 days at 92% RH, although lesion numbers relative to control plants were not quantified. Results of my study suggest that B, squamosa conidia survive well on leaf surfaces for 2-3 days after deposition in the absence of dew or rain without a significant loss of viability, although survival could be shorter on leaves exposed to full sunlight. Lesion production after a minimum 6 hour dew period was consistent with the observations of Shoemaker and Lorbeer (1977) who, using aqueous conidial suspensions, reported a 6 hour minimum leaf wetness period for lesion development. We observed that relatively few lesions were pro- duced using only 6 hours dew, and a sharp increase in lesion numbers occurred with 12 hours of dew at 20 C (Figure 2.2), which also agreed with Shoemaker and Lorbeer (1977). However, my study more clearly and quantitatively defined the influence of temperature and dew period on lesion numbers induced by B, squamosa. The slower rate of lesion production at 15 C compared with 20 C could aid in understanding lesion production under field conditions. Dew periods shorter than 16 hours could result in significantly fewer lesions at 15 C than at 20 C (Figure 2.2), and temperatures of 25 C of greater with any dew period could also result in fewer lesions (Figure 2.2)(McDonald, 1981; Shoemaker and Lorbeer, 1977). Shoemaker and Lorbeer (1977) reported that spore germination on glass slides declined from 80% at 21 C to 20% at 24 C, while McDonald (1981) and Swanton (1977) reported optimal germination at 24 C on glass 29 slides. I observed a sizable reduction in spore germination on leaves at 25 C compared to 20 C. The reduced spore germination and even greater reduction in production of infection hyphae could account for the reduced lesion numbers noted at 25 C. Extent of pathogen development within lesions was dependent, at least in part, upon length of continuous dew period.‘ After 4 days of continuous dew some lesions were expanding, initiating the leaf blight phase of the disease (Table 1), similar to observations of Clark and Lorbeer (1976). Hyphal length observations revealed that hyphae contin- ued to grow in 5-15% of the original lesions and were responsible for the rapid and destructive leaf blighting phase of the disease. The reason for some lesions expanding and others remaining static in size is not understood as yet, nor is the effect of extended dew periods on expansion of a larger proportion of lesions understood. It is possible that biochemical or physiological characteristics, which could aid in overcoming host resistance mechanisms may be inherent in 5-15% of the conidia. Quantitative studies using other isolates of B, squamosa, or mutants of B, squamosa may identify cultures which differ in ability to induce expanding lesions. Such cultures may be useful in elucidating why some lesions expand and other do not. _30 REFERENCES Clark, C. A. and J. W. Lorbeer. 1976. Comparative histopathology of Botrytis squamosa and B, cinerea on onion leaves. Phytopathology 66:1279-1289. Clinton, G. P. 1903. Report of the station botanist. 27th Annual Report, Conn. Agric. Exp. Stn. pp. 334-336. Lacy, M. L. and G. A. Pontius. 1983. Prediction of weather mediated release of conidia of Botrytis squamosa from.onion leaves in the field. Phytopathology 73:670-676. Lorbeer, J. W. 1966. Diurnal periodicity of Botrytis squamosa conidia in air. Phytopathology 56:887 (Abstr.). McDonald, M. R. 1981. Effect of environmental and host factors on Botrytis leaf blight of onion. M. Sci. Thesis, Univ. Guelph, Guelph, Ontario. 108 pp. McLean, D. M. and B. Sleeth. 1959. Tip and leaf blight of onions in the lower Rio Grande Valley. Journal of the Rio Grande Valley Horticultural Society 13:152-154. Page, 0. T. 1955. Botrytis leaf spot on onions and its control. Can. J. Agric. Sci. 35:358-365. Segall, R. H. and A. G. Newhall. 1960. Onion blast or leaf spotting caused by species of Botgytis. Phytopathology 50:76-82. Shoemaker, P. B. and J. W. Lorbeer. 1977. The role of dew and temperature in the epidemiology of Botrytis leaf blight of onion. Phytopathology 67:1267-1272. Sutton, J. C., C. J. Swanton, and T. J. Gillespie. 1978. Relation of weather variables and host factors to incidence of airborne spores of Botrytis squamosa. Can. J. Bot. 56:2460-2469. Swanton, C. J. 1977. Influence of environmental factors on development and control of Botrytis leaf blight of onions. M. Sci. Thesis, Univ. Guelph, Guelph, Ontario. 89 pp. Tanner, M. R. and J. C. Sutton. 1981. Effect of leaf wetness duration and temperature on infection of onion leaves by Botrytis squamosa. Phytopathology 71:565 (Abstr.). CHAPTER III INFLUENCE OF INTERRUPTIONS OF DEW PERIOD 0N NUMBERS 0F LESIONS PRODUCED 0N ONION BY BOTRYTIS SQUAMOSA INTRODUCTION Botrytis leaf blight is a leaf spotting and blighting disease ‘ which is especially severe under prolonged moist conditions at temper- atures of 15-24 C (Lacy and Pontius, 1983; Segall and Newhall, 1960; Sutton et al., 1978; Swanton, 1977). Studies relating dew period and temperature to leaf blight development indicated that lesion production was optimal at 18-20 C and that lesion frequency increased with increasing leaf wetness duration through 24-48 hours (McDonald, 1981; Shoemaker and Lorbeer, 1977; Swanton, 1977; Tanner and Sutton; 1981). Under field conditions, leaves are wet or dry for variable time periods, depending on dew or rain conditions. The influence of the length of and timing of interruptions in leaf wetness on lesion development in Botrytis leaf blight has not been well defined. McDonald (1981) reported reductions in lesions when leaf wetness periods were interrupted for 4 hours after a 4 or 8 hour leaf wetness period compared with those given a 24 hour leaf wetness period. Swanton (1977) examined the influence of interruptions in leaf wetness after 2 and 8 hours. His data suggested that interruptions longer than 10 hours after 8 hours leaf wetness resulted in reduced lesion numbers. Dzikowski (1980) observed greater reductions in percent leaf area 31 32 diseased when plants were interrupted for 1-4 hours after 5 hours of leaf wetness than after 2 hours leaf wetness. The timing of dew period interruptions and influence of interruption duration needs further clarification, and the influence of humidity during interruption has not been examined. The objectives of this study were to determine the influence of timing of initiation of post-inoculation interruptions in leaf wetness, the influence of duration of the interruptions, and the influence of humidity during the interruption, on lesion numbers produced on onion leaves by Botrytis squamosa. MATERIALS AND METHODS Botrytis squamosa was grown and spores were collected as described in Chapter II. In all experiments l-mo-old onion plants (cv. Spartan Banner, Granada, or Yellow Sweet Spanish) sprouted from bulbs were used. For inoculation, plants were positioned within a cylindrical 61 cm diameter by 77 cm deep settling tower with a rotating base. Dry conidia (2.5 mg, ca. 1.25 X 106) were dispersed near the top of the tower by directing a low velocity stream of air from a pipet tip over conidia on a piece of weighing paper, while the plants rotated in the chamber at 5-6 r.p.m. After the conidia were dispersed, a cover was placed over the top of the tower for about 5 minutes to reduce external air currents and allow the spores to settle on leaf surfaces. Dew was produced on plants within 1 hour after placing them in a commercial dew chamber (Percival Mfg. Co., Boone, IA 50036). 33 Influence of Timingipf Dew Period Interruption on Lesion Production Forty-two onion plants were inoculated in each of 2 groups of 21 plants, then were incubated within the dew chamber at 20 C for 2, 4, 6, 8, 10, 12, or 24 hours. These incubation periods were followed by 2 hours without dew in a 20 C growth chamber at 65 i 10% relative humid- ity (RH), then plants were moved back to the dew chamber for the remainder of the 24 hour incubation period. Inoculated and noninoc- ulated controls remained in the dew chamber for 24 hours. Numbers of lesions per plant were determined after an additional 6 hours in the growth chamber. The experiment was conducted three times. Influence Of Interruption Duration on Lesion Production Thirty-six onion plants were inoculated in each of 2 groups. Plants were incubated in the dew chamber for 6 hours then were trans- ferred to a growth chamber at 20 C. After 0.3, 0.7, 1.0, 1.3, or 1.7 hours in the growth chamber, 6 replicate plants for each interruption period were returned to the dew chamber for the remainder of the 24 hour incubation period. Noninoculated controls remained in the dew chamber for 24 hours. Lesions were counted after an additional 6 hours in the growth chamber. In a separate set of experiments 24 onion plants were inoculated in each of two groups. Plants were incubated in the dew chamber at 20 C for 6 hours, then were transferred to a growth chamber at 20 C and 65 i 10% RH. After 4, 8, or 24 hours in a growth chamber, 6 replicate plants from each interruption period were returned to the dew chamber for an additional 44 hour incubation period. Noninoculated controls remained in the chamber for 44 hours. Experiments were conducted twice. 34 Influence of Humidity Duringgan Interruption Onion plants were inoculated with 2.5 mg conidia. After 6 hours in the dew chamber, 6 replicate plants were removed and placed in a growth chamber at 30, 60, or 90 i 10% RH for 20 minutes, then returned to the dew chamber for the remainder of the 24 hour period of the experiment. Six control plants remained in the dew chamber continuously for 24 hours. Humidity and temperature were monitored with a recording hygrothermograph. The experiment was conducted three times. Spore Germination Rate on Leaf Surfaces Two groups of fourteen onion plants were inoculated and placed in the dew chamber at 20 C. After 1, 2, 4, 6, 8, 10, 12, or 24 hours, the 3rd or 4th youngest leaves of 4 replicate plants were sampled, since lesions produced on these leaves were the most uniform in size. Four 1-cm2 leaf tissue pieces were removed from each leaf, fixed in formalin- 50% ethanol-glacial acetic acid (1:18:1, v/v), stained with cotton blue in lactic acid solution (28 mg aniline blue, 20 ml water, 10 m1 glycerol, and 10 ml 85% lactic acid), and examined using light microscopy. Conidia which washed off leaves during fixation were collected on 13- mm-diameter membrane filters (pore size 8 0.33 um), stained, and counted as above. RESULTS Influence of Timinggof Dew Period Interruptions on Lesion Production Plants given an interruption in dew period of 2 hours following 2—12 hours of dew had fewer lesions than those continuously in the dew chamber for 24 hours prior to interruption (Figure 3.1). Fewest lesions were produced on those plants provided with 6 hours dew, 2 hours of 35 400 t' 300 LESIONS/ PLANT B O IOO 2 4 6 8 I0 I27" 24 LEAF WETNESS DURATION PRIOR TO INTERRUPTION (HR) Figure 3.1. Influence of leaf wetness duration prior to a 2-hr interruption (dry period), followed by rewetting plants for a total of 22 hr total wetness period, on lesions/plant. Mean values were derived from three replicate experiments. LSD (Bf0.05)-74. 36 interruption (dry period), and 18 hours of dew after the interruption. Numbers of lesions decreased with increasing initial wetness duration from 2 through 6 hours, then increased as time of wetness increased through 24 hours. Since maximum lesion production occurred by 24 hours (Chapter II), plants which received 24 hours dew prior to removal from the chamber served as control plants. Influence of Interruption Duration on Lesion Production Onion plants provided 6 hours dew followed by interruption dura- tions of 0.3-1.7 hours, within a 24 hour incubation period, displayed fewer lesions than uninterrupted control plants (Table 3.1). An abrupt drop in lesion numbers was apparent after a 0.3 hour (20 minute) inter- ruption, and there was a tendency toward decreasing lesion numbers as the length of dry period increased, but differences were not significant. Table 3.1. Effect of interruption duration of dew period on infection Of onion by Botrytis squamosa. Lesions/Plant~ Treatment Duration (hour) Trial Number Wet Dry Wet I II 6 0.0 18.0 x343 1: 119 450 :I: 90 6 0.7 17.7 229 :I: 72 316 :1: 20 6 0.7 17.3 233 :l: 75 294 :I: 51 6 1.0 17.0 209 :I: 24 266 :I: 23 6 1.3 16.7 174 :I: 105 274 :l: 55 6 1.7 16.3 140 :I: 43 216 i 101 xMean of 6 replicate onion plants. 37 Lesion numbers were greatly reduced on plants incubated under conditions of dew for 6 hours, then interrupted for 4 or 8 hours, com- pared to noninterrupted controls (Table 3.2). Significant reductions in lesionnumbers were evident on those plants which were interrupted for 24 hours following 6 hours dew. Table 3.2. Influence of interruption after a 6 hour dew period at 20 C followed by an additional 24 hour dew period. Lesions/Plant Treatment Duration (hour) Trial Number Wet Dry Wet I II 6 0 24 X413 :1: 64 380 :t 105 6 4 24 152 i 76 126 t 60 6 8 24 147 i 55 167 t 62 6 24 24 107 i 55 90 t 25 xMean of 6 replicate onion plants. Influence of HumidityBDuring Interruptions There were fewer lesions on plants where dew period was inter- rupted by incubations at 90, 60, or 30% RH as humidities decreased (Table 3.3), although differences in most cases were not statistically different from each other or from those where dew period was not inter- rupted, according to Duncan's multiple range test (B_- 0.05). Fewer lesions were produced at 30% RH than under continuous dew in one experiment. 