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IM' " «9,31031'3. ’ “A" 11:5er ?f"¢m‘t.§ ' - . fl "'5; ,M.:'& r'lfj- {Mg- :meuefifi .41 45m ,.zm~§§xj;tkw&21g:£zmzf; . “M , -11». 41:5 v v‘ ‘ni b .‘u'! ' ~. ‘ h u“. . - .M 4 ‘.~. ”3:523 as: H.» u n. «4; .1. f at it . u' " {if [m I 33,. ."rz-grzf'f r‘ .‘ «x V 5’ 3&5? [‘1 fr, V W 1‘59" T “ .‘t ”1"“: , 3:. I”. ‘-‘!‘I;?‘;t'z‘.'~,<- Dyhu‘ ’1‘. ' WAIVER . r -V a {O I r , 3 1293 00605ll This is to certify that the dissertation entitled Biology and Control of Fusarium oxysporum f. sp. apii Race 2 presented by Karen Faye Ireland Toth . has been accepted towards fulfillment of the requirements for PhD degree in BotanLancLPlant Pathology gfi7 4%% Xecg/ / Méén' professor MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771 __._———~A—_ ”£23": mi Mchigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE IT— MSU Is An Affirmative Adlai/Equal Opportunity lnditutlon BIOLOGY AND CONTROL OF FUSARIUM OXYSPORUM F. SP. APII RACE 2 By Karen Faye Ireland Toth 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 1989 903QQQG ABSTRACT BIOLOGY AND CONTROL OF FUSARIUM OXYSPORUM F. SP. APII RACE 2 W Karen Faye Ireland Toth Fusarium oxysporum f. sp. apii race 2, the causal agent of Fusarium yellows of celery, is the limiting factor in celery production in Michigan. First identified in Michigan in 1981, Fusarium yellows has spread to all the celery-growing regions of the state. Control measures are presently inadequate for celery fields highly infested with race 2. Determination of soil populations of F. oxysporum f. sp. apii race 2 is difficult with present methods. Vegetative compatibility and protein banding patterns were investigated as alternatives. F. oxysporum f. sp. apii race 2 isolates from Michigan, California, and New York were placed within a single vegetative compatibility group based on heterokaryon formation between chlorate-induced nitrate auxotrophics. F. oxysporum f. sp. apii race 1 and 11 other form species of F. oxysporum were not vegetatively compatible with race 2. Protein banding patterns on polyacrylamide gels were similar for all form species of F. oxysporum tested. Vegetative compatibility more accurately identified race 2 cultures isolated from soil than gel electrophoresis or pathogenicity tests . Somaclones regenerated from cell suspensions derived from a moderately resistant celery cultivar expressed high levels of resistance to race 2. The high resistance was passed onto some progeny via self- fertilization. These lines could provide a source of resistance for celery growers. Seven celery cultivars and 21 experimental lines were screened for resistance to F. oxysporum f. sp. apii race 2 in field trials in Michigan. Lines highly resistant to race 2 were identified, but most were horticulturally inferior to susceptible cultivars. Soil temperatures above 30° C reduced celery growth and resistance to race 2. Rotation to onions, leeks, and other vegetables reduced F. oxysporum f. sp. apii race 2 populations in Michigan muck fields. Incorporation of dried or green residues of various crops into infested muck fields did not reduce Fusarium yellows severity on celery. A race 2- suppressive field was discovered in Michigan. A disease management strategy involving resistant cultivars. rotation with other crops, and assessment of soil populations of F. oxysporum f. sp. apii race 2 was evolved using data generated in this study. ACKNOWLEDGEMENTS I would like to sincerely thank Dr. Melvyn Lacy, my major professor, for all his advice, direction, understanding, and friendship during my graduate career at Michigan State. An appreciation is also extended to my guidance committee, Drs. John Lockwood, Christine Stephens, and Amy Iezzoni, for their interest and advice. A special thanks goes to Dr. Raymond Hammerschmidt for offering many helpful suggestions and for substituting at my dissertation defense. A very special thanks goes to Mr. Richard Crum, Mr. Brian Cortright, and numerous undergraduate students who provided helping hands for all aspects of this project. Much appreciation is also extended to Celery Research, Incorporated for providing the funds for this study, and to Mr. Brian Willbrandt, Mr. Tom Willbrandt, and Mr. Gordon Martinie for generously providing space on their commercial farms and added technical assistance for my field research. My parents, Jane and Croswell Ireland, deserve a special round of applause for supporting me in every way possible to allow me to pursue my goals. Most of all, I give my thanks, appreciation, and love to my number- one editor and husband, Dr. Peter P. Toth, for believing in me and always encouraging me to do my best. iv For Peter TABLE OF CONTENTS LIST OF TABLES .................................................... LIST OF FIGURES ................................................... LIST OF ABBREVIATIONS ............................................. CHAPTER 1. INTRODUCTION .......................................... INTRODUCTION ...................................................... Fusarium yellows of celery .................................... History and biology ....................................... Control ................................................... Objectives .................................................... LITERATURE CITED .................................................. CHAPTER 2. POTENTIAL OF NITRATE NONUTILIZING MUTANTS AND SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS IN IDENTIFYING FORMAE SPECIALES OF FUSARIUM OXYSPORUM .... INTRODUCTION ...................................................... Vegetative Compatibility Grouping .............................. Polyacrylamide Gel Electrophoresis ............................. MATERIALS AND METHODS ............................................. Fungal Strains ................................................. Media .......................................................... Nitrate Nonutilizing Mutants ................................... Selection of nitrate nonutilizing mutants ................... Complementation tests ....................................... Classification of mutants ................................... Polyacrylamide Gel Electrophoresis ............................. Cultures .................................................... Protein isolation ........................................... Electrophoresis procedure ................................... Greenhouse Pathogenicity Tests ................................. Isolation and Identification of Unknown Fungal Isolates ........ RESULTS ........................................................... Vegetative Compatibility Test .................................. Complementation tests between F. oxysporum f. sp. apii race 2 and other formae speciales of F. oxysporum ............ Identification of unknown isolates with vegetative complementation .............................................. vi H papa Cannaauwuwna l6 l7 18 23 26 26 27 27 27 29 29 31 31 32 32 34 34 36 36 36 38 Electrophoresis ................................................ 44 Banding patterns for F. oxysporum f. sp. apii race 2 and other formae speciales of F. oxysporum ....................... 