38 Table 3.3. Influence of a 6 hour dew period, a 20 minute dew inter- ruption at 30, 60, or 90% RH, followed by an 18 hour dew period, on infection of onion by Botrytis squamosa. xLesions/Plant Trial Number Mean Humidity (%) I II III Response no interruption y388 a y426 a y376 a y397 a 90 i 10% 284 ab 326 a 306 a 317 ab 60 i 10% 270 be 321 a 273 a 288 ab 30 i 10% 104 c 303 a 231 a 213 b xMean of 6 replicate onion plants. yValues in a column followed by the same letter do not differ significantly (B_= 0.05) according to Duncan's multiple range test. Spore Germination on Leaf Surfaces Spore germination on leaves began after 2 hours in the dew cham- ber, and percent germination increased rapidly between 6-12 hours dew with maximum germination occurring by 20 hours (Figure 3.2). DISCUSSION Timing of and duration of interruptions of the dew period influ- enced lesion production in onion by B, sfiuamosa. Humidity during the interruption also influenced lesion production significantly in one experiment. Our results indicated that B, squamosa was most sensitive to a desiccation period after 6 hours dew. Dzikowski (1980) also found greater reductions in lesion numbers after a 5 hour initial leaf wetness period than after a 2 or 22 hour leaf wetness period. However, he did not quantify treatment responses in terms of lesion numbers and it is 39 gang 3 b O I SIGERNHNMNKN' 8% O L n a n n O 4 3 I2 IE 20 24 LEAF‘NEflflflfliDURAWKNIGflD Figure 3.2. Percent germination of Botrytis squamosa conidia on leaf surfaces at 20 C and continuous leaf wetness. 40 not clear how his treatment differences, quantified in terms of percent leaf area diseased, were obtained. Fungi such as Stemphylium botryosum f. sp. lycopersici (Bashi and Rotem, 1974), Alternaria porri (Bashi and Rotem, 1974), Cercospora mnsicola (G008 and Tschirch, 1963) or Botrytis cinerea (Goods and Zathurecky, 1967) can withstand a dry period prior to their establish- ment within the host. 2, sguamosa appeared to be sensitive to desic- cation during a dry period occurring prior to penetration into host tissue. Sensitivity to a dry period.was also reported for germinating conidia of such fungi as Coccomyces hiemalis (Eisensmith et al., 1982), Glomerella cingulata (Leben and Daft, 1968), and Alternaria longipes (Norse, 1973). Germination of g, sguamosa conidia began after 2 hours and increased through 20 hours on wet leaf surfaces at 20 C. After 6 hours, which is the postinoculation wetness period when conidia were most sensitive to a dry period, less than 20% of the conidia had visible germ tubes, suggesting that the conidia were sensitive to a dry period at a stage prior to the formation of germ tubes. Previous studies concerning survival of g, guamosa conidia on onion leaf surfaces (Chapter II) and in soil (Ellerbrock and Lorbeer, 1977) indicated that g, sguamosa can survive for several days on contin- ually dry leaf surfaces or for several weeks in continually moist soil. However, conidia placed in soil and subjected to wet-dry regimes exhib- ited enhanced mortality over those exposed to continually moist or dry treatments. In my studies, very few lesions developed on plants pro- vided with 6 hours dew, then interrupted for 24 hours (5-10 per plant). Placing these plants subsequently in the dew chamber for 24 hours 41 resulted in increased lesion numbers, suggesting that the ungerminated spores had survived on leaf surfaces and could still cause lesions. Under field conditions g, sguamosa conidia probably survive movement by air currents and deposition during the day, and are stimr ulated to germinate under conditions of leaf wetness at night. Conidia probably do not survive more than one or two periods of dew insuffi- cient for infection. As previously defined (Chapter II; Shoemaker and Lorbeer, 1977) minimal leaf wetness for infection would be 6-12 hours. It is possible that a lower rate of germination on onion leaves may occur under some conditions in nature. For example, Clark and Lorbeer (1977) reported less germination in the presence of some bacterial isolates in the phyllosphere of onion leaves. In addition, other factors such as solar irradiation may also influence the survival of conidia or their ability to infect onion leaves. 42 REFERENCES Bashi, E. and J. Rotem. 1974. Adaptation of four pathogens to semi-arid habitats as conditioned by penetration rate and germinating spore survival. Phytopathology 64:1035-1039. Clark, C. A. and J. W. Lorbeer. 1977. The role of phyllosphere bacteria in pathogenesis by Botrytis squamosa and B, cinerea on onion leaves. Phytopathology 67:96-100. Dzikowski, P. A. 1980. The use of weather information for timing fungicide applications to control leaf blight in onions. M. Sci. Thesis, Univ. Guelph, Guelph, Ontario. 62 pp. Eisensmith, S. P., A. L. Jones, and C. E. Cress. 1982. Effects of interrupted wet periods on infection of sour cherry by Coccomyces hiemalis. Phytopathology 72:680-682. Ellerbrock, L. A., and J. W. Lorbeer. 1977. Survival of sclerotia and conidia of Botrytis squamosa. Phytopathology 67:219-225. Gooda, H. M. and P. G. M. Zathurecky. 1967. Effects of drying on the viability of germinated spores of Botrytis cinerea, Cercospora musae, and Monilinia fructicola. Phytopathology 57:719-722. 6005, R. D. and M. Tschirch. 1963. Greenhouse studies on the Cercospora leaf spot on banana. Trans. Br. Mycol. Soc. 46:321-330. Lacy, M. L. and G. A. Pontius. 1983. Prediction of weather mediated release of conidia of Botrytis squamosa from onion leaves in the field. PhytOpathology 73:670-676. Leben, C. and G. C. Daft. 1968. Cucumber anthracnose: influence of nightly wetting of leaves on numbers of lesions. Phytopathology 58:264-265. McDonald, M. R. 1981. Effect of environmental and host factors on Botrytis leaf blight of onion. M. Sci. Thesis, Univ. Guelph,. Guelph, Ontario. 108 pp. Norse, D. 1973. Some factors influencing spore germination and penetration of Alternaria longipgs. Ann. Appl. Biol. 74:297-306. Segall, R. H. and A. G. Newhall. 1960. Onion blast or leaf spotting caused by species of Botrytis. Phytopathology 50:76-82. Shoemaker, P. B. and J. W. Lorbeer. 1977. The role of dew and temperature in the epidemiology of Botrytis leaf blight on onion. Phytopathology 67:1267-1272. Sutton, J. C., C. J. Swanton, and T. J. Gillespie. 1978. Relation of weather variables and host factors to incidence of ariborne spores of Botrytis squamosa. Can. J. Bot. 56:2460-2469. 43 Swanton, C. J. 1977. Influence of environmental factors on development and control of Botrytis leaf blight on onions. M. Sci. Thesis, Univ. Guelph, Guelph, Ontario. 89 pp. ~ Tanner, M. R. and Sutton, J. C. 1981. Effect of leaf wetness duration and temperature on infection of onion leaves by Botrytis squamosa. Phytopathology 71:565 (Abstr.). CHAPTER IV INFLUENCE OF LEAF POSITION AND MATURITY ON DEVELOPMENT OF BOTRYTIS §QUAMOSA IN ONION LEAVES INTRODUCTION Botrytis leaf blight, caused by Botrytis squamosa, is recognized as a leaf spotting and blighting disease of onion foliage (Hickman and Ashworth, 1943; Page, 1955; Hancock and Lorbeer, 1963). Leaf spots are discrete, desiccated spots, 1-10 mm long by 1-2 mm.wide. Under prolonged moist conditions, some lesions may expand and girdle leaves, causing collapse of tissues and leaf blighting. Leaves die from the tip down- ward. Greater lesion numbers and blighting occur on outer leaves than on younger, inner leaves (Hickman and Ashworth, 1943; Page, 1955; Small, 1971; Shoemaker and Lorbeer, 1977). Histopathology of infection was studied by Clark and Lorbeer (1976). They defined 2 phases in lesion formation: (i) collapse and separation of mesophyll from the epidermis, resulting in cavity formation and (ii) subsequent collapse and degeneration of adjacent tissues. However, little is known regarding growth ole, squamosa within lesions, espe- cially relative to leaf position or leaf maturity. The objectives of this study were to examine the effects of leaf position, lesion size, and leaf maturity on develOpment of infection hyphae within lesions on onion leaves. 44 45 MATERIALS AND METHODS In all experiments isolate BSS-4 was used since this isolate was highly virulent and sporulated more prolifically than other isolates. One-month-old onion plants, sprouted from bulbs, were inoculated using a dry spore inoculation technique as described in Chapter II. Following inoculation, plants were incubated in a commercial dew chamber (Percival Mfg. Co., Boone, Iowa 50036) at 20 C for the specified duration of leaf wetness. The position of leaves on plants was defined using a number system from 1 through 6 where 1 represented the innermost (youngest) leaf and 6 represented the outermost (oldest) leaf. Leaf Position vs. Lesion Numbers The influence of leaf position on the number of lesions per unit area of leaf induced by a standardized number of conidia was examined. Six replicate onion plants were inoculated, placed in a dew chamber at 20 C for 24 hours, then held in a growth chamber at 20 C for 6 hours. Leaf area was determined and lesions were counted for each leaf. Since calculated leaf area, using the equation for area of a right circular cone (A - nrh, where A - area,1r= 3.146, r - radius of cone base, and h 8 cone height), was generally within 1-2 cm2 of leaf area determined by using a leaf area meter (Li-Cor model 3100, Li-Cor, Lincoln, NB 68504), this equation was used for computing leaf areas. This represented a simple, nondestructive method of estimating leaf area. Leaf Position vs. Lesion Size The relationship between leaf position and mean lesion size was examined. Four replicate onion plants were inoculated and incubated in 46 the dew chamber at 20 C for 24 hours. Four 1-cm2 tissue segments were excised from each of 4 replicate leaves. The segments containing lesions were fixed in FAA and stained with trypan blue prior to examin- ation. Relative lesion area was estimated by multiplying length by width. A minimum of 20 lesions per leaf were examined. The experiment was repeated twice.‘ Leaf Position vs. Infection Hyphae Development The relationship between leaf position and mean length of infection hyphae within lesions was examined. Four onion plants were inoculated with 2.5 mg conidia, then incubated in the dew chamber for 24 hours. Four 1-cm2 tissue segments containing lesions were excised from each of 4 replicate leaves. Segments were fixed in FAA and stained with trypan blue prior to examination. Infection hyphal lengths were measured using a microscope equipped with an ocular micrometer. Lesion Size vs. Infection Hyphae Development The relationship between lesion size and length of infection hyphae within lesions was examined. Onion plants were inoculated, then incubated in the dew chamber at 20 C for 1, 3, or 5 days. Lesions in the leaf positions 1 through 6 were sampled. Four l-cm2 tissue segments were excised from each of 4 replicate leaves. Lesions were fixed (and stored) in FAA and stained with trypan blue at room temperature, then were rinsed and mounted in water prior to examination. Infection hyphal lengths were measured using a light microscope equipped with an ocular micrometer. 47 Rate of Lesion Development The rate of increase in lesion size with time on leaves of differ- ent ages was determined. Sixteen onion plants were inoculated with 2.5 mg B. squamosa conidia, then placed in the dew chamber. After 8, 12, 16, and 24 hours, four replicate plants were removed from the cham- ber. Lesion sizes on each leaf were estimated using a rating scale from 1-4 where 1 represented small (551 mm long) lesions and 4 represented large (2 4 mm long) lesions. Influence of Tissue Maturity on Infection Hyphae Development The influence of tissue maturity (senescence) on infection hyphae development was examined. Four onion plants were inoculated with single 5-ul drops of a 1 X 103 conidia per ml suspension at 4 sites on each leaf. Plants were incubated in a dew chamber at 20 C for 24 hours. Leaf pieces from inoculated senescing (yellow) and healthy (green) tissues were excised, fixed in FAA, and stained in trypan blue. Hyphal lengths were measured using a microscope fitted with an ocular micrometer. RESULTS Leaf Position vs. Lesion Numbers Numbers of lesions per square centimeter of leaf tissue on onion plants uniformly inoculated with conidia of B, squamosa increased from the youngest to the oldest leaf (Figure 4.1). Significantly (£30.05) higher numbers of lesions were produced on the outermost leaf (position 5) compared with leaves one through four (LSD test, Pf0.05). Greater number of lesions were also produced on leaf four compared with one and two . 48 5.0 r 4.0 VT T “U 5 .. . o 3.0 \ (D Z .9 F— a) 2.0 [LI .. .J _l. 1.0 * h A l 2 3 4 5 LEA F POSITION Figure 4.1. Influence of leaf position on lesion numbers on onion after inoculation with B, squamosa and incubation in a dew chamber at 20 C for 24 hours (leaf position 1 - youngest, innermost leaf). 49 Leaf Position vs. Lesion Size The mean size of Botrytis leaf blight lesions of the same age increased with an increase in age of onion leaves (Figure 4.2). Mean lesion areas were significantly greater on leaf six compared with one through four. Leaf five contained greater lesion areas than leaf one (LSD test, §=0.05). Leaf Position vs. Infection Hyphae Development Mean infection hyphal lengths within lesions increased with increase in age of onion leaves (Figure 4.3). Significantly greater mean infection hyphal lengths were observed on leaves five or six com? pared with one through four. Greater mean lengths were also observed on leaves three or four compared with leaf one (LSD test, £?0.05). Lesion Size vs. Infection Hyphae Lengths Lesion area correlated poorly with length of infection hyphae within lesions after 24 hours incubation in the dew chamber. Correlation coefficients (r) for leaves in positions 1-6 were 0.67, 0.36, 0.36, 0.42, 0.36, and 0.27, respectively. After one day in the dew chamber infection hyphae were <100 um in length, although lesions had developed to near the maximum size for nonexpanding lesions (2-5 X 1-4), indicating that lesions developed well in advance of the hyphae within. Rate of Lesion Development The rate of lesion appearance and expansion within the dew chamber was slower on the innermost leaf than on the outermost leaf (Table 4.1). Rates of development were similar in leaf positions three through six. 50 :5 9 l J 9‘ 9 no 9 I l ._g LESION AREA (MMZ) '9 .1 O r . . . r I 2 3 4 5 6 LEAF POSITION Figure 4.2. Influence of leaf position on lesion size on onion after inoculation with B, sguamosa and incubation in a dew chamber at 20 C for 24 hours. 51 IOOd 3 1. 3 E 80- w 1 "I z "” I —l 60. r- .J ' < :I: .I o. r ). I 40. z _- 9. v 2; I 1 Lu 20' . ‘ u. I- ; III C T' l l l I T I 2 3 4 5 6 LEAF POSITION Figure 4.3. Influence of leaf position on infection hyphal lengths within lesions on onion after inoculation with B, squamosa and incubation in a dew chamber at 20 C for 24 hours. 52 Table 4.1. Relationship between leaf position and incubation period in a dew chamber on lesion size following inoculation of onion plants with B, guamos . Lesion sizea Dew Leaf position Period (hour) 1b 2 3 4 5 6 8 0.0c 0.3 0.9 0.5 0.7 0.8 12 0.9 1.0 1.4 1.6 1.7 1.6 16 0.6 1.0 1.4 1.4 1.8 2.0 24 1.0 2.0 2.3 2.3 2.5 2.4 aLesion size was visually rated using a rating scale of 1-4 where 1 represented less than 1 mm diam and 4 represented lesions equal to or greater than 4 mm long. bYoungest leaf. cBased on 20 lesions per leaf from 3 replicate leaves. Influence of Tissue Maturity on Infection Hyphae Development Hyphae grew almost three times as fast in senescent as in healthy tissue. Mean infection hyphal lengths of B, squamosa growing in senes- cent tissues at 20 C for 24 hours after inoculation were 207 t 9 um compared with lengths of 77 i 15 in nonsenescent tissues. DISCUSSION Although both lesion size and lengths of infection hyphae increased with increasing age of leaves on any given leaf in position 1-6, a poor correlation was found between lesion area and lengths of infection I hyphae. Lesions developed rapidly on both young and old leaves. By 24 hours lesions were near their maximum size yet infection hyphae were only 1-100 um in length (Chapter II). This suggests that lesions development 53 may result from penetration rather than hyphal development within the lesion, with leaf maturity being the primary determinant of lesion size. Hancock and Lorbeer (1963) believed that lesions developed from enzymes released during spore germination, but prior to penetration. In my observations of spore germination on leaf surfaces, germinated spores were observed which were not associated with lesions. Lesions were gen- erally associated with those conidia which germinated and penetrated. We further observed that most infection hyphae remained restricted in their development within lesions (Chapter II), except on the outermost leaves where expanding lesions occurred under conditions of prolonged leaf wetness. In the initial lesion there is a rapid and extensive collapse of cells beneath the epidermal layer which results in an empty cavity extending across most of the leaf. The epidermal layer generally remains intact. Twenty four to 72 hours after lesion onset some wall thickening can be observed under the light microscope in cells bordering the lesion. When the leaf senesces and the green leaves begin to fade and turn yellow, a green ring is often observed surrounding the lesion. After the initial 24 hour period, the cavity area does not further expand. In some lesions, hyphae grow beyond the lesion border toward healthy tissues. As hyphae grow toward and into the healthy tissues, there is water-soaking and softening of the tissues in advance of the hyphal progression. Upon drying, these tissues tend to collapse, have a blighted appearance, and may become desiccated ("expanding" lesions). Increasing the leaf wetness period following inoculation resulted in an additional increase in numbers of expanding lesions, primarily on the outermost leaves. Although numbers of expanding lesions were small, even 54 under extended periods of wetness, only a few of these lesions could cause extensive collapse and blighting. Since onions normally produce 15-16 leaves in a growing season, but rarely have more than 10 at any one time (Jones and Mann, 1963), older leaves begin to senesce and die naturally, even without the effects of disease, as plants begin to mature and bulbs form. The senescing leaves provide a substrate where expanding lesions and sporulation occurs commonly. If large amounts of inoculum are produced, even young leaves can be severely damaged with very large numbers of small lesions. Clark and Lorbeer (1976) reported develOpment of expanding lesions 72-96 hours after inoculation. This study demonstrated that extent of development of B, squamosa depended mostly on tissue maturity. Greatest development of B. squamosa was evident on senescing tissues. Botgytis squamosa also grew well in dead onion leaves. Clark and Lorbeer (1967) reported greater superficial growth of B, squamosa when plants were inoculated with conidia in water containing nutrients than in water. Thus, greater availability of nutrients and lack of a resistance response may account for the aggressive colonization of senescent tissues by B, squamosa. We observed longer germ tubes and more stomatal penetrations on the senescing tissues. Very short germ tubes were observed on young leaves. This study suggests that B, sguamosa acts as a weak pathogen on young, healthy onion foliage, inducing a host reaction resembling that of the hypersensitive response. However, B, squamosa growing within senes- cent tissues or expanding lesions can result in destructive leaf blighting. 55 REFERENCES Clark, C. A. and J. W. Lorbeer. 1976. Comparative histopathology of Botrytis squamosa and B, cinerea on onion leaves. Phytopathology 66:1279-1289. Hancock, J. G. and J. W. Lorbeer. 1963. Pathogenesis of BotEytis cinerea, B, squamosa, and B, allii on onion leaves. Phytopathology 63:669-673. Hickman, C. J. and D. Ashworth. 1943. The occurrence of BotEytis app. on onion leaves with special reference to B, squamosa. Trans. Br. Mycol. Soc. 26:153-157. Jones, H. and L. K. Mann. 1963. Onions and their allies. Leonard Hill Limited, London. 286 pp. Page, 0. T. 1955. Botrytis leaf spot on onion and its control. Can. J. Agric. Sci. 35:358-365. Shoemaker, P. B. and J. W. Lorbeer. 1977. The role of dew period and temperature in the epidemiology of Botrytis leaf blight of onion. Phytopathology 67:1267-1272. Small, L. W. 1970. The epidemiology of leaf blight disease of onions incited by B, squamosa. M. Sci. Thesis, McGill Univ., Montreal, Quebec. 249 pp. CHAPTER V INFLUENCE OF TEMPERATURE AND WATER POTENTIAL ON GROWTH AND SPORULATION OF BOTRYTIS SQUAMOSA INTRODUCTION Environmental parameters important in leaf blight development include temperature, leaf wetness duration (MeDonald, 1981; Tanner and Sutton, 1981; Shoemaker and Lorbeer, 1977; Swanton, 1977), and air humi- ity, expressed as relative humidity (RH) (Swanton, 1977; Sutton et al., 1978) or as vapor pressure deficits (Lacy and Pontius, 1983). Lesion production is Optimal at 18-20 C and lesion numbers and leaf blighting is generally more severe an outer, older leaves (Hickman and Ashworth, 1943). Sporulation occurs only on senescent or necrotic tissues (McDonald, 1981; Small, 1970; Swanton, 1977). Growth of B, sguamosa.was reported by Swanton (1977) to be most rapid between 15-33 C. Shoemaker and Lorbeer (1977) reported that the optimal temperature for radial growth was 24 C. Segall and Newhall (1960) reported maximum growth as dry weight at 21 C. The influence of polychromatic light, carbohydrate source, and pH on conidiation of B, sguamosa was studied by Berquist et a1. (1972). Maximum sporulation occurred at Ph 5.5. Starch, dextrin, or potato extract were more effective than mono- or disaccharides in stimulating conidial production. A 14 hour photoperiod of fluorescent near UV was optimal for conidial produciton. 56 57 No studies of which I am aware have examined the influence of water potential on growth and sporulation of B, squamosa. Since air humidity and temperature are important in spore production in the field (Lacy and Pontius, 1983), and understanding of the influence of these parameters on growth of B, squamosa could be epidemiologically important. The objectives of this study were (i) to examine the influence of moisture and temperature on growth and sporulation of B, squamosa, and (ii) to determine the influence of moisture period and interrupted moisture period on sporulation of B, squamosa from onion leaf tissues. MATERIALS AND METHODS Influence of Temperature and water Potential on Growth Rates on PLYiéEEE Prune extract-lactose—yeast extract agar (PLY) was used as the basal medium, the osmotic potentials of which were adjusted using various concentrations of KCl, NaCl, sucrose, or polyethylene glycol (PEG) 8000. Measurements of the water potentials were made using a wescor deWpoint hygrometer and C-52 sample chambers (wescor, Inc., Logan, Utah 84321). Agar plates were inoculated by placing a 4-mm-diameter plug from.the advancing margin of a 72-hour-old culture of B, squamosa onto the center of each PLY agar plate. 'Four replicate plates per treatment were then incubated at 20, 25, or 30 C. Colony diameters were measured daily over 4 days and growth rates were calculated over days 2-4 since growth was linear during this time. Influence of Temperature and Water Potential on Dry weigBt in PLY Broth Flasks containing 50 ml of PLY broth adjusted to various water potentials with KCl, sucrose, NaCl, or PEG 8000 were each inoculated with three 4-mm-diam X S-mm plugs from.the advancing margin of 72-hour-old 58 cultures of B. squamosa. Flasks were incubated at 20, 25, or 30 C in still culture for 4 days. Dry weights were determined by collecting the contents of each flask on separate 7-cm-diameter glass microfiber filter papers (Whatman Ltd., England). Each filter was rinsed with 1000-1500 ml water to ensure complete removal of media and osmotica, then air dried in a forced air drier at 60 C until a constant weight was obtained. Influence of Temperature and Water Potential on Growth in Onion Leaves Water potentials of sterilized (autoclaved for 85 minutes, 20 p.s.i.) green onion leaves were adjusted by positioning leaves over dis- tilled water, in sealed cahmbers, containing various concentrations of NaCl. Seven days were allowed for equilibration prior to inoculation of each leaf piece with a 4-mm-diameter plug from the advancing margin of a 3-day-old culture of B, squamosa. Radial growth on leaves was measured after 3 days incubation at 20, 25, or 30 C. Water potentials of the leaves were determined using a Wescor dewpoint hygrometer. Influence of Desiccation Period and Temperature on Sporulation The influence of desiccation period and temperature on sporulation of B, squamosa was examined using infected leaf tissue segments. One- cm2 onion leaf segments were placed on water agar and a 4-mm-diameter plug from the advancing margin of B, squamosa growing on PLY agar was transferred to each leaf segment. After 3 days incubation at 20 C in darkness, the agar plugs were removed, leaves were transferred from the agar to a sterile plastic surface, air dried for two hours in a laminar flow hood, then were placed in sealed chambers over CaClz. 