44 Identification of unknowns with SDS-PAGE ..................... 49 DISCUSSION ........................................................ 51 LITERATURE CITED .................................................. 55 CHAPTER 3. SOMACLONAL VARIATION AS A MEANS FOR INCREASING RESISTANCE IN CELERY TO FUSARIUM OXYSPORUM F. SP. APII RACE 2 ........................................... 60 INTRODUCTION ...................................................... 61 MATERIALS AND METHODS ............................................. 70 Genetic terminology ........................................... 70 Plant growth conditions ....................................... 70 Tissue culture media .......................................... 71 Callus initiation ............................................. 72 Somaclone regeneration ........................................ 73 Screening somaclones for resistance to F. oxysporum f. sp. apii race 2 ................................................... 74 Vernalization and self pollination ............................ 74 Screening somaclone progeny for resistance .................... 75 F. oxysporum f. sp. apii race 2 ........................... 75 Foliar pathogens .......................................... 76 RESULTS ........................................................... 79 Somaclone regeneration and screening for resistance to F. oxysporum f. sp. apii race 2 ...................................... 79 Screening somaclone progeny for resistance .................... 81 F. oxysporum f. sp. apii race 2 ........................... 81 Foliar pathogens .......................................... 84 DISCUSSION ........................................................ 87 LITERATURE CITED .................................................. 90 CHAPTER 4. EVALUATION OF CELERY GERM PLASM FOR RESISTANCE TO FUSARIUM OXYSPORUM F. SP. APII RACE 2, AND THE EFFECT OF HIGH SOIL TEMPERATURES ON THE EXPRESSION OF THE RESISTANCE ..................................... 97 INTRODUCTION ...................................................... 98 Host resistance ............................................... 99 Temperature effects on resistance to Fusarium ................. 101 MATERIALS AND METHODS ............................................. 105 Field screening ............................................... 105 Fungal population study ....................................... 108 Temperature effects on disease resistance ..................... 109 RESULTS ........................................................... 111 Field trials .................................................. 111 Temperature effects on resistance ............................. 123 DISCUSSION ........................................................ 130 LITERATURE CITED .................................................. 135 vii CHAPTER 5. BIOLOGICAL CONTROL OF FUSARIUM OXYSPORUM F. SP. APII RACE 2 ........................................... 139 INTRODUCTION ...................................................... 140 Rotation crops and organic amendments effects on soil microbial competition ......................................... 142 Suppressive soils ............................................. 143 MATERIALS AND METHODS ............................................. 146 Media ......................................................... 146 Crops ......................................................... 146 Soil populations .............................................. 147 Soil dilutions ............................................ 147 Identification of isolates ................................ 148 Fusarium yellows severity rating scale ........................ 149 Rotational crops .............................................. 149 Onions and other vegetables ............................... 149 leeks ..................................................... 150 Crop residues ................................................. 150 Dried amendments .......................................... 150 Green amendments .......................................... 151 Cover crops ................................................... 152 Suppressive soil .............................................. 153 RESULTS ........................................................... 154 Effect of crop rotation on Fusarium yellows ................... 154 Onions and other vegetables ............................... 154 Leeks ..................................................... 154 Effect of cover crops on Fusarium yellows severity in the greenhouse .................................................... 157 Effect of crop residues on Fusarium yellows severity in the field ......................................................... 157 Dried onions and mint ..................................... 157 Green amendments .......................................... 157 Suppressive soil .............................................. 161 DISCUSSION ........................................................ 165 LITERATURE CITED .................................................. 172 viii Table LIST OF TABLES CHAPTER 2 Classification of nitrate-nonutilizing mutants based on growth on different nitrogen sources ...................... Heterokaryon formation between nitrate-nonutilizing mutants from 14 Fusarium oxysporum f. sp. apii race 2 isolates and one F. oxysporum f. sp. apii race 1 isolate ... Comparison of disease severity caused by wild-type and nitrate-nonutilizing mutants for 5 isolates of Fusarium oxysporum f. sp. apii race 2 .............................. Comparison of disease severity on celery by different formae speciales of Fusarium oxysporum .................... Complementation between nitrate-nonutilizing mutants from Fusarium oxysporum f. sp. apii race 2 and F. oxysporum f. sp. apii race 1 strains with 11 other formae speciales of F. oxysporum .......................... Identification by vegetative compatibility and greenhouse pathogenicity tests of F. oxysporum f. sp. apii race 2 cultures isolated from muck soil .............. Identification by SDS-PAGE, vegetative compatibility, and greenhouse pathogenicity tests of F. oxysporum f. sp. apii race 2 cultures isolated from muck soil .......... CHAPTER 3 Mean disease ratings for somaclone progeny screened for resistance to F. oxysporum f. sp. apii race 2 in the field ..................................................... Disease ratings for somaclonal R1 progeny screened for resistance to Septoria apiicola and Pseudomonas cichorii in the greenhouse ................................ ix Page 30 37 39 4O 41 43 50 83 85 CHAPTER 4 Disease ratings of celery in F. oxysporum f. sp. apii race 2 field trials in Michigan during 1986 and 1987 ....... Disease ratings of celery in F. oxysporum f. sp. apii race 2 field trials in Michigan during 1988 ................ Yields of celery lines in 1986 and 1987 Michigan celery field trials screening for resistance to F. oxysporum f. sp. apii race 2 ........................................ Yields of celery lines in 1988 Michigan celery field trials screening for resistance to F. oxysporum f. sp. apii race 2 ............................................... Results of ratings for horticultural characteristics for celery cultivar trials in Decatur, Michigan in 1986 ....... Disease ratings for lines derived from celery X parsley crosses and screened for resistance to F. oxysporum f. sp. apii race 2 at Hudsonville, Michigan in 1988 .......... Disease ratings for somaclonal progeny screened for resistance to F. oxysporum f. sp. apii race 2 at Decatur, Michigan in 1987 .......................................... CHAPTER 5 Fusarium yellows disease ratings for celery planted in muck soil collected from a field naturally infested with Fusarium oxysporum f. sp. apii race 2 and cropped continuously to celery or rotated to leeks for one year .... Effect of cover crops on Fusarium yellows disease potential of muck soil in the greenhouse ................... Disease ratings for moderately resistant celery cultivar Tall Utah 52-70 HR plants grown in F. oxysporum f. sp. apii race 2 infested soil amended with dried onion and mint residues ............................................. Effect of green crop amendments on Fusarium yellows disease potential of muck soil in the field ................ Properties of a soil suppressive and a soil conducive to F. oxysporum f. sp. apii race 2 ........................... 112 113 114 115 117 121 122 156 158 160 164 LIST OF FIGURES Figure Page CHAPTER 1 1 Counties in Michigan with confirmed F. oxysporum f. sp. apii race 2-infested celery fields as of 1987 .............. 7 CHAPTER 2 1 Nitrate assimilation pathway in fungi ...................... 21 2 Heterokaryon formation between complementary nitrate- nonutilizing mutants from two vegetative compatible Fusarium oxysporum isolates ................................ 28 3 Sodium dodecylsulfate polyacrylamide gel electrophoresis of soluble proteins from Fusarium oxysporum f. sp. apii isolates and molecular weight standards in 12% polyacrylamide gels ........................................ 45 4 Sodium dodecylsulfate polyacrylamide gel electrophoresis of soluble proteins from F. oxysporum isolates and molecular weight standards in 12% polyacrylamide gels ...... 46 5 Sodium dodecylsulfate polyacrylamide gel electrophoresis of soluble proteins from F. oxysporum isolates and molecular weight standards in 12% polyacrylamide gels ...... 47 6 Sodium dodecylsulfate polyacrylamide gel electrophoresis of soluble proteins from F. oxysporum isolates and molecular weight standards in 12% polyacrylamide gels ...... 48 CHAPTER 4 1 Mean weekly temperatures for June, July and August during 1986 to 1988 for Paw Paw - Decatur and Hudsonville, Michigan ................................................... 102 2 Fusarium yellows severity rating scale ..................... 107 xi Mean Fusarium yellows severity ratings for moderately resistant celery cultivar Tall Utah 52-70 HR and susceptible cultivar Tall Utah 52-70 R grown at different soil temperatures ................................ Mean Fusarium yellows severity ratings for celery cultivars Pilgrim (Peto) (susceptible) and Pilgrim (MSU) (moderately resistant) grown at different soil temperatures ............................................... Mean height of plants from susceptible celery cultivars Pilgrim (Peto) and Tall Utah 52-70 R, and moderately resistant cultivar Tall Utah 52-70 HR grown at different temperatures in muck soil naturally infested with F. oxysporum f. sp. apii race 2 and steamed muck soil ......... Mean weight of plants from susceptible celery cultivars Pilgrim (Pete) and Tall Utah 52-70 R, and moderately resistant cultivar Tall Utah 52-70 HR grown at different soil temperatures in muck soil infested with F. oxysporum f. sp. apii race 2 and steamed muck soil ......... CHAPTER 5 F. oxysporum f. sp. apii race 2 populations in two fields from a Willbrandt Farms near Muskegon, Michigan from the spring of 1986 through the fall of 1987 ........... Mean disease ratings for celery cultivars Florida 683 (susceptible to F. oxysporum f. sp. apii race 2) and Tall Utah 52-70 HR (moderately resistant) grown in various ratios of Fusarium yellows-conducive and Fusarium yellows-suppressive muck field soil ........................ Mean disease ratings for celery cultivars Florida 683 (susceptible to F. oxysporum f. sp. apii race 2) and Tall Utah 52-70 HR (moderately resistant) grown in various ratios of Fusarium yellows-conducive soil and autoclaved muck field soil ................................. xii 124 127 128 155 162 LIST OF ABBREVIATIONS cm centimeters C degrees celsius g grams ha hectares LSD least significant difference 1 liters m meters uM micromoles m1 milliliters mm millimeters mM millimoles M moles RF electrophoretic mobility of protein on SDS-PAGE relative to bromophenol dye front rpm rotations per minute t metric tonnes Tris tris(hydroxymethyl)aminomethane v/v volume for volume w/v weight per volume xiii CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW INTRODUCTION Celery (Apium graveolens L. var dulce (Mill.) Pers.) probably originated in marshlands around the Mediterranean Sea (Ryder 1979). Celery was first grown for medicinal purposes, most likely because its bitter flavor and odor suggested curative properties (Ryder 1979) and because celery seed contains an opiate (Hart 1977). During the late 1500's to early 1600's celery began to be cultivated as a food product to add flavor to cooked foods (Ryder 1979, Ware and McCollum 1975). As celery began to be cultivated as a fresh vegetable in the 1700's, its horticultural characteristics began to change (Ryder 1979). Wild celery was a leafy plant, probably resembling the leafy vegetable smallage which is still planted in gardens as a cooking herb. Selection and breeding lead to the development of the large, succulent, and highly edible petioles for which commercial celery is grown today. Celery belongs to the family Umbelliferae, which also contains celeriac, parsley, carrot, parsnip, and several weeds (Ryder 1979). Celery grows by producing a close rosette of petioles on a compressed stem forming a tight or loose head. Commercial celery cultivars are biennials, but will bolt and flower in the first year with a vernalization treatment (below 10° C for 6 to 8 weeks). Bolting occurs when the main stem elongates to form a branched flower stalk containing 3 numerous umbels of small white perfect flowers which have 5 petals and 5 stamens. Celery seeds are actually fruits made up of two compressed carpels which contain the true seed. Like most Apium species, celery plants are diploid with 2n - 2x - 22. Celery cultivars are generally open-pollinated. Celery plants can be self-pollinated, although individual flowers do not self-pollinate because the styles remain immature until after the pollen from that flower is shed (Ryder 1979). Commercial celery cultivars fall into three categories: yellow types which contain some chlorophyll when blanched, easy-blanching or self-blanching which are white when blanched, and green types which are not blanched (Hart 1977). The yellow and self-blanching cultivars were grown in the United States until the mid 1940's. Growers turned to green cultivars during World War II because of the lack of field hands for blanching. After World War II, celery growers continued growing the green cultivars because consumers had accepted them and because the green cultivars were highly resistant to the fungal disease Fusarium yellows and some diseases caused by mineral deficiencies (Hart 1977). Yellow celery cultivars are still most common in Europe (Ryder 1979). There are three recognized types of green celery: Pascals, Utahs, and Crystal Jumbos (Hart 1977). Utahs are yellow-green in color and have a more open growth habit than the other two types. Pascals are also yellow-green with petioles which curve in towards the top. Crystal Jumbos are more gray-green in color with a closed, upright growth habit. Green celery is one of the most used salad vegetables in the United States (Ryder 1979). Celery is 94% water and contains small amounts of lg. carbohydrates and fat, which is why it is a popular diet food (Schneider 1982). One cup of chapped celery contains approximately 20 calories, 1.1 g protein, 0.1 g fat, 4.7 g carbohydrate, 47 mg calcium, 151 mg sodium, 320 mg vitamin A, 409 mg potassium, 11 mg vitamin C, and trace amounts of other vitamins and minerals. Celery production in the United States began near Kalamazoo, Michigan in the 1800's (Hart 1977). Michigan remains the third largest celery-producing state, producing 6.4% of the total United States production in 1987 (Shapley and Dudek 1989). Other celery producing states include California, Florida, Texas, Ohio, Wisconsin, and New York. In Michigan, celery is grown on highly organic muck soils. Almost all of the celery produced in Michigan is grown in the western half of central Michigan (Shapley and Dudek 1989). Most of the celery farms are family owned and operated, and an average-sized farm would contain approximately 75 acres of tillable muck (Shapley and Dudek 1989). Celery seedlings are started in greenhouses or outdoor beds, transplanted into the fields from May until July, and harvested from late June until October. Celery is an intensively managed crop, requiring a high input expenditure compared to net return in order to produce a profitable crop (Shapley and Dudek 1989). Celery is generally monocropped, and few vegetables are as profitable as celery to grow (Lorenz and Maynard 1988, Ryder 1979, Ware and McCollum 1975). The climate in Michigan is favorable for the production of high quality processing and fresh market celery (Shapley and Dudek 1989), yet 5 insect pests and diseases take their toll. The disease problems on celery in Michigan include: late blight caused by Septoria apiicola, early blight incited by Cercospora apii, bacterial blight caused by Pseudomonas apii and P. cichorii, Sclerotinia pink rot, cucumber mosaic and common celery mosaic viruses, and aster yellows (Lacy and Grafius 1980). Black heart caused by calcium deficiency and cracked stem due to boron deficiency are two nutritional problems common in Michigan celery (Lacy and Grafius 1980). Fusarium yellows caused by F. oxysporum f. sp. apii (Lacy and Elmer 1985) is the most important disease of celery in Michigan and is the topic of this dissertation. I. FUSARIUM YELLOWS OF CELERY History and biology, Fusarium oxysporum f. sp. apii (R. Nels. & Sherb.) Snyd. and Hans., the causal agent of Fusarium yellows of celery was the limiting factor in production of yellow and self-blanching celery cultivars grown during the first half of this century (Nelson et a1. 1937, Ryker 1935), but the disease vanished after the introduction of highly resistant green celery cultivars in the l940-l950's (Opgenorth and Endo 1985). In 1978, Fusarium yellows of celery reappeared in California due to a new race of the fungus (race 2) (Hart and Endo 1978). Race 2 attacked previously resistant green cultivars as well as yellow and self-blanching celery cultivars, whereas the original Fusarium yellows pathogen attacked only the yellow and self-blanching cultivars (Schneider and Norelli 1981). F. oxysporum f. sp. apii race 2 was identified in Michigan by 1981 (Elmer and Lacy 1984) and had spread to all the celery growing regions 6 in Michigan by 1987 (Figure 1) (Toth unpublished). Fusarium yellows is also an important disease of celery in New York (Awuah et al. 1986), Ohio (R. Rowe, personal communication), Wisconsin (Elmer and Lacy 1984), and Texas (Martyn 1987). Fusarium yellows was observed on celery grown in soil from Florida (Toth unpublished), but has not caused important losses in that state. The most common diagnostic symptom of Fusarium yellows is a red to brown discoloration in the vascular tissue of the roots and crowns of infected plants (Awuah et a1. 1986, Hart and Endo 1978, Lacy and Elmer 1985). The discoloration may extend into the petioles in severely infected plants, and be accompanied by rotting of the central crown area. As the disease progresses, plants become stunted and chlorotic. Severely infected plants can wilt and die, some of them because of bacterial soft rot which develops in the diseased crowns. Even slightly infected plants are of low quality because they are often stunted and bitter tasting (Awuah et a1. 1986, Toth unpublished). F. oxysporum f. sp. apii race 2 infects celery roots through the root tips (Hart and Endo 1981). Although numerous infections were required for severe disease to develop (Hart and Endo 1981), a F. oxysporum f. sp. apii race 2 population of only 42 propagules per gram of muck soil was predicted to be sufficient to incite severe disease in susceptible celery (Elmer and Lacy 1987b). F. oxysporum f. sp. apii race 2 is a soil-borne pathogen. F. oxysporum isolates are imperfect fungi which produce single-celled, oval-shaped microconidia in false heads on relatively short monophialides (Nelson et a1. 1983). Infectious spores of F. oxysporum memo». new Bl¢ orstco none mm cum oscooa Aucom ) [mt wetter-o mssauxtt m oczmw moo / m W m! osceou M Gum-I w av 0cm # woos" Isa-nu unouuo # w.“ W nus-(teal smut: women.» came? WW # 100‘!!! men 5? CW“ mum sear Iona m must! I L # nae»: mv urea m meu MW WI mu W catnouu «arson 1'th WAYNE stun-cu as: suoseru m \ mum: Ltmwu Figure 1. Counties in Michigan (jg apii race 2-infested celery fields as of 1987. ) with confirmed F. oxysporum f. sp. 8 include chlamydospores and macrospores; the latter are slightly sickle shaped and have an attenuated apical cell and a foot-shaped basal cell. F. oxysporum f. sp. apii race 2 produces abundant microconidia and less abundant chlamydospores, but rarely produces macroconidia on commonly used culturing media. Gonorogl,‘L F. oxysporum f. sp. apii race 2 is difficult to control on celery. Soil fumigation is expensive, and has not yet proven to be a viable means of controlling the pathogen (Awuah et a1. 1986, Greathead 1988, Otto et al. 1976). Likewise, no commercially available fungicides have controlled this pathogen (Awuah et a1. 1986). Host resistance is the most practical method of controlling F. oxysporum f. sp. apii race 2 (Lorenz and Maynard 1988, Opgenorth and Endo 1985). Field trials in California (Hart and Endo 1978, Opgenorth and Endo, 1979 & 1985), Michigan (Elmer et a1. 