59 To determine the influence of desiccation period on sporulation, dried, infested leaf segments were incubated over CaCl2 for 3, 6, 9, or 12 days at 22 C then were transferred to water agar. The plates were incubated at 20 C under a 12 hour light phot0period in fluorescent light and examined for sporualtion over a subsequent S-day incubation period. Since under field conditions temperature fluctuations would govern rate of sporulation, the influence of temperature on sporulation from the infested leaf segments was examined. After 3 days under desiccation at 22 C, leaf segments were transferred to water agar plates and incu- bated at 15, 20, 25, or 30 C under a 12 hour photoperiod in fluorescent light. After 3 days incubation, conidia were collected from each of 4 replicate plates for each temperature. Since sporulation from leaf segments occurred over a period of several days, the dynamics of sporulation was examined to determine when the bulk of conidia were produced. Onion leaf segments were inoculated, incubated, and dried as described above, then were transferred to water agar plates. The plates were incubated at 15 C and conidia were collec- ted daily through 6 days. The influence of cyclic wet-dry-wet periods on sporulation was examined. Colonized, dried leaf segments were placed on water agar and incubated at 20 C under a 12 hour fluorescent light-12 hour dark regime. At the beginning of each 24 hour period, leaves were removed and dried for 1-2 hours in a laminar flow hood, then placed over CaCl2 in sealed chambers. After drying periods (flow hood + sealed chambers) of 4, 8, 12, or 16 hours, leaves were placed on water agar and returned to the incubator for the remainder of the 24 hour cycle. Conidia were collec- ted immediately prior to each drying period, on each of six consecutive 60 days. For each treatment 4 replicate plates were used and conidial numbers were quantified as conidia/cm? colonized leaf tissue. Under field conditions onion leaves supporting sporulation fre- quently lie on the ground and are influenced by moisture conditions at the soil-air interface. The influence of moisture in such a corres- ponding situation was examined using agar plates adjusted to various water potentials as the model system. Colonized, dried leaf segments were placed on water agar adjusted to various water potentials with KCl. Potentials of the agar were determined using a Wescor dewpoint hygrometer as previously described. After incubating plates at 20 C for 5 days under a 12 hour fluorescent light-12 hour dark regime,conidia were removed as previously described (Chapter II) and quantified using a hemacytometer. RESULTS Influence of Temperature and Water Potential on Growth Rate on PLY A335 Growth of B, squamosa on PLY agar adjusted to various water poten- tials with osmotica were similar at 20 or 25 C but much lower at 30 C (Figure 5.1). In the presence of KCl, sucrose, or PEG at 20, 25, or 30 C growth rates of B, sguamosa increased from -0.9 through -5 to -20 bars, then declined to zero below -100 bars. In the presence of NaCl growth rates increased from 0.9 through -5 to -10 bars only at 20 C, then declined and ceased by -60 bars. At 25 and 30 C growth, in the presence of NaCl, declined and was not observed at lower than -60 bars. Below -30 bars, there was no significant differences in growth at the three temepratures used, with any osmoticum. 61 '5 ' m -—-zo'c Ono-25.0 Modfl’fi IZ- 1:: O '8 \ e . e U U h ‘ 8 8 h g -—-zo’c 12 » 3 o-mzs‘c u WIIEII POT! IITIAl Hla rs) Figure 5.1. Growth rate of B, squamosa at 20, 25, and 30 C on PLY agar adjusted to various water potentials with KCl, sucrose, NaCl, and PEG 8000. 62 Influence of Temperature and Water Potential on Dry Weight in PLY Broth Growth ole. squamosa in PLY broth adjusted to various water potentials with osmotica were similar at 20 or 25 C but much lower at 30 C (Figure 5.2). Growth was stimulated when potentials were lowered -5 to -20 bars but over lower potentials growth declined. In the pres- ence of KCl, sucrose, or PEG 800 growth was not detected at -70 to -95 bars and with NaCl growth was not detected below -40 bars. Influence of Temperature and Water Potential on Growth in Onion Leaves Growth of B, sguamosa in onion leaves was similar at 20 or 25 C and greatly reduced at 30 C (Figure 5.3). At 20 or 25 C radial growth was similar from -0.9 through -24 bars, then declined through -90 bars (Figure 5.3). At 30 C radial growth was similar to that at 20 or 25 C at -9 to -79 bars. Influence of Desiccation Period and Temperature on Sporulation When leaf segments, infested with B, squamosa, were desiccated for 3-12 days, then placed on water agar plates at 20 C for 3 days, B. squamosa was observed growing equally well from all of the leaf segments. The duration of the desiccation period through 12 days did not appear to influence the extent of sporulation. Sporulation of B, squamosa from.infested leaf segments placed on water agar for 4 days was greater at 15 or 20 C, less at 25 C, and nil at 35 C (Figure 5.4). The sporulation dynamics of B, squamosa was monitored over 6 days. No sporulation was evident from leaf segments placed on water agar at 15 C for 24 hours. Large numbers of conidia were produced after 2 or 3 days on leaf segments placed on agar (Figure 5.5). Conidial production 63 25. I61 ‘ zoo , , —- zo'c o : : 25‘ success 8 m --2o'c m I" 9‘ ° myucm um 70! o 20 u so ' so WATER WIEIITIM. Hum Figure 5.2. Dry weight of B, squamosa at 20, 25, and 30 C after 4 days in PLY broth, adjusted to various water potnetials with KCl, sucrose, NaCl, and PEG 8000. 64 25 A20“ 2 E E I5- 5 m {D _| IO‘ 5 3 5.. “‘ ”" ““““ Gnu—é ‘U‘ig” ' T ' 0 20 4O 60 90 I00 WATER POTENTIAL I-BARS) Figure 5.3. Radial growth of B, squamosa in onion leaves at 20, 25, and 30 C at various water potentials. 65 5.0 4.0 cu 2 o \ S 3.0 9 z o 0 2.0 L9 0 _n LO 0.0 IS 20 25 30 35 TEMPERATURE (t) Figure 5.4. Numbers of conidia of B, squamosa collected from onion leaf segments after 4 days incubation on water agar at various temperatures. 66 9’ ‘f' CONIBIA ICMZ x :04 9‘ | If I I V r ' 2 3 4 5 6 7 INCUBATION PERIOD (DAYS) Figure 5.5. Numbers of conidia collected after 1-6 days from B. squamosa-colonized onion leaf segments incubated at 20 C. 67 declined during the period from day 3 to day 6. Additional leaf seg- ments contained on separate plates, from which conidia were not collec- ted until after 6 days, yielded numbers of spores per cm2 similar to the sum of conidia daily collected over days 2-6 (7.8 X 105 vs. a sum of 7.0 x 105). Interruption duration studies revealed that for each 4 hour time period added to the dry duration, conidial numbers were reduced by about half (Figure 5.6). Sporulation with a 16 hour dry period within each 24 hour period resulted in relatively few conidia per cmz. Similar observations were reported in spore trap studies in field onions (Lacy and Pontius, 1983) Sporulation of B, squamosa from onion leaf segments placed on water agar adjusted to various water potentials revealed greatest spor- ulation at 20 C, less at 25 C, and almost none at 30 C. At 20 and 25 C growth declined from -10 through -95 bars (Figure 5.7)- DISCUSSION Growth and development of B, sguamosa was favored at temperatures of 15 or 20 C, but was much less at 30 C or above. In the Northeastern United States, where leaf blight is important, prolonged periods above 30 C are not common, especially during evening hours, so high (30 C) temperatures may not normally be a limiting factor in leaf blight development in this part of the country. Growth of B, squamosa was not evident at water potentials below -95 to -100 bars within a 4 day period, indicating that growth of B, squamosa is sensitive to moisture stresses which could be common in the aerial environment of the plant. Leaf wetness or very high humidities 68 comom / cm?- x I04 '9 9‘ 4? '9' 03 1‘ 09 . o ' O V 4 8 We. I2 l6 INTERRUPTION DURATION (hr) Figure 5.6. Influence of interruption duration within each 24 hour period on sporulation of B, squamosa from colonized leaf segments. Means and fitandard deviations were calculated from mean daily conidia/cm leaf area and measurements were made over 5 days. 69 .HQM Sues maoausouon Houo3 msoauo> ou woumsfivm Homo Hours :0 vooman muooawom mama soaco vmuaaoaoo aoum 0 on no .mN .om no mmoaonmm am no sofiuoaouonm .m.m ounwam m> . ON a I I0 5 ,0: x ,wo / VIOINOO I '32 Om 70 would be necessary to supply moisture in senescing or necrotic leaf tissues for growth of B, squamosa. In addition, moist soil, especially in combination with a dense canopy of onion leaves to retain high humidities would be highly favorable for B, squamosa growing in leaves on or near the soil surface. Sporulation of B, squamosa was evident within 2 days when dried, colonized leaves were incubated under moist conditions. Sporulation from leaves colonized for 3 days, dried, then incubated under moist conditions for two days was similar to sporulation following a 5 day period when leaves were inoculated and maintained under continually moist conditions, suggesting that a drying period did not cause any delay or reduction in sporulation. Field observations 0f.§: squamosa indicated that large numbers of conidia are produced after 3 days of favorable environmental conditions (Lacy and Pontius, 1983). It is probable that much of this sporulation arose through previously colon- ized tissues. Survival of B, sguamosa in desiccated leaf tissue segments may have important implications in understanding leaf blight epidemiology. Previous studies have demonstrated that long term survival is by means of sclerotia (Ellerbrock and Lorbeer, 1977a; Walker, 1926) and conidial survival is short term (Ellerbrock and Lorbeer, 1977a). Conidia of B. squamosa were collected from overwintered cull onions by Ellerbrock and Lorbeer (1977b) and were observed by McDonald (1981). Survival of B, sguamosa as mycelia within leaf tissues during a growing season may occur, as evidenced in this study, for at least a number of days. Field observations of B, squamosa revealed that sporulation aften occurs as a large spore release, followed by smaller releases (Lacy and 71 In my study, when infected leaf segments were placed on water agar, abundant conidial production was observed after 2 or 3 days, followed by lesser production through 6 days. Fluctuating wet-dry conditions greatly influenced the intensity of spore release. Amount of sporulation was related to duration of moisture period (or dry period). Thus, as observed under field condi- tions (Lacy and Paontius, 1983), continually moist conditions are most favorable for spore release, although fluctuating conditions with long moist periods will support lesser sporulation. Since conditions required for infection also require prolonged moist conditions, B. squamosa appears to be well adapted for conidial release under the environmental conditions most favorable for infection and continued leaf blight development. 72 REFERENCES Berquist, R. R., R. K. Horst, and J. W. Lorbeer.. 1972. Influence of polychromatic light, carbohydrate source, and pH on conidiation of Botryotinia squamosa. Phyt0pathology 62:889-895. Ellerbrock, L. A. and J. W. Lorbeer. 1977a. Survival of sclerotia and conidia of Botrytis squamosa. Phytopathology 67:219-225. Ellerbrock, L. A. and J. W. Lorbeer. 1977b. Sources of primary inoculum of Botrytis squamosa. Phytopathology 67:363-372. Hickman, C. J. and D. Ashmorth. 1943. The occurrence of Botrytis app. on onion leaves with special reference to B, squamosa. Trans. Br. Mycol. Soc. 26:153-157. Lacy, M. L. and G. A. pontius. 1983. Prediction of weather mediated release of conidia of Botrytis squamosa from.onion leaves in the field. Phytopathology 73:670-676. McDonald, M. R. 1981. Effect of environmental and host factors on Botrytis leaf blight of onion. M. Sci. Thesis, Univ. Guelph, Guelph, Ontario. 108 pp. Segall, R. H. and A. G. Newhall. 1960. Onion blast or leaf spotting caused by species of Botrytis. Phytopathology 50:76-82. Shoemaker, P. B. and J. W. Lorbeer. 1977. The role of dew and temperature in the epidemiology of Botrytis leaf blight on onion. Phytopathology 67:1267-1272. Small, L. W. 1970. The epidemiology of leaf blight disease on onions incited by Botrytis squamosa. M. Sci. Thesis, McGill Univ., Montreal, Quebec. 249 pp. Sutton, J. C., C. J. Swanton, and T. J. Gillespie. 1978. Relation of weather variables and host factors to incidence of airborne spores of Botrytis squamosa. Can. J. Bot. 56:2460-2469. Swanton, C. J. 1977. Influence of environmental factors on development and control of Botrytis leaf blight on onions. M. Sci. Thesis. Univ. Guelph, Guelph, Ontario. 89 pp. Tanner, M. R., and J. C. Sutton. 