1986), and New York (Awuah et a1. 1986) demonstrated that most yellow and green celery cultivars were susceptible to F. oxysporum f. sp. apii race 2. The green celery cultivars grown today were all derived from a single plant designated number 52-70 selected from the cultivar Crystal Jumbo for resistance to F. oxysporum f. sp. apii race 1 (Hart 1977, Opgenorth and Endo 1985). Although 52-70 was very heterozygous (Hart 1977), further selections from 52-70 which lead to a number of green cultivars narrowed the genetic pool in celery which possibly was the basis for the nearly uniform susceptibility of celery cultivars to F. oxysporum f. sp. apii race 2. Cultivars Tall Utah 52-70 HR and Florida 683 K were selected from line 52-70 H for possible resistance to F. oxysporum f. sp. apii race 2 9 (Hart 1977). Tall Utah 52-70 HR was rated as moderately resistant and Florida 683 R as susceptible to F. oxysporum f. sp. apii race 2 in field trials conducted in Michigan (Elmer et a1. 1986). Deacon is another cultivar which has a moderate level of resistance to F. oxysporum f. sp. apii race 2 (Elmer et a1. 1986). UC-l was a breeding line released from the University of California at Davis in 1984 because of its high resistance to F. oxysporum f. sp. apii race 2 (Orton et a1. 1984). UC-l was derived from crosses between several race 2-resistant celeriac (A. graveolens var. rapaceum) accessions, race 2-resistant celery, and susceptible celery. Although tall and vigorous, UC-l contains many undesirable characteristics like hollow petioles and numerous side shoots carried over from the celeriac parents. UC-l is currently being used in celery breeding programs as a source of resistance to F. oxysporum f. sp. apii race 2. Researchers were able to increase resistance in celery to F. oxysporum f. sp. apii race 2 (Heath-Pagliuso et a1. 1988, Wright and Lacy 1988), Septoria apiicola, Cercospora apii, and Pseudomonas cichorii (Wright and Lacy 1988) by regenerating celery plants from cell suspensions. In some somaclones, the increased resistance was stable and passed onto the progeny via self-fertilization (Heath-Pagliuso et a1. 1988). Heath-Pagliuso et a1. (1989) recently released a highly resistant celery line, UC-T3, which was derived from a somaclone regenerated from cells from susceptible cultivar Tall Utah 52-70 R. The mechanisms of resistance in the moderately resistant celery cultivars that are commercially available is not known. F. oxysporum f. sp. apii race 2 colonized the roots and crowns of susceptible and 10 moderately resistant celery equally, but colonized the petioles of susceptible plants to a greater extent than moderately resistant plants (Elmer and Lacy 1987a). Likewise, vascular discoloration and rotting readily occurs in petioles of susceptible cultivars, but are rarely observed in resistant plants (Elmer and Lacy 1987a). Researchers have explored other strategies for controlling F. oxysporum f. sp. apii race 2 in celery. Endo (1982) reported that increasing the pH of soil to 7.5-7.8 decreased severity of Fusarium yellows in the greenhouse. In celery fields, F. oxysporum f. sp. apii race 2 propagules were found to exist and remain viable at soil depths of at least 30 cm. The amount of lime needed to raise the pH from the normal average of 6.6 for the California celery fields examined to a slightly alkaline pH to a depth of 30 cm proved to be economically prohibitive. Schneider (1982,1985) examined the effects of different forms of nitrogen and their associated ions on the development of Fusarium yellows in celery in sandy loam soils in California. Ammonium salts were more conducive to Fusarium yellows than nitrate salts, regardless of the accompanying ions. A regime of RC1 with Ca(N03)2 almost completely controlled Fusarium yellows in the greenhouse and in some field trials. Disease suppression was related to a R:Cl ratio of 1:1 in treated petioles, rather than total amount of each ion. Awuah and Lorbeer (1982) reported that nitrogen and potassium fertilizers were ineffective in controlling Fusarium yellows in muck fields in New York. Likewise, nitrate fertilizers did not reduce the severity of Fusarium yellows in celery grown in a muck field in Michigan (Lacy 1982). 11 Crop rotation is another control tactic used for many soil-borne pathogens. Fusarium yellows incidence and severity increased when celery residues were added to soils infested with F. oxysporum f. sp. apii race 2 (Elmer and Lacy 1987a, Opgenorth and Endo 1981) suggesting that monocropping celery, especially when celery trimmings are incorporated back into the fields, would rapidly increase populations of F. oxysporum f. sp. apii race 2 in celery fields. Unfortunately, F. oxysporum f. sp. apii race 2 readily colonizes the roots of a number of plant genera (including weeds) without causing obvious symptoms (Elmer and Lacy 1987a). This leaves a potential source of inoculum even after rotation out of celery. Cr0p rotation could reduce F. oxysporum f. sp. apii race 2 populations if the pathogen was unable to colonize the roots of the crop of choice, or if residues left after the crop was harvested were deleterious to the pathogen. Roots of onion and lettuce were colonized by a UV-light induced color mutant of F. oxysporum f. sp. apii race 2 to a significantly lesser extent than were roots of susceptible celery (Elmer and Lacy 1987a). No mutant colonies were recovered from parsley or crabgrass roots. Less severe Fusarium yellows developed on susceptible celery grown in muck soil infested with F. oxysporum f. sp. apii race 2 and to which onion residues were added than when grown in soil with celery residues (Elmer and Lacy 1987a). Likewise, F. oxysporum f. sp. apii race 2 populations decreased more rapidly in soils with onion residues than soils with celery residues (Elmer and Lacy 1987a). 12 II. OBJECTIVES The main objectives of this doctoral project were to a develop laboratory technique for identifying F. oxysporum f. sp. apii race 2 cultures and to examine various methods for controlling the pathogen. Chapter 2 examines the potential of laboratory procedures involving vegetative compatibility or gel electrophoresis in identifying F. oxysporum f. sp. apii race 2 cultures isolated from soil dilutions. Chapters 3 to 5 encompass various strategies examined for controlling F. oxysporum f. sp. apii race 2. The control methods studied include the use of tissue culture techniques in an attempt to increase resistance in celery to F. oxysporum f. sp. apii race 2 (Chapter 3) and the testing of experimental celery lines for resistance in field trials under Michigan growing conditions (Chapter 4). The effects of crop rotation, cover crop choice, and organic amendments on F. oxysporum f. sp. apii race 2 populations in celery fields are presented in Chapter 5. Chapter 5 also covers some preliminary studies on a possible race 2-suppressive field in Michigan. 13 LITERATURE CITED Awuah, R. T. and Lorbeer, J. W. 1982. Occurrence of Fusarium oxysporum f. sp. apii race 2 in New York and attempts to control Fusarium yellows of celery caused by this pathogen. National Celery Workshop Proceedings. 1981-82 Annual Report, California Celery Research Program. California Celery Research Advisory Board, Dinuba, CA. 207 PP- Awuah, R. T., Lorbeer, J. W., and Ellerbrock, L. A. 1986. Occurrence of Fusarium yellows of celery caused by Fusarium oxysporum f. sp. apii race 2 in New York and its control. Plant Dis. 70:1154-1158. Endo, R. M. 1982. The biology and control of the Fusarium yellows disease of celery. 1980-81 Annual Report, California Celery Research Program. California Celery Research Advisory Board, Dinuba, CA. 83 pp. Elmer, W. H. and Lacy M. L. 1984. Fusarium yellows (Fusarium oxysporum f. sp. apii race 2) of celery in Michigan. Plant Dis. 68:537. Elmer, W. H. and Lacy, M. L. 1987a. Effects of crop residues and colonization of plant tissues on propagule survival and soil populations of Fusarium oxysporum f. sp. apii race 2. Phytopathology 77:381-387. Elmer, W. H. and Lacy, M. L. 1987b. Effects of inoculum densities of Fusarium oxysporum f. sp. apii in organic soil on disease expression in celery. Plant Dis. 71:1086-1089. Elmer, W. H., Lacy M. L., and Honma, S. 1986. Evaluations of celery germ plasm for resistance to Fusarium oxysporum f. sp. apii race 2 in Michigan. Plant Dis. 70:416-419. Greathead, A. 1988. Control of Fusarium yellows. California Celery. California Celery Research Advisory Board, Dinuba, California. 2:3-5. Hart, L. P. 1977. History of celery according to Gene Hill. Unpublished. Hart, L. P. and Endo R. M. 1978. The reappearance of Fusarium yellows of celery in California. Plant Dis. Reptr. 62:138-142. Hart, L. P. and Endo, R. M. 1981. The effect of time of exposure to inoculum, plant age, root development, and root wounding on Fusarium yellows of celery. Phytopathology 71:77-79. l4 Heath-Pagliuso, S., Pullman, J., and Rappaport, L. 1988. Somaclonal variation in celery: screening for resistance to Fusarium oxysporum f. sp. apii. Theor. Appl. Genet. 75:446-451. Heath-Pagliuso, S., Pullman, J., and Rappaport, L. 1989. UC-T3 somaclone: celery somaclone resistant to Fusarium oxysporum f. sp. apii race 2. HortScience 24:(In press). Lacy, M. L. 1982. Reappearence of Fusarium yellows disease in Michigan Celery. National Celery Workshop Proceedings. 1981-82 Annual Report, California Celery Research Program. California Celery Research Advisory Board, Dinuba, CA. 207 pp. Lacy, M. L. and Grafius, E. J. 1980. Disease and Insect Pests of Celery. Extension Bulletin E-1427. Cooperative Extension Service, Michigan State University. 8 pp. Lacy, M. L. and Elmer, W. H. 1985. Fusarium yellows of celery in Michigan. Michigan Extension Bulletin E-1823. 2 pg. Lorenz, 0. A. and Maynard, D. N. 1988. Rnott's Handbook for Vegetable Growers. Third edition. John Wiley and Sons, New York. 456 PP- Martyn, R. D. 1987. Fusarium yellows of celery in Texas. Plant Dis. 71:651. Nelson, R., Coons, G. H., and Cochran, L. C. 1937. The Fusarium yellows disease of celery. Mich. Agric. Exp. Sta. Tech. Bull. #155. 74 PP- Nelson, P. E., Toussoun, T. A., and Marasas, W. F. 0. 1983. Fusarium species: An Illustrated Manual for Identification. The Pennsylvania State University Press. University Park, Pennsylvania. 193 pp. Opgenorth, D. C. and Endo, R. M. 1979. Sources of resistance to Fusarium yellows of celery in California. Plant Dis. Reptr. 63:165-169. Opgenorth, D. C. and Endo, R. M. 1981. Competitive saprophytic ability (GSA) of Fusarium oxysporum f. sp. apii. (Abstr.) Phytopathology 71:246-247. Opgenorth, D. C., and Endo, R. M. 1985. Additional sources of resistance to race 2 of Fusarium oxysporum f. sp. apii. Plant Dis. 69:882-884. Orton, T. J., Hulbert, S. H., Durgan, M. E., and Quiros, C. F. 1984. UCl, Fusarium yellows-resistant celery breeding line. HortScience 19:594. Otto, H. W., Paulus, A. 0., Snyder, M. J., Endo, R. M., Hart, L. P., and Nelson, J. 1976. A crown rot of celery. Calif. Agric. 30:10-11. 15 Quiros, C. P., D'Antonio, V., and Ochoa, 0. 1988. Breeding celery for disease resistance and improved quality. Annual Report of California Celery Research Advisory Board, Dinuba. California. 2:1-3. Ryder, E. J. 1979. Leafy Salad Vegatables. Avi Publishing Company, Inc., Westport, Connecticut. 266 pp. Ryker, T. C. 1935. Fusarium yellows of celery. Phytopathology 25:578-600. Shapley, A. E. and Dudek, T. A. 1989. The cost of producing celery, West Central Michigan: 1990. Staff Paper, Department of Agricultural Economics, Michigan State University. 8 pp. Schneider, R. W. 1982. Use of potassium and competitive root colonizers in the control of Fusarium yellows of celery. 1980-81 Annual Report, California Celery Research Program. California Celery Research Advisory Board, Dinuba, CA. 83 pp. Schneider, R. W. 1985. Suppression of Fusarium yellows of celery with potassium, chloride, and nitrate. Phytopathology 75:40-48. Schneider, R. W. and Norelli, J. L. 1981. A new race of Fusarium oxysporum f. sp. apii in California. (Abstr.) Phytopathology 71:108. Ware, G. W. and McCollum, J. P. 1975. Producing Vegetable Crops. The Interstate Printers and Publishers, Inc. Danville, Illnois. 599 pp. Wright, J. C. and Lacy M. L. 1988. Increase of disease resistance in celery cultivars by regeneration of whole plants from cell suspension cultures. Plant Dis. 72:256-259. CHAPTER 2 POTENTIAL OF NITRATE NONUTILIZING MUTANTS AND POLYACRYLAMIDE GEL ELECTROPHORESIS IN IDENTIFYING FORMAE SPECIALES OF FUSARIUM OXYSPORUM. 16 17 INTRODUCTION Fusarium oxysporum (Schlect.) emend. Snyd. & Hans. is a soil-borne pathogen of a wide variety of agricultural crops (Snyder and Hansen 1940). F. oxysporum f. sp. apii (R. Nels. & Sherb.) Snyd. and Hans., the cause of Fusarium yellows of celery (Apium graveolens L. var dulce (Pers.) Miller), was the limiting factor in production of yellow and self-blanching celery cultivars grown during the first half of this century (Nelson et a1. 1937, Ryker 1935), but the disease vanished after the introduction of highly resistant green celery cultivars in the 1950's (Opgenorth and Endo 1985). In 1975, Fusarium yellows of celery reappeared in California (Hart and Endo 1978). The reappearance of Fusarium yellows appeared to be caused by a new race of the fungus which was designated race 2 because it caused disease in both race 1-resistant green cultivars and race 1- susceptible self-blanching celery cultivars (Schneider and Norelli 1981). F. oxysporum f. sp. apii race 2 has also been isolated from the celery growing states of Michigan (Elmer and Lacy 1984), New York (Awuah et a1. 1986), Ohio (R. Rowe, personal communication), Wisconsin (Elmer 1985), and Texas (Martyn 1987). FUSarium yellows is currently the most important disease of celery in Michigan, making its study a high priority in celery research. Most of the 122 strains and races of F. oxysporum are morphologically indistinguishable from each other (Nelson et a1. 1983, 18 Snyder and Hansen 1940), and from saprophytic F. oxysporum (Nelson et a1. 1983). The most common technique used to identify a particular pathogenic strain or race of F. oxysporum is to carry out a pathogenicity test with the appropriate host(s) (Armstrong and Armstrong 1968). These procedures are laborious and time-consuming since it usually takes several weeks for symptoms to appear, and, in the case of celery, require rather exacting greenhouse conditions for symptom expression (Endo et a1. 