1981. Effect of leaf wetness duration and temperature on infection of onion leaves by Botrytis squamosa. Phytopathology 71:565 (Abstr.). Walker, J. C. 1926. Botrytis neck rots of onions. J. Agric. CHAPTER VI FIELD OBSERVATIONS CONCERNING SPREAD 0F BOTRYTIS LEAF BLIGHT INTRODUCTION Botrytis leaf blight epidemics have been associated with periods of prolonged moist conditions and moderate temperatures (Segall and Newhall, 1960; McLean, 1960; Small, 1970; Swanton, 1977). Spore pro- duction was promoted by leaf wetness periods >13 hours, 14-20 C temper- atures, and leaf dieback, but was restricted by leaf wetness periods 512 hours and temperatures 512 C (Sutton et al., 1978). Large spore releases were preceded by a 2 to 3 day period of 12-20 C temperatures and vapor pressure deficits of 0-5 mb (air close to saturation) (Lacy and Pontius, 1983). McDonald (1981) observed Sporulation of B, squamosa under condi- tions of high relative humidity (RH) on necrotic leaves on moist soil. Small (1971) and Swanton (1977) also reported that sporulation of B, squamosa occurred only on necrotic or senescent tissues and that the latent period was shorter when necrotic tissues were present on plants. A diurnal periodicity in spore release was reported by Lorbeer (1966), Small (1970), Sutton et al. (1978), and Lacy and Pontius (1983). Data relating spore release in the field to lesion numbers were published by Small (1970) and Sutton et al. (1978). Small (1970) reported that the logit of lesions per plant increased linearly with 73 74 the log of cumulative spores. He implied that a threshold value of 1,700 cumulative spores were needed before appreciable disease resulted. The purpose of this study was to investigate the behavior of B, squamosa in an onion field plot at the M. S. U. Muck Farm, which would compliment aerobiology studies also being conducted at this location. MATERIALS AND METHODS Microscopic Examination of Lesions Since lesions on onion can be induced by other fungi, such as Alternaria, lesions were examined for the presence of B, squamosa or other fungi. During August, 1980, onion leaves were sampled from an onion field plot at the M. S. U. Muck Farm. Fifty-five small and 59 large lesions were excised, stained in cotton blue in lactophenol, and examined under a light microscope for the presence of Botrytis and other fungi. Botrytis squamosa was identified by characteristics and morphology of the conidium, and to a lesser extent, infection hyphae. Lesion Counts The number of lesions on plants within a field plot at the M. S. U. Muck Farm.was counted on 50 randomly selected plants on 7 occasions between August 11 and September 8, 1981. Conidial popula- tions of B, squamosa in the air were monitored using a Burkard 7 day recording spore trap (Burkard Mfg. Co. Ltd., Rickmanswirth, Hertford- shire, England) (Lacy and Pontius, 1983). 75 Development of a Leaf Blight Epidemic The progress of a leaf blight epidemic was followed at 6 sites along each of 38 rows in a field plot at the M. S. U. Muck Farm on August 11 and August 20. Plants at each site were rated visually using a scale of 0-5 where 0 represented no leaf spotting and 5 repre- sented very severe leaf dieback; 1, 2, 3, or 4 represented trace, slight, moderate, and severe spotting and dieback, respectively. RESULTS Microscopic Examination of Lesions In microscopic observations of Botrytis leaf blight lesions B, squamosa was observed within 22-42% of the lesions (Table 6.1). B, squamosa identification was based on infection hyphal diameter and conidial shape and size. Additional fungal genera associated with lesions included Alternaria and Stemphylium. These fungi were iden- tified based on conidial morphology. Lesions containing infection hyphae with no associated spores were catagorized as unknown. Fungi were not observed in about half of the lesions examined. Lesion Counts Lesion numbers on August 11 were about 43 per plant (Table 6.2). Lesion numbers were not observed to increase between August 11 and August 31. Spore releases were very low during this period. Increases in lesion numbers closely followed large spore releases on September 2 and September 8 (Table 6.2). 76 Table 6.1. Percentage of various fungi observed within small and large lesions collected from onions grown at the M. S. U. Muck Farm during 1981. Z of lesions in which indicated fungi were observed Lesion Total Botrytis Alternaria Stemphylium.Unidentified typea observed squamosa spp. spp. others none Small 55 22 18 4 51 5 Large 59 42. 15 0 45 0 aLesions were grouped as small (<2mm diameter) or large (22 mm diameter). Table 6.2. Lesions per plant and cumulative spores trapped in an onion field plot at the M. S. U. Muck Farm during 1981. Lesions per Cumulative spores Date plant trapped per 0.6 M3 of.air August 11 433 0.5 x 102 17 22 4.2 x 102 24 . 22 8.6 x 102 31 23 3.8 x 103 September 2 32 3.3 X 104 4 61 7.7 x 104 8 500 1.3 x 105 aMean derived from 50 plants 77 Development of .a Leaf Blight Epidemic The progress of a leaf blight epidemic was monitored on August - 11 (Figure 6.1) and August 20 (Figure 6.2) by means of visual ratings. On August 11 leaf spotting was observed throughout the plot, although it was most severe at the west end. On August 20, leaf blighting was more extensive. Greatest severity was observed in the west half of the plot and disease appeared to have increased uniformly across the remainder of the plot. DISCUSSION Examination of lesions from onions grown at the M. S. U. Muck Farm revealed that B, sguamosa was the predominant organism associated with typical leaf blight lesions. This confirms reports of Hickman and Ashworth (1943), Viennot-Bourgin (1953), Page (1955), and Hancock and Lorbeer (1963) that B, sguamosa is the predominant organism assoc- iated with leaf blight. Although Alternaria pprri has been associated with leaf spotting (Nolla, 1927; Skiles, 1953; Bock, 1964), B, p255i_was not observed in lesions collected at the Muck Farm during 1981. The Alternaria observed in lesions for the Muck Farm onions resembled B, tenuis and it was probably associated with dead leaf debris. Observations of dead onion leaves collected at the M. S. U. Muck Farm during 1980 and incubated in a moist chamber yielded abundant sporulation of A. tenuis. The observation of many "sterile" lesions is also similar to observations of Yarwood (1938) and Hickman and Ashworth (1943). Some of the lesions may have been caused by other injuries (eg. chemical spray injuries). It is also possible that lesions were induced by 78 Uh .wsHuanHn oum>om I m can mGOHmoH on u o muons mlo mo oHoom waHuou HosmH> m wawms roomy mp3 huHuo>om monomHn .omaH .HH uo=w=< so 89mm 30:: .=.m.z_o£u on uqu nflnfim noon cam x om 8 cans“: nonsn mNN nn Uganda mama nfinaunom Lo nnfinm>mm .n.o manage I—IMNNv-II—I HNNNNH H .H H H .H m N N EV m M“ m N N m“ H .H H .9 o H o H H I—IH I—I I—lr-IHO H H H H F4 04 01 04 04 F4 F4 0: OJ F4 F4 F1 F4 04 ca ea r4 F4 94 F4 F4 F4 F4 F4 F4 r4 F4 01 04 F4 I-II-INMMN F4 F1 F4 04 01 F4 F1 F4 F4 F4 01 F4 F4 04 F1 01 «3 o: 04 r4 r4 ed 00 F4 F4 r4 F4 F4 04 F4 F4 F1 F4 r4 r4 r4 r4 F4 01 P4 F4 F4 r4 r4 ,4 F4 F4 F4 r4 F4 04 r4 r4 F4 F4 F4 F1 94 F4 r4 F4 F4 F4 F1 F4 F1 F4 F4 F4 F4 r4 r4 F4 F4 F4 c> F1 F1 94 F4 F4 F4 F4 F1 F4 F4 F4 C) CD r4 r4 r4 r4 F4 F4 F4 Fa r4 r4 :3 F4 F4 F1 r4 r4 r4 r4 c> C) c: c: :3 r4 :3 r4 r4 r4 -4 <3 F1 F1 F1 r4 :3 c3 F4 r4 r4 :3 r4 F4 F4 F4 04 r4 :3 F4 9 H 79 Uh m maHms woumu mmB huHuo>om mmmomHo .wsHuanHn ouo>om u m use maonmH o: u o ouoga mlo mo oHoom msHumu HosmH> .omnH .o~ nnnmn< no spam son: .=.m.z man on noHn cana“ none oou x on 8 cans“: nnnnn mmm an unmade «and nansnnom Lo snanopom .~.e mnnmfim NNNNNNMNNmMNNNNNNNNNNNmNNNNNNNNNHHNNNH m mm m .N m m“ m MN m .V c .u m .u e m” e mu a M“ m “N m m“ m mu m Mn m m“ N mH.H N M“ m m“ N m mm.“ m .V.q m .V q in a mu m mu.q q A” m mm m m“ N mu_c m MN m mm m H MW_N H “N N mm,“ m m m mum“ e m mule q q..u m m M“ c m mu,“ m m mn.m N m mu.“ N a_mw N HH.H N N mum“ N N .V a m .V_c n mn.q q mn.¢ e .V q m..~ q mu.q m MH.N m muuN m MN m Nan_H N MN.N m MN m H HmHNNNNNMNmNMNNNmNmNmHHHHNHNNHHHHHNNHH 8O penetration ole, sguamosa and then spores were dislodged from the leaf surface before infection hyphae became established. Numbers of lesions per plant relative to cumulative spores suggests that large spore releases are required to induce detectable differences in lesion numbers. Similar observations were published by Swanton (1977) and Small (1970). On August 11 lesions were present on the older foliage, but these leaves suffered natural senescence and death by August 17. Thus, reductions in lesions per plant were observed from August 11 through August 17. Although additional lesions probably occurred on August 24 and August 21, senescence of older leaves continued and therefore lesions per plant remained relatively constant. Lesions were observed on Home ram :0 HHHHo oHummwom mo nusouw .H.m ouanm 8.. $322.25 on m.~ 3 w... o. '10 (AVG! NW) BIVH HLMOMO 90 15» re: A ' o—zoc 11F tun-025C 9 ‘1- ’1 .\w o . t 15 sucaou B ‘2 ’1 A :22 1r . . , GROW“! IA“ (MM/MY) D 0 00 00 “Huntsman-ms: Figure 7. 2. Growth of B. a_J_._____lii at 20, 25, and 30 C on PLY medium adjusted to various water potentials with KCl, NaCl, sucrose and PEG 8000. 91 at 30 C maximum growth occurred at -30 to -40 bars; growth was halted at -95 to -100 bars. In the presence of NaCl or PEG 8000 at 20 or 25 C, growth was optimal at -10 to -15 bars, while at 30 C growth rates were Optimal at -10 to -15 bars; growth was halted at -50 to -60 bars. The optimal water potential for growth was observed to shift from higher to lower potentials with increasing temperature. Influence of Temperature and Water Potential on Dry Weight in PLY Broth Dry weights of B, eiiii_grown in PLY broth at various water potentials adjusted with KCl, sucrose, NaCl, or PEG 8000 generally corresponded to growth rates on PLY medium. Greatest dry weights were produced at -5 to -10 bars (Figure 7.3a-d). In the presence of KCl or sucrose at 20 or 25 C growth ceased at -90 to -95 bars, while in the presence of NaCl or PEG 8000 at 20 or 25 C growth ceased at -30 to -45 bars. Influence of Temperature and Water Potential on Growth in Onion Leaves Growth ole, eliii in onion leaves was similar at 20 and 25 C and very low at 30 C over a 3 day period (Figure 7.4). At 20 or 25 C growth decreased linearly with decreasing potentials from -30 to -95 bars. At 30 0.2:.Ellii failed to grow at potentials of 0 to -5 bars; some growth occurred between -5 and -55 bars and ceased near -75 bars. Influence of Inoculum Concentration on Lesion Development Lesion diameters on onion bulbs increased linearly with logarith- mic increases in spore concentration. Mean lesion diameters on onion bulbs inoculated with 10 ul of conidial suspension (0, 102, 103, 104, 5 10 , or 106 conidia per ml) were 0.0, 0.2, 0.43, 0.62, 0.92, and 1.17- cm-diameter, respectively, after 9 days (Figure 7.5). 92 KCI. A 75 "—20 C coo-«.25: 0" was"! and 75 —'ZOC MZ5C ”30C 0 20 '40 ' 80 80 WATER POTENTIAL I'M. 5) Figure 7.3. Dry weight of B, allii at 20, 25, and 30 C after 4 days in PLY broth, adjusted to various water potentials with KCl, NaCl, sucrose, and PEG 8000. 93 C A ‘ °.. 0c :12 . ..-... 30%: z I -. 4 E 9 - O a: £9 2' 6 - 5 < m 3 . ., 00...-..‘0- . -0-.-... -........... '5“... o " 1 ”g..." l 0mm.....::::h. 0 2° 4° 5° 90 100 . WATER POTENTIAL FEARS) Figure 7.4. Radial growth of B. allii in onion leaves at 20,25, and 30 C at various water potentials. 94 :- a) m 5 5 I I I I I lESION DIAMETER (mm) B) o [{4} l L l 1 ° 102 103 104 105 106 INOCULUM CONCENTRATION (conidia/ml) Figure 7.5. Lesion diameters in onion bulbs inoculated with various concentrations of B, allii conidia, after 9 days at 20 C. 95 Influence of Temperature on Rate of Disease Development Growth of B, eiiii in onion bulbs increased linearly through time at each temperature (Figure 7.6). Most rapid development was observed at 20 C. Rate of lesion expansion was much greater within a bulb scale than between scales. After 3 weeks lesions were 4-8 cm in diam- eter, yet the fungus only penetrated 1-2 bulb scales deep. After 6 weeks, the lesions were 8-12 cm in diameter and sclae to scale penetra- tions were more evident near the neck of the bulbs. After 9 weeks many of the bulbs were completely decayed. Effect of High Temperature on Survival of B. allii Survival of B, eilii in leaf segments was greater at 32 C than at 37 C (Figure 7.7). At 37 C viability of B, eBBii_declined after 3 days and the fungus was not recovered after 6 days. At 32 C viability declined after 9 days. ‘B,.eBlii.was not recovered from.onion bulbs incubated at 37 C for 6, 9, or 15 days or from bulbs incubated at 32 C for 15 days. B, eBBii_was recovered from about 5% of the bulbs incu- bated at 32 C for 3, 6, 9, or 12 days. At temperatures of 40 or 50 C viability of B, eliii_in agar plugs declined as the duration of expo- sures increased (Figure 7.8). DISCUSSION Results of these studies suggest that parameters important in the development of B, eilii in onion tissues include temperature, initial inoculum concentration, water potential, and whether or not the infec- tions are symptomless. Understanding the relationship between the parameters is epidemiologically important. 96 I6 2 8 l2: :2 § I185 o z 7 -49 a U! I 3 _, 1' 0 a? e‘ “’4'. 3‘ v9? 4» ”J”; . 0 I 25 20 15 10 5 TEMPERATURE (C) Figure 7.6. Influence of temperature and incubation period on lesion diameter in onion bulbs inoculated with B,_allii. 97 IOO‘ 90- 80, 320 704 60' “I. SURVIVAL TIME (DAYS) Figure 7.7. Survival of Botrytis allii in onion leaf segments after various incubation durations at 32 or 37 C. 98 25 20 ‘6': °L S U RVIVA L 3 O 4 3O 4O 50 60 TEMPERATURE (C) Figure 7.8. Survival of Botrytis allii in agar plugs at various temperatures after 24, 48, or 72 hours. 