1978). Other characteristics used to identify fungi include vegetative compatibility (Puhalla 1985) and electrophoretic separation of proteins (Micales et a1. 1986), which were investigated in this work as alternatives to pathogenicity tests. I. VEGETATIVE COMPATIBILITY GROUPING Heterokaryosis and the resulting parasexual cycle are mechanisms for gene exchange in fungi (Alexopoulus and Mims 1979). Heterokaryosis is the only means known for genetic recombination in imperfect fungi such as F. oxysporum f. sp. apii race 2. Heterokaryosis with resulting genetic recombination was reported for auxotrophic mutants of the imperfect fungi F. oxysporum f. sp. pisi (Buxton 1956), F. oxysporum f. sp. cubense (Buxton 1962), Verticillium albo-atrum (Hastie 1962, Hastie 1964), and V. dahliae (Puhalla and Mayfield 1974). Parasexual recombination was also reported for Aspergillus niger (Pontecorvo et a1. 1953) and Penicillium chrysogenum (Pontecorvo and Sermonti 1954). Vegetative compatibility is the ability of genetically similar fungal strains to anastomose and form a stable heterokaryon. Mutually compatible strains are assigned to one vegetative compatibility group 19 (VCG). Strains which cannot form a heterokaryon are said to be vegetatively incompatible. Vegetative compatibility is often controlled by several genes, and two strains must have the same alleles at each locus in order for a stable heterokaryon to form (Anagnostakis 1982, Croft and Jinks 1977, Perkins et al. 1982, Puhalla and Spieth 1983, Puhalla and Spieth 1985). Vegetatively incompatible strains of imperfect fungi would be genetically isolated from one another. Thus, vegetatively compatible strains should be more alike in other morphological and biochemical traits than vegetatively incompatible strains. Assignment of strains to VC groups was positively correlated with pathogenicity tests for F. oxysporum (Ratan and Ratan 1988, Ploetz and Correll 1988, Puhalla 1979), Gibberella fujikuori (- F. moniliforme) (Puhalla and Spieth 1983), and for V. dahliae (Corsini et al. 1985, Puhalla 1979). Vegetatively compatible strains of F. oxysporum were also similar for colony size (Correll et al. 1986a) and isozyme banding patterns (Bosland and Williams 1987). Strains of Aspergillus nidulans placed in the same VCG had similar growth patterns and antibiotic production (Croft and Jinks 1977). Forced heterokaryosis between different auxotrophic or color mutants is the primary technique used to study vegetative compatibility. Vigorous growth or complementary pigmentation from such pairings is taken as evidence of a forced, balanced heterokaryon. Prototrophic growth confirming heterokaryosis has been reported between UV-light or mutagenically-induced auxotrophic mutants of V. albo-atrum ( Hastie 1962, Hastie 1964, Typas and Heale 1976), V. dahliae (Puhalla 1979, Puhalla and Mayfield 1974, Typas and Heale 1976), G. zeae (- F. 20 graminearum) (Leslie 1987), F. oxysporum f. sp. gladioli (Buxton 1954), F. oxysporum f. sp. lycopersici (Sanchez et a1. 1976), F. oxysporum f. sp. pisi (Buxton 1956), and G. fujikuori (- F. moniliforme) (Puhalla and Speith 1983, Puhalla and Speith 1985). Heterokaryosis between UV- induced color mutants has been demonstrated for Neurospora (Huang 1964), V. dahliae (Puhalla 1979), A. nidulans (Croft and Jinks 1977), and F. oxysporum f. sp. apii (Puhalla 1984). The generation of mutants by UV light and chemical mutagen treatment is too laborious to be useful in screening populations of field isolates for vegetative compatibility. Likewise, a researcher has no control over the type of mutations induced, and often several genes may be affected by the mutagenic treatment. Mutants induced by these techniques must be carefully characterized to determine what mutations are present. The number of mutated genes is virtually impossible to determine for fungi which have no sexual stage. Recently, researchers have employed a technique which produces nitrate nonutilizing (nit) mutants without the use of a mutagen for studying heterokaryosis (Bosland and Williams 1986, Correll et a1. 1986, Elmer and Stephens 1989, Jacobson and Gordon 1988, Joaquim et a1. 1987, Ratan and Ratan 1988, Ploetz and Correll 1988, Puhalla 1985, Puhalla and Spieth 1985). Fungi like F. oxysporum f. sp. apii race 2 are normally able to utilize nitrate as a nitrogen source by reducing it to ammonium via the enzymes nitrate reductase and nitrite reductase (Figure 1). Two proteins which regulate the nitrate-assimilation pathway (Cove 1976, Rlittich et a1. 1986) and a molybdenum-containing cofactor required by 21 Pathway-specific regulatory protein Major nitrogen regulatory protein NITRATE :: NITRITE r: AMMONIUM nitrate reductase nitrite reductase molybdenum co-factor HYPOXANTHINE :; XANTHINE :: URIC ACID purine dehydrogenase Figure 1. Nitrate assimilation pathway in fungi (Cove 1979, Marzluf 1981) 22 nitrate reductase and xanthine dehydrogenase (Cove 1976, Rlittich et a1. 1986, Pateman et al. 1964) are also required for nitrate reduction (Figure 1). To produce nit mutants, wild type fungal strains are grown on a medium containing 1.5% potassium chlorate (R0103) (Puhalla 1985). Chlorate, a nitrate analogue, is presumably reduced within fungal cells by nitrate reductase to toxic chlorite which severely restricts colony growth (Cove 1976, Rlittich et a1. 1986). Mutants resistant to chlorate display a faster, more expansive growth on chlorate medium, and most chlorate-resistant mutants are unable to utilize nitrate as a nitrogen source (Correll et al. 1987, Cove 1976, Rlittich et a1. 1986, Puhalla 1985). Exposure to chlorate selects for mutations in the nitrate reductase locus, any of the 4 to 5 loci which code for the molybdenum- containing cofactor, or in the locus coding for the pathway regulatory protein (Cove 1976, Rlittich et a1. 1986). Individual nits usually possess only one mutation. Nits which are produced from an individual fungal isolate and which have mutations in different areas of the nitrate-assimilation pathway are able to complement one another and form a nitrate-utilizing heterokaryon when their hyphae anastomose (Correll et al. 1987), unless the isolate is self-incompatible (Bosland and Williams 1987, Correll et al. 1988, Jacobson and Gordon 1988). Complementary nit mutants from vegetatively compatible fungal strains form nitrate-utilizing heterokaryons (Correll et a1. 1987, Puhalla 1985). Strains whose complementary nit mutants do not form a nitrate-utilizing heterokaryon are vegetatively incompatible (Puhalla 1985). 23 Nits have been recovered from a number of different fungi including various form species of F. oxysporum (Bosland and Williams 1987, Correll et al. 1986a, Elmer and Stephens 1989, Jacobson and Gordon 1988, Ratan and Ratan 1988, Larkin et al. 1988, Ploetz and Correll 1988, Puhalla 1985), G. fujikuori (Puhalla and Spieth 1985), V. albo-atrum (Correll et al. 1988), and V. dahliae (Joaquim et al. 1987, Joaquim and Rowe 1988). Division of isolates into VCG corresponded to division by host range in most cases (Bosland and Williams 1987, Correll et a1. 