99 At temperatures of 5-20 C radial growth of B, eiiii increased with increasing temperature. At temperatures above 30 C, B, eilii was restricted in its development within onion bulbs. Long term expo- sures to temperatures near 35 C have been shown to be lethal to B, elBii (Harrow and Harris, 1969; Vaughan et al., 1964). This has been the basis of high temperature postharvest treatment to control neck rot in storage. Improper curing of onions, or storage of onions in humid environ- ments has been shown to be favorable for neck rot development (Harrow and Harris, 1969; Munn, 1917; Vaughan et al., 1964). Since water potentials of substrate will tend to equilibrate with surrounding water potentials of the air, the lower the atmospheric potential, the lower the substrate potential of dead onion tissues. Thus under humid storage conditions B, eilii_would be expected to continue its growth and devel- opment. B, ellii_was found to increase its growth rate at lower substrate water potentials as temperatures increased through 30 C. Similar trends were reported for Fusarium (Cook and Christen, 1976; Manandhar and Bruehl, 1973) and Verticillium (Cook and Christen, 1976). Cook and Christen (1976) suggested that this may play an adaptive role. Such an adaptation would be favorable for growth of B, eBBii under field conditions where warm day temperatures with high atmospheric potentials (high humidities) were followed by warm night temperatures and dew formation. At 20 C onion leaves equilibrated to atmospheric humidities below 95% probably would restrict the growth of B, eBBii. The initial inoculum concentration applied to onion bulbs was found to determine the amount of lesion expansion. Thus, it is possible 100 that the rate of bulb decay in storage may reflect the degree of , infection occurring prior to harvest or the amount of inoculum present at harvest. 101 REFERENCES Cook, R. J. and A. A. Christen. 1976. Growth of cereal root rot fungi as affected by temperature-water potential interactions. Phytopathology 66:193-197. Harrow, K. M. and S. Harris. 1969. Artificial curing of onions for control of neck rot (Botrytis allii Munn). New Zealand J. Agric. Res. 12: 592-604. Kaufman, J. and J. W. Lorbeer. 1967. Control of Botrytis neck rot of onions by fungicidial dusts and chemicals. Plant Dis Rep. 51: 696-699. Manandhar, J. B. and G. W. Bruehl. 1973. In virto interactions of Fusarium and Verticillium wilt fungi with water, pH, and temper- ature. Phytopathology 63:413-419. Munn, M. T. 1917. Neck rot disease of onions. New York State Agric. Exp. Stn. Bull. 437:363-455. Newhall, A. G., W. W. Gunkel, and D. C. Ludington. 1959. Artificial curing for control of onion neck-rot. New York Agric. Exp. Stn. Farm Res. Quart. Bull. 25:7. Rosberg, D. W. and H. B. Johnson. 1959. Artificial curing of Texas onions. Texas Agric. Exp. Stn Misc. Publ. 395. pp. 1-7. Talboys, R. W. 1960. A culture-medium aiding the identification of Verticillium albo-atrium and X, dahlae. Plant Path. 9:57-58. Vaughan, E. K., M. G. Cropsey, and E. N. Hoffman. 1964. Effects of field curing practices, artificial drying, and other factors in the control of neck rot in stored onions. Oregon Agric. Exp. Stn. (Corvalis) Tech. Bull. 77. 22 pp. walker, J. C. 1919. Onion diseases and their control. USDA Farmers Bulletin 1060. Walker, J. C. 1926. Botrytis neck rots of onions. J. Agric. Res. 33:893-928. Wallace, E. R. and C. J. Hickman. 1955. The influence of date of lifting and method of storing on loss of onion bulbs harvested in 1943. Ann. Appl. Biol. 32:200-205. CHAPTER VIII FIELD OBSERVATIONS CONCERNING BOTRYTIS ALLII INTRODUCTION Botrytis neck rot, caused by Botrytis allii, is a bulb decay disease which generally appears in bulbs in storage. Infections are believed to occur near to or during harvest, through dead leaves near bulbs, or through exposed succulent neck tissues when onions are topped (Munn, 1917; Walker, 1926). Wet conditions are believed to favor the spread and development of B, eiiii under field conditions (Mann, 1917; Maude and Presly, 1977a, 1977b). MeKeen (1951) found that Spanish onion seedlings were predisposed to infection by §:.2£l££ at low soil temp- eratures. Maude and Presly (1977a, 1977b) showed that bulbs grown from seed internally infected with B, eilii, had more neck rot incidence in stor- age than bulbs from seed treated with benomyl. Neck rot was signifi- cantly reduced in storage by the use of benomyl (1 g/ kg seed) prior to planting. This control strategy has not been evaluated with onion seed produced in the United States. It may also be possible to reduce bulb rot from infected seedlings using similar treatments of U. S.- grown seed. Tichelaar (1967) and Maude and Presly (1977a, 1977b) reported that B, eiéii_conidia may spread the disease among field onions by infecting leaves asymptomatotically, then sporulating from.dead leaf tips. If symptomless infections result from conidial inoculum in the field, then 102 103 fungicidal sprays might reduce such infections on leaves. This in turn could reduce neck rot incidence in storage. Reports by Kaufman et al. (1964) and Kaufman and Lorbeer (1967) revealed that fungicidal sprays applied at or near harvest reduced neck rot incidence in storage. Tichelaar (1967) felt that regular applications of fungicides may keep the inoculum density of B, allii low. MATERIALS AND METHODS Seed Treatments for Control of Neck Rot Seed borne infections could imply that current control strategies for neck rot, which revolve around the harvest period should be reexam- ined. An experiment was initiated to examine the relationship between seed infestation and fungicides on neck rot incidence in storage. The experiment, outlined in Table 8.1 was designed as a randomized block containing 2 blocks and was conducted at the M. S. U. Muck Farm, Bath, MI, during the summer of 1980. Onion seeds were hand planted on May 9th. Onions were harvested on September 10th. At harvest, bulbs were collec- ted in 10 foot sections of row in each treatment combination, counted and weighed. Bulbs were placed in a greenhouse until the necks were dry, then were stored in a cool (3.5 C) storage room. Bulbs were examined for neck rot in March, 1981. Botgytis-infested seed remaining from the seed treatment experiment of 1980 were stored at 5 C. During August, 1980, 100 seeds for each treatment were surface-disinfested in 10% bleach for 1-3 minutes, rinsed in sterile water, then placed on PLY agar (25 seeds/plate). Plates were examined after 5-6 days for sporulating colonies of B, allii 104 During August, 1980, a minimum of 10 dead onion leaves were sampled from seven of the treatments. Leaves were placed in glass petri plates containing moist paper towels. After 3-5 days incubation at 20-25 C leaves were examined for conidiOphores and conidia of B, .Elll$9.§° cinerea, or B, squamosa. Effect of Inoculating Onion Leaves With B. allii Conidia on Neck Rot Incidence in Storage. To asses the importance of foliar infection by B, eBBii_conidia on neck rot incidence in storage, a field experiment was conducted during the summer of 1980, at the M. S. U. Muck Farm. The experiment contained 4 blocks and treatments included spraying either an aqueous suspension of B, eilii_conidia onto the foliage. Conidial inoculum was produced by growing B, eilii on PLY agar at 20 C for 7-12 days. Conidia were suspended in water using Tween 20 (polyoxyethylene sorbitan mono- laurate) (1 drop/100 ml water) and then sprayed onto onion foliage on August 13, 1980. At harvest, bulbs were collected in 10 foot sections of row from each treatment combination, then counted and weighed. Bulbs were placed on a greenhouse bench until the necks were dry, then were placed in cool (3.8 C) storage. Bulbs were examined for neck rot in March, 1981. During 1981, an experiment was set up to examine the timing of inoculation of onion foliage witth, eiiii conidia on neck rot incidence in storage. Onions were inoculated O, 4, 8, 12, or 16 weeks after planting. A conidial suspension was also applied to freshly cut necks of an additional group of onions after harvest. Bulbs were air dried in the greenhouse, then placed in cool storage. Bulbs were assessed for neck rot incidence in March, 1982. 105 During 1982, an experiment was set up at the M. S. U. Muck Farm in which late season fungicide applications were examined as a possible control of B, 59$}; in storage. Onions were planted on May 4. Bravo or Benlate, at two rates, and a combination of the two chemicals, as listed in Table 8.5, were applied on a seven day schedule beginning in August. A conidial suspension ofl , eiBii conidia were applied to onions on a similar schedule. Onions were harvested on September 15 and dried at the Muck Farm in storage racks for 1-2 weeks. Onion bulbs were stored at 3.5 C and examined for neck rot in March, 1983. Incidence of B. allii in Onion Seed During 1980, seed lots from Asgrow Seed Co, were examined for B, egiii, Seeds were surface disinfected for 1-3 minutes in 0.5% NaClO + 1 drop/100 ml Tween 20, rinsed in sterile water then placed in PLX medium (25 seeds/plate). A total of 300 seeds per variety were examined. During 1981 a sample of seed was obtained from Dr. James Zalewski (Ore Ida Foods, Inc.) in Oregon. One hundred seeds were placed in 102 bleach for 3, 6, or 9 minutes, rinsed in sterile water, then placed on PLY agar. Onion seed was also obtained from Dr. R. B. Maude in England. These seeds were surface disinfested in 0.5% NaClO for 1-3 minutes then placed on PLY agar. RESULTS Seed Treatments for Control of Neck Rot No significant reductions in bulb weight were evident as a result of preplanting inoculations with B, allii conidia (Table 8.1). Neck 106 rot was not detected among bulbs in any of the treatments after 5 months storate (Table 8.1). Table 8.1. Effect of fungicidal seed treatments on bulb weight and neck rot incidence (1980). Rate Mean bulb Total # (g/kg Botrytis weight bulbs Z Neck Treatment seed) infested (g) examined rot Benlate 1 + Arasan 1 yes 352 110 O Benlate 1 + Arasan 1 no 320 114 0 Arasan 1 yes 320 107 O Arasan 1 no 287 101 O Benlate 2 '+ Arasan 2 yes 364 100 0 Benlate 2 + Arasan 2 no 324 88 0 Arasan 2 yes 295 102 0 Arasan 2 no 337 101 0 Nontreated - yes 353 106 0 Nontreated - no 373 101 0 Botrytis allii was recovered from non-fungicidal-treated and Arasan-treated seed from Asgrow Seed Co. stored at 5 C for 3 months. Recovery of B. allii was greater for artificially infested than for noninfested seed. B, allii was nOt recovered from seed treated with benomyl (Table 8.2). 107 Table 8.2. Effect of artificial infestation and fungicides on recovery of B. allii from onion seed (Asgrow Seed Co., cv. Spartan Banner) after three months storage at 5-C (1980). ' Seed Rate Infested with Mean recovery treatment (g/kg seed) Botgytisa from seed (%) Benlate 1 + Arasan 1 yes - 0 t 0 Benlate 1 + Arasan 1 no 0 i 0 Arasan 1 yes 18 i 2 Arasan 1 no 1 t 1 Arasan 2 I -no 1 i 1 Nontreated - yes 23 i 9 Nontreated - no 4 i 3 aTwenty ml of a conidial suspension of B, allii (108 conidia/ml) were applied to 60 grams of onion seed, then seeds were air dried. Botrytis allii, B. cinerea, and B, squamosa all were recovered from dead onion leaves collected from the 1980 neck rot trial. Botrytis leaf blight was present within the neck rot trial and accounts for the relatively high recovery of B, squamosa from.the leaves (Table 8.3). Effect of Foliar Application of B. allii on Neck Rot Incidence in Storage Foliar inoculations of onion leaves with B, eliii conidia two weeks prior to harvest during 1980 greatly enhanced the incidence of neck rot in storage. Fifty percent neck rot was observed in inoculated bulbs compared with 28% in control bulbs. Botrytis allii infections were not evident in either foliage or bulbs at harvest, or when bulbs 108 were initially placed in storage. Bulb weights at harvest (0.15 lb/bulb inoculated vs. 0.14 lb/bulb control) did not differ signifi- cantly as the result of conidial applications to the foliage two weeks prior to harvest. Table 8.3. Recovery of Botrytis fungi from dead onion leaves collected from the seed treatment experiment at the M.S.U. Muck Farm (1980). Seed Botrytis allii Recovery (%) treatment infesteda eiiii. cinerea squamosa Benlate + Arasan yes 1 2 2 Benlate ,+ Arasan no 0 0 10 Arasan yes 0 1 I 7 Arasan no 2 1 6. Nontreated yes 0 1 5 Nontreated no 0 4 5 aTwenty ml of a conidial suspension of B, eilii.(108 conidia/ml) were applied to 60 grams of onion seed, then seeds were air dried. Timing of conidial inoculation of foliage experiments suggested that B. eBBii_infections in bulbs in storage were greatest after foliar inoculations near harvest (16 weeks after planting). Infections were also high after foliar inoculations, 4 weeks after planting. The greatest incidence of neck rot was observed in those plants inoculated on a fresh cut made in the neck after harvest (Table 8-4-)- Fungicidal spray applications on B, eilii inoculated foliage prior to harvest did not significantly reduce the neck rot level in storage over nonsprayed controls (Table 8.5). 109 Table 8.4. Effect of timing of inoculation with B, allii conidia of foliage of field onions on neck rot incidence in storage. Timing of inoculation Mean bulb (weeks after planting) weight (grams) % Neck rot 0 375 2.0 4 320 11.6 8 381 5.7 12 408 3.1 16 441 21.0 non inoculated . 417 1.1 post inoculateda - 81.2 aConidia were applied after harvest to a fresh cut in the neck area. Table 8.5. Effect of fungicides applied to onion foliage on neck rot incidence in storage Fungicide Lbs. AI/A % Neck rot Benlate 0.25 46.3 i 7.9 Benlate 0.50 48.7 i 13.6 Bravo 0.75 51.4 i 10.3 Bravo 1.50 59.2 t 2.5 Benlate 0.25 + Bravo 0.75 43.2 i 10.3 Control - 53.4 i 6.7 110 DISCUSSION In 1977 Maude and Presly (1977b) reported that seeds infected with B, eBBii_resulted in increased neck rot incidence in storage. In the seed treatment experiment of 1980 no neck rot developed in storage. The seed used by Maude and Presly (1977a, 1977b) was grown in Europe and was internally infected, while that used in the seed treatment experiment described above was surface-infested. Thus seedling infec- tions as observed by Tichelaar (1967) and Maude and Presly (1977a, 1977b) may not have occurred in our trials where California-grown seed was used. A repeat of this experiment during 1981, which also included seed obtained from.England, was lost due to heavy rain and flooding near harvest. Examination of seed from Asgrow Seed Co., Kalamazoo, MI, and Desert Seed Co., Brooks, Oregon , yielded very low levels of B, eBBii. Maude (personal communication), who also examined American seed, stated that he found a very low incidence of B, eilii_in American seed. Thus, in the United States, seed borne infections may not be contribu- ting significantly to neck rot incidence in storage. It is possible that greater incidences of seed borne B,qulii_may occur during periods of wet weather if fungicidal sprays are not effectively used. The increase in neck rot following foliar applications of B, ellii supports evidence from.Maude and Presly and Tichelaar that B, eilii infections can occur from an aerial deposition of conidia. There were gross symptoms of neck rot at harvest. This supports observations of Maude and Presly (1977a, 1977b) and Tichelaar (1967) that symptomless infections can occur in bulbs prior to harvest. The increased incidence of neck rot when conidia were applied at 4 and 16 weeks after planting 111 may have been due to favorable conditions for infection after inocu- lation. Additional studies concerning the aerial dessimination of B, allii and its ability to colonize onion tissues are needed. 112 REFERENCES Kaufman, J. and J. W. Lorbeer. 1967.- Control of Botrytis neck rot of onions by fungicidial dusts and desiccant chemicals. Plant Dis. Rep. 51:696-699. Kaufman, J., J. W. Lorbeer, and B. A. Friedman. 1964. Relationship of fungicides and field spacing to Botrytis neck rot of onions grown in New York. U. S. Dept. Agric. A. R. S. 51-1. 6 pp. Maude, R. B. and A. H. Presly. 1977a. Neck rot (Botrytis allii) of bulb onions I. seed-borne infection and its relationship to the disease in the onion crop. Ann. Appl. Biol. 86:163-180. Maude, R. B. and A. H. Presly. 1977b. Neck rot (Botrytis allii) of bulb onions II. seed-borne infections in relationship to the disease in store and effect of seed treatment. Ann. Appl. Biol. 86:181;188. McKeen, C. D. 1951. An occurrence of rot of Spanish onion seedlings caused by Botrytis allii. Sci. Agric. 31:541-545. Munn, M. T. 1917. Neck rot disease of onions. New York State. Agric. EXP. Stn. BUllo 4370 pp. 363-4550 Tichelaar, G. M. 1967. Studies on the biology of Botrytis allii on Allium cepa. Neth. J. Plant Path. 73:157-160. Walker, J. C. 1926. Botrytis neck rots of onions. J. Agric. Res. 33:893-928. APPENDICES APPENDIX A SIMULATION MODELS OF BOTRYTIS LEAF BLIGHT OF ONION PREFACE: These models were completed in association with Systems Science 843 during spring term, 1983. The following individuals assisted in the preparation of the models: J. A. Biernbaum, M. Hoffhines, D. M. Timberlake, D. P. welch, and R. O. Barr. A description of the model, written in manuscript format follows. INTRODUCTION Botrytis leaf blight is a leaf spotting and blighting disease of onion (Allium cepa L.), caused by the fungus Botrytis squamosa walker. The disease is especially severe under prolonged moist conditions at temperatures of 15-24 C. Presently, as many as 10-14 fungicidal Sprays are applied annually to prevent Botrytis leaf blight epidemics. Since the disease is weather- dependent, an understanding of how weather conditions influence disease development could be applied in disease predictive systems. Using such systems, onion growers could judiciously apply fungicides only when needed, thus conferring substantial savings in production costs, and reducing the impact of pesticides on the environment. Disease predictive models have been developed for sour cherry (9), apple scab (11), potato late blight (12,15), and tomato early blight (14,17). These models have increased knowledge of the biology of the pathogens, have proven to be effective in lowering disease control costs 113 114 and in increasing production of these important food crops, and have decreased the impact of pesticides on the environment. Similar systems could be derived for Botrytis leaf blight predictions in onion. This report summarizes modeling efforts with the onion-Botrytis leaf blight system.and defines area of future research needed for further development and refinement of the models. MATERIALS AND METHODS Description of the Botrytis Leaf Blight Prediction Model (BOLEB) A Botrytis leaf blight prediction model (BOLEB) was constructed which uses inputs of ambient temperature and air humidity, expressed as vapor pressure deficit (VPD), to predict B, squamosa spore population dynamics. The model also provides estimates of lesion numbers and infected leaf area (blighted tissue). and simulates the influence of fungicidal sprays on blight epidemics. There are four basic components of BOLEB. They are: a) spore release; b) lesion production; c)lesion expansion and tissue blighting; and d) fungicidal spray option. a. The release of Spores. Lacy and Pontius (12) showed that parameters most closely associated with sporulation of B, squamosa were temperature and VPD. They expressed air humidity as vapor pressure deficit (VPD) (saturation vapor pressure - ambient vapor pressure) because of greater linearity between VPD and spore release at various temperatures than between relative humidity and spores released at various temperatures, resulting in better regression fits. A sporulation predictive index was constructed using 3-day averages of temperature and VPD, which estimated the probability of spore release on the following (4th consecutive) day. 115 The index value ranged from 0-1, where 1 represented the greatest prob- ability of spore release. In field testing, the model has proven valuable in predicting large spore releases (unpublished data). Thus the model of Lacy and Pontius (12) represented an appropriate starting point in our modeling efforts. In fact, BOLEB uses their predictive index, which we have designated average index value (AIV). Quantity of spores trapped within an onion field/24 m3 air/day/cm2 infected leaf area per plant available to support sporulation (SPORREL) was estimated, using environmental conditions favorable for spore release, as determined by the AIV value, the infected leaf area (cm2/ plant) which was available to support sporulation, and potential spores corresponding to the infected leaf area which could be expected in 24 m3 air sampled by a Burkard recording spore trap within a 24 hour period from a 50 X 200 foot onion field area (equation 1). Equation 1. SPORREL - AIV * AILA * MAX where: AIV - average index value AILA.- available infected leaf area in cmZ/plant Max = 225 ’SPORREL estimates the-quantity of spores trapped under various environmental conditions in a 24 hour period per 24 m3 air sampled divided by the total leaf area available to support sporulation in cm2 from a single plant. It is expressed in terms of a single plant for simplicity and convenience in model derivation and because of limitations in available data. The value of MAX was derived using spore release and blighted leaf length data published by Swanton (18), and from equations of Bolgiano (5) 116 which related leaf length to surface area of onion leaves. MAX is a constant which represents the average number of conidia (225 conidia) per 24 m3 air sampled/day/cmz of available infected leaf area of a single representative plant, under conditions highly favorable for blight development. It is also expressed in terms of a single plant, but also represents a field of onions at a given density. The relation- ship between planting density and conidial numbers possible is not known. AIV is identical to the index value described by Lacy and Pontius (12). Its value ranges from 0-1, where 1 represents highly favorable conditions for sporulation. Under conditions less than optimal for sporulation, only a portion of possible conidia would be formed and released (1). Thus a remaining portion of infected leaf area (RILA) would support sporulation when favorable conditions resumed. Such cyclic sporulation in response to environmental conditions were reported by Alderman (1). The remaining infected leaf area (RILA) was calculated according to equation 2. Equation 2. RILA - (1 - AIV) * AILA The remaining infected leaf area available to support spore forma- tion-and release (RILA) was then added to newly produced infected leaf area (NILA) according to equation 3. Equation 3. AILA - NILA + RILA This AILA would then be recalculated and utilized in equation 1 each day, and represented infected leaf area which could support spore production on that day. 117 b. Lesion Production. Using the data of Sutton et al. (17), a relation- ship between conidial numbers under field conditions and resulting lesions was derived. From this relationship, linear regression models were formulated to estimate lesion numbers resulting from.eumulative spores trapped. Reports of Alderman and Lacy (2) and Shoemaker and Lorbeer (16) established that lesions develop within 24 hours following arrival of the spores on the leaf surface under highly favorable condi- tions. BOLEB estimates lesion numbers on the day following spore release. Since Alderman and Lacy (2) and Ellerbrock and Lorbeer (8) reported poor survival of conidia after their release and impaction on leaves, under conditions not favorable for germination, a conidial survival component was not incorporated into lesion formation of subse- quent days. c. Infected Leaf Area. Usually lesions caused by B, squamosa reach a size of 1-2 x 3-5 mm, and most are restricted from developing further (2). Alderman and Lacy (2) reported that some lesions may continue to expand under favorable environmental conditions, and that the number of expanding lesions increases with increasing duration of favorable condi- tions (high AIV). Expanding lesions lead to blighting and leaf tissue death, and since sporulation of the causal fungus occurs only on dead or dying tissue, blighting caused by expanding lesions can greatly increase the area of leaf tissue on which the fungus can produce spores (AILA). Relationships between numbers of expanding lesions, infected leaf area, and spores released were estimated using spore release data from.the Michigan State Univeristy Muck Farm during 1981. Equations were derived which take the following discrete time form (equation 4.) 118 E ation 4. ELES = * Q“ (t + 3) a LES(t) where ELES = expanding lesions contributing to blighted leaf area, t = current day, a - a constant which is the slope parameter, and LES - the current day's lesions. B, squamosa developing in expanding lesions creates infected leaf area, but this area is not utilized in the model for 3 days. On the fourth day the value of ELES is redefined as newly infected leaf area (NILA) and is utilized in equation 3. The 3 day delay is equivalent to the latent period and is consistent with observations of Lacy and Pontius (12) who found that three days of favorable conditions preceded large spore releases from blighted tissue. The pr0portion of lesions which will become expanding lesions depends on the favorability of environmental conditions. Conditions favorable for formation of expanding lesions are also favorable for Spore production (1). Thus, environmental conditions leading to a high AIV would yield a greater proportion of expanding lesions. Several linear regression models are used to calculte ELES in BOLEB, and the particular model used in a given day depends on the AIV values The value of ELES is the current day's contribution of lesions on the plant and is calculated using the spore release value of the prece- ding day and linear regression models. Several regression equations are used which take into account the reduction in healthy leaf area as lesions accumulate on the leaves. d. Spray Component. An optional Spray component was added to examine the influence of simulated fungicidal sprays on simulated blight epidemics. Assumptions incorporated in the spray option wer that i) sprays would 119 remain effective for 7 days; ii) sprays would reduce lesion numbers by 75%; and that iii) sprays would reduce spores released by 25%. A seven day interval between sprays was chosen as a fairly conservative estimate of the length of protection provided. The 75% reduction in lesions per plant resulting from a fungicidal spray was derived from 8 years of unpublished field data by J. W. Lorbeer (Cornell Univeristy) who quantified reductions in lesions per leaf in fungicide-sprayed vs. unsprayed plots. The 25% reduction in spores released as a result of fungicide sprays was derived from a 1981 field data set from the Bolthouse farm, Sheridan, MI. The fungicide spray option was triggered when the AIV was greater than 0.5 and when a spray was not applied within the previous 7 days. The 0.5 AIV "trigger" value is arbitrary, based on field observation and experience (7), and may be altered up or down to make the model more or less conservative. Onion Growth Model As a further refinement of the onion blight model, blighted leaf area predicted by the blight model was used in an onion growth model to simulate yield losses due to reduction in leaf area by blighting. Our goal in development of an onion growth model was to design a simple, yet biologically realistic model which would imitate yield reductions recorded in earlier artificial defoliation studies (3,9). In particular, Hawthorn (9) and Baker and Wilcox (3) found that yield reductions were most acute at the time of bulb initiation, or approximately five weeks prior to harvesting. We felt that destruction of phtosynthetically active tissues by B, squamosa would result in an effect similar to that achieved when removal of such tissues was done in defoliation studies. 120 The onion model treats the onion plant as consisting of two components: 1) the leaf tissues per plant in square centimeters, and ii) the bulb in grams dry weight. Each component was modeled as a discrete system.with the following form: GLSA ' GLSA + f(DEGDAY) (t + 1) (t) -- BLBWT + g(GLSA) BLBWTG + 1) (t) where GLSA a green leaf surface area per plant in cm?; BLBWT - bulb dry weight in grams; t - current day; and DEGDAY - cumulative degree days. The f and 3 functions relate growth of leaves and bulb to cumulative degree days and green leaf surface area, respectively, and are the best fit linear regression coefficients based on data generated using an onion model constructed by Bolgiano (5). Degree days are calculated using a base of 5.6 C which is the lower growth limit implied for onion (5,6). Diurnal temperature curves were approximated as sine curves from maximum and minimum temperatures (4). The experiment of Baker and Wilcox (3) was simulated using our onion model and Baker and Wilcox's data derived from actual defoliation studies. Baker and Wilcox (3) found 30, 45, and 60% reductions in yield after removal of 30, 60, or 90% of the leaf area respectively. Our model accurately mimicked the effects of artificial defoliation on yield in the latter portion of the growing season (Figure A.l) so we felt confident that defoliation caused by B, squamosa would be similar to results obtained in artificial defoliation experiments. 121 100 90- 6’ 0: 80- .- 12 C) 0 70- m . . o ' 60.. ....- I' 1- ......---°" /' 5 . .I' 8 50.. 7’ m . O. 'l I 40- a. ooooooooooo -opo-o-o-e-.-e-J 0 20 40 6° 3° '00 '20 DAYS AFTER GERMINATION Figure A.1. Simulation of yield as percent of control after defoliation of 30, 60, or 90% of the leaf tissue on various days after germination of onion. 122 Model Runs Model runs were conducted using BOLEB, and ONLEG (BOLEB joined to the onion growth model). The initial inputs of BOLEB were i) the initial infected leaf area, which could be estimated from the number of lesions pergplant, and the area per lesion, and ii) the average daily temperatures in Farenheit and the average VPD, which were converted to AIV. Temperature and relative humidity (converted to VPD) were collec- ted at the M. S. U. Muck Farm, Bath, MI, during 1978 and 1981 and at the Bolthouse farm, Sheridan, MI, during 1981 and 1982. The initial inputs of ONLEG, in addition to those of BOLEB were i) the day after seed germination that one lesion per leaf was first observed, ii) the duration of model simulation run, in terms of either degree days of days; and iii) frequency of output. Output included day, degree day, green leaf surface area, and bulb dry weight. RESULTS AND DISCUSSION Leaf Blight Model (BOLEB) Simulations were run using data from the Muck farm during 1978 and 1981 (Figure A.2a,b) and from the Bolthouse farm, 1981 and 1982 (Figures A.20,d). Weather conditions at the M. S. U. Muck Farm during 1978 were very unfavorable for leaf blight development. On only two occasions during the season did AIV values rise above 0.5 (the condition where fungicidal sprays were advised). By the end of the season 22,000 cumula- tive spores/ 24 m3 air/ day were trapped using a Burkard recording spore trap (Figure A.2a), compared with 274,000 during 1981 (Figure A.2b). No sprays were applied to these plots in either year. During 1981, conditions were favorable for leaf blight epidemics. Numbers of 123 mHvHsoo vouoHsaHm van .voHaEmm uHo >3 24.: 80 . am“ 9N . m NOON s 0% 6 >40 8<.§ OUN DON 2N On“ CNN . - . N v 1‘ 03.0...0...’ ... [0 I. he} 3:! etc-03:3. 92‘ IO- ; 0c .3353. 0:... .azzx.asex.qua a. HQQH Nth 3 a ' VIOINOO. -3I\I.LV‘|0I'II'IO II sOHuao woken on» usonufis use nqu uonaom a \ oHoHcou 0>HuoHsaso Hanuo< .vaN.< ousth >40 25.11. sou own emu . n 1 0 0 I. W! I. .m ....\.e' ......R U. - e‘o‘ 0.0 1' o ... ..... .... 10 m a V i l a: N a 3 . ..c.. l O 9 0‘0. 0 ... . 3.9 5.3 .9253...- 1..0 r 9W ..... as... 8 .3525. .... .... E... s; .32.... .3... .... ... ~82 E 02.3: u ... N. >40 ON. 00. Do on or ON 0 h b n b '0 a m 3 W. N am r _ I o d. M m m .s r». X W m 3 O V 8 ask- 2. .233 0.... '.0" fl 2...... s. I 32 Eek 33226 e ow 292.12.qu cur: m>I 1879 EITHER NOW 0R AT SOME TIME IN ITS PAST HEREPR(6) I .TRUE. BULBWT-BULBWT+(NEWGLSAIGLSA)*.00871 ENDIF ENDIF GLSAINEWGLSA RETURN END ************************************************************************ ************************************************************************ * FUNCTION DGRDY(JJ) * * DGRDY RETURNS THE TOTAL NUMBER OF DEGREE DAYS FOR A GIVEN DAY. * u-a-a-N-a- 30 20 40 141 ENTRY:TMAX, MAXIMUM TEMP FOR THE DAY, AND, TMIN, MINIMUM TEMP FOR THE DAY IN COMMON BLOCK EXIT :DGRDYS CALCULATED IMPLICIT REAL (A92) COMMON/TEMP/TMIN,TMAX DATA TPI/6.283185308/,HPI/1.570796327/,BASE/5.6/ IF (TMAX.GT.BASE) GOTO 10 DGRDY-.OOOOI RETURN CONTINUE ZITMAXITMIN XMITMAX+TMIN IF (TMIN.LT.BASE) GOTO 20 DGRDYIXM/2.-BASE IF (DGRDY.GT.O.) GOTO 30 DGRDY-.OOOOI RETURN CONTINUE TBASEIBASE*2. AIASIN((TBASE-XM/Z) DGRDY-(2.*COS(A)-(TBASE-XM)*(HPI—A))/TPI IF (DGRDY.GT.O.) GOTO 40 DGRDYI.00001 RETURN END it*********************************************************************** *3!**********************M********************************************** * SUBROUTINE DEFAULT IMPLICIT REAL (A92)- COMMON/DEFLT/DEF(120) DEF(1)-116. DEF(2)I131. DEF(3)-147. DEF(4)I161. DEF(5)-177. DEF(6)-188. DEF(7)-198. DEF(8)-211. DEF(9)I225. DEF(10)I230. DEF(11)-240. DEF(12)-252. DEF(13)I270. DEF(1A)-282. DEF(15)I297. DEF(16)I312. DEF(17)-325. DEF(18)-341. DEF(19)-354. DEF(20)I367. DEF(21)I379. DEF(22)I393. DEF(23)I408. DEF(24)I428. DEF(25)I450. DEF(26)I463. DEF(27)I474. DEF(28)I490. DEF(29)I507. DEF(30)I520. DEF(31)I537. DEF(32)I551. DEF(33)I564. DEF(34)I580. DEF(3S)I593. DEF(36)I602. DEF(37)I612. DEF(38)I626. DEF(39)I646. DEF(40)I662. DEF(41)I676. DEF(42)I693. DEF(43)I709. DEF(44)I727. DEF(45)I744. DEF(46)I762. DEF(47)I781. DEF(48)I802. DEF(49)I825. DEF(50)I839. DEF(51)I856. DEF(52)I877. DEF(53)I897. DEF(54)I910. DEF(56)I924. DEF(57)I958. DEF(58)I977. DEF(59)I995. DEF(60)I1012 DEF(61)I1025 DEF(62)I1036 DEF(63)I1047 DEF(64)I1062 DEF(65)I1079 DEF(66)I1096 DEF(67)I1109 DEF(68)I1119 DEF(69)I1132 DEF(70)I1144. DEF(71)I1157. DEF(72)I1173 DEF(74)I1207. DEF(75)I1225 142 DEF(76)I1240. DEF(77)I1257. DEF(78)I1274. DEF(79)I1289. DEF(80)I1305. DEF(81)I1322. DEF(82)I1336. DEF(83)I1349. DEF(84)I1366. DEF(85)I1381. DEF(86)I1397. DEF(87)I1409. DEF(88)I1414. DEF(89)I1420. DEF(90)I1428. DEF(91)I1440. DEF(92)I1454. DEF(93)I1467. DEF(94)I1480. DEF(95)I1494. DEF(96)I1506. DEF(97)I1520. DEF(98)I1535. DEF(99)I1553. DEF(100)I1570. DEF(101)I1587. DEF(102)I1602. DEF(103)I1620. DEF(104)I1635. DEF(105)I1651. DEF(106)I1665. DEF(107)I1679. DEF(108)I1693. DEF(109)I1705. DEF(110)I1716. DEF(111)I1722. DEF(112)I1732. DEF(113)I1747. DEF(114)-176o.' DEF(115)I1774. DEF(116)I1788. DEF(118)I1812. DEF(119)I1824. DEF(120)I1836. RETURN END 143 APPENDIX C SUBROUTINE BEGBLYT SUBROUTINE BEGBLYT * k * ************************************************************************ * THIS SUBROUTINE CALCULATES : NUMBER OF SPORES / 24 M**3 / DAY * : CUMULATIVE SPORES * : LESIONS / PLANT * : CUMULATIVE LESIONS / PLANT * : TOTAL INFECTED LEAF AREA IN CM**2 * : INFECTED LEAF AREA CM**2 / DAY * VARIABLES INCLUDE: * AILA - ADJUSTED INFECTED LEAF AREA * AIV = AVERAGE INDEX VALUE OCER LAST THREE DAYS. * AIVS = TABLE OF AVERAGE INDEX VALUES * Ava . x COORDINATE OF AIV TABLE * AIVY - Y COORDINATE OF AIV TABLE * ALNNES . NATURAL LOG OF LESION NUMBERS * ALNSPOR . NATURAL LOG OF SPORE NUMBERS * ATEMP - THREE DAY AVERAGE OF AVERAGE TEMPERATURES *» AVETEMP - AVERAGE TEMPERATURE OVER THREE DAY PERIOD. * AVPD - THREE DAY AVERAGE OF VAPOR PRESSURE DEFICITS * DLTALES - LESIONS ATTRIBUTED TO CURRENT DAY * ELES - NUMBER OF EXPANDING LESIONS (BLIGHT) / DAY * HIGHTMP . HIGH TEMPERATURE FOR THE DAY IN CENTIGRADE. * ILA = INFECTED LEAF AREA IN CM**2 / PLANT * LES - TOTAL NUMBER OF LESIONS / PLANT * LESSUM a SUM OF DLTALES * LOWTEMP = LOW TEMPERATURE FOR THE DAY IN CENTIGRADE. * NTOTILA - NATURAL LOG OF TOTILA INFECTED LEAF AREA * RILA - REMAINING INFECTED LEAF AREA WHICH HAS POTENTIAL To * RELEASE SPORES * RH - RELATIVE HUMIDITY. * SEVEN . COUNTER FOR DETERMINING WHEN A SPRAY IS NO LONGER * EFFECTIVE * SPORREL . SPORES RELEASED * SPORSUM - SUM OF SPORES RELEASED * TEMPDTA - ARRAY 0F TEMPERATURES AND RELATIVE HUMIDITIES * TILA . ARRAY OF TOTAL INFECTED LEAF AREAS OVER 4 DAY PERIOD. * TOTILA . TOTILA INFECTED LEAF AREA * VPD - VAPOR PRESSURE DEFICIT. * VPDFCT = SUBROUTINE THAT CALCULATES VAPOR PRESSURE DEFICITS 144 145 FUTURE IS USED TO COMPUTE (TODAY + 3) POSITION FOR THE REVOLVING FOUR ELEMENT ARRAY INFECTED LEAF AREA. LASTVAL IS USED TO COMPUTE YESTERDAYS INFECTED LEAF AREA). *********************************************************************** *********************************************************************** IMPLICIT REAL (AAZ) COMMON/TEMPS/HIGHTMP,LOWTMP,RH DIMENSION TILA(4),INDXVAL(3),TEMPDTA(3,2) INTEGER AIVS(32,17),TEMPLOC INTEGER TODAY,LASTVAL,FUTURE,SEVEN,IVCNT,INDX,J,ANS,Ava,AIVY RILAI0.0 TOTILAI0.0 ELESI0.0 DLTALESI0.0 YESTLESI0.0 LESIO.0 SPORSUMI0.0 SEVENIO LESSUMI0.0 TODAYI-l INDx=o SRCMILAI0.0 SPORREL-0.0 TEMPLOCIO MAXIZZS ************************************************************************ *** READ IN TABLE OF AIV VALUES *** ************************************************************************ DO 50 I-1,32 READ(1,'(1714)')(AIVS(I,J),JI1,17) 50 CONTINUE iii-31'8- ************************************************************************ . *** READ IN INITIAL INFECTED LEAF AREA *** ************************************************************************ PRINT*,'INITIAL INFECTED LEAF AREA IS ' READ*,TILA(1) ILAITILA(1) ************************************************************************ *** ZERO OUT THE REST OF THE INFECTED LEAF AREA ARRAY *** ************************************************************************ DO 75 JI2,4 TILA(J)-0.0 75 CONTINUE ************************************************************************ *** INITIALIZE TEMPDTA ARRAY TO THE SAME FIRST VALUE OF AVETEMP AND VPD ************************************************************************ AVETMPI(HIGHTMP+LOWTMP)/2 CALL VPDFCT(AVETMP,RH,VPD) DO 85 J-1,3 TEMPDTA(J,1)IAVETMP TEMPDTA(J,2)IVPD 85 CONTINUE RETURN 146 ENTRY BLIGHT (DUMMY) *** CALCULATE AVERAGE TEMPERATURE FOR THE DAY *** AVETMPI(HIGHTMP+LOWTMP)/2 *** CALCULATE VAPOR PRESSURE DEFICIT *** CALL VPDFCT(AVETMP,RH,VPD) TEMPLOCITEMPLOC+1 IF (TEMPLOC.EQ.4) TEMPLOCII TEMPDTA(TEMPLOC,1)-AVETMP TEMPDTA(TEMPLOC,2)-VPD ************************************************************************ *** CALCULATE SPORULATION INDEX VALUE *** tt*******************************************t************************** ATEMP-O.o AVPD-0.0 DO J-1,3 ATEMPéATEMP+TEMPDTA(J,1) AVPD-AVPD+TEMPDTA(J,2) 10 CONTINUE ATEMP-ATEMP/a AVPD-AVPD/3 AIVXININT(AVPD*4.0)+1 AIVY-NINT(AVETMP)-7 AIv-AIVS (Ava ,AIVY) ************************************************************************ *** ROTATE THE INFECTED LEAF AREA ARRAY *** ************************************************************************ TODAY-MOD(TODAY+1,5) IF (TODAY.EQ.O) TODAY-1 LASTVAL=MOD