1988, Ratan and Ratan 1988, Larkin et al. 1987, Ploetz and Correll 1988, Puhalla 1985), although isolates from some form species and races of F. oxysporum (Elmer and Stephens 1989, Jacobson and Gordon 1988, Ploetz and Correll 1988) and isolates of V. dahliae from potato (Joaquim et al. 1987) were divided into more than one VCG. Saprophytic isolates were also placed within different VCG's than pathogenic isolates of the same species (Correll et al. 1986a, Ratan and Ratan 1988, Larkin et al. 1988). II. GEL ELECTROPHORESIS Gel electrophoresis techniques were shown to be reliable for classifying fungi (Micales et al. 1986). Enzymes coded by different alleles or separate genetic loci possess different electrophoretic mobilities due to variations in their respective amino acid content and molecular weight (Chrambach and Rodbard 1971, Micales et al. 1986). Banding patterns from different fungal strains can be compared by counting the number of bands, looking for bands present in some strains 24 which are absent in others, or by staining for different enzymes and comparing relative number and position of the bands for each isozyme (Micales et a1. 1986). Comparing protein banding patterns on polyacrylamide and starch gels has proven useful in distinguishing between some species of fungi, and even between different strains within a species. Banding patterns on starch gels of 103 pathogenic F. oxysporum isolates from crucifers divided them into 4 groups comparable to separation by host specificity and vegetative compatibility (Bosland and Williams 1987). Considerable variation was observed in protein banding patterns from 11 species of Puccinia, but less variation was noted between formae speciales within a species (Burdon and Marshall 1981). Differences were noted in protein banding patterns for different species of Phytophthora (Bielenin et al. 1988, Erselius and De Vallavieille 1984, Gill and Powell 1968a), but within a species banding patterns were similar or identical (Bielenin et al. 1988, Gill and Powell 1968b). Based on protein patterns it was possible to separate 3 species of Neurospora (Chang et al. 1962), and F. oxysporum from 13 other Fusarium species, V. albo-atrum, and Graphium (Glynn and Reid 1969). This study was undertaken to determine if vegetative compatibility and electrophoretic separation of proteins have potential as techniques to identify F. oxysporum f. sp. apii race 2. Vegetative compatibility was studied by complementation, or lack thereof, between nit mutants of several F. oxysporum f. sp. apii race 2 isolates and other form species of F. oxysporum. Soluble proteins from these same isolates were separated in sodium dodecyl sulfate-polyacrylamide gels and their 25 banding patterns compared. F. oxysporum f. sp. apii race 2 isolated from muck soils were identified using these two techniques, and the results compared with those obtained from greenhouse pathogenicity tests. 26 MATERIALS AND METHODS I. FUNGAL ISOLATES The same fungal isolates were used in all experiments. Isolates Foa 2, 3-9, 11, 12, 18 and W31 were Fusarium oxysporum f. sp. apii race 2 isolates obtained from symptomatic celery collected throughout Michigan and tested for pathogenicity in the greenhouse. Foa Al and A2 were race 2 isolates obtained from celery in New York and provided by J. Lorbeer (Cornell University). Foa P13 and NR-l were race 2 which originated in California and were compliments of J. Puhalla (University of California at Berkeley). Fba A8 was a F. oxysporum f. sp. apii race 1 isolate (pathogenic only on yellow celery cultivars) originally obtained from France and was also provided by J. Puhalla. F. oxysporum f. sp. melonis race 2, F. oxysporum f. sp. lycopersici, F. oxysporum f. sp. cepae, and F. oxysporum f. sp. conglutinans were provided by W. Elmer (Michigan State University). F. oxysporum f. sp. chrysanthemi, F. oxysporum f. sp. redolans, F. oxysporum f. sp. glycine, F. oxysporum f. sp. medicaginis, F. oxysporum f. sp. gladioli, F. oxysporum f. sp. tuberosi, F. oxysporum f. sp. dianthi, and F. solani f. sp. phaesoli were from the collection of T. Isakeit (Michigan State University). F. solani f. sp. pisi was previously collected from an unknown location in Michigan. 27 II. MEDIA Potato carrot agar (PCA) was prepared by autoclaving 20 g carrots and 20 g potatoes in 500 ml distilled water for 30 min. The autoclaved broth was filtered through cheesecloth and brought to a final volume of l l with distilled water. 20 grams of agar were added, then the medium autoclaved for 20 min, and then dispensed into Petri plates. Minimal medium (MM) (Puhalla 1985) contained 0.9 M sucrose, 0.2 M NaN03, 7.3 mM RH2P04, 2.0 mM Mg804-7H20, 6.7 mM RC1, 0.0002% (v/v) sterile trace elements solution, and 20 g agar/liter medium. Sterile trace elements solution contained 0.3 M citric acid, 0.2 M ZnSOA-7H20, 0.2 M Fe804-7H20, 0.03 M Fe(NH4)2(804)2-6H20, 1.0 mM CuSOa~5H20, 0.21 mM MnSOQ-HQO, 0.85 mM H3B03, and 0.25 mM NagMoOa-HZO. The trace elements solution was stored at 4' C. Minimal medium broth contained the same ingredients except agar. III. NITRATE-NONUTILIZING MUTANTS e e ion of nit ate- onuti in m ants Nitrate-nonutilizing (nit) mutants were selected for on a potato-sucrose medium containing 15 g RC103/ liter medium (RPS) (Puhalla 1985). A sterile flattened needle was scraped across colonies on PCA and then used to stab-inoculate RPS medium. The isolates first exhibited slow, restricted growth on RPS, but within 5 to 15 days faster growing, chlorate-tolerant sectors were observed. Aerial mycelium from some sectors was transferred to MM which contained nitrate as the sole nitrogen source. Nitrate-nonutilizing mutants were distinguished by their thin, but normally expansive growth and lack of aerial mycelium on MM (Figure 2). 28 Figure 2. Heterokaryon formation between complementary nitrate- nonutilizing mutants from two vegetatively compatible Fusarium oxysporum isolates. Note thin, expansive growth of mutants, and dense, wild-type growth of heterokaryon (middle of right side of culture). 29 Eemnleaentetien_testsi Heterokaryons were forced on MM with nitrate as the sole nitrogen source. Blocks approximately 0.5 cm2 cut from nit cultures on MM were placed approximately 1.0 cm apart on fresh MM plates and kept in an incubator at 25 to 28° C and a 12 hr photoperiod. Prototrophic heterokaryons were identified as wild-type growth where the hyphae from two nit mutants intergrew (Figure 2). Heterokaryons were first observed after 1 week and no new heterokaryons were observed after 3 weeks following transfer to MM. Nit mutants obtained from an individual fungal strain were grown together on MM in all possible combinations. Two nit auxotrophs with complementary mutations were selected for pairing (complementation testing) with all other F. oxysporum f. sp. apii isolates. The complementary nits for each F. oxysporum f. sp. apii race 2 isolate and the race 1 isolate were paired by growing them together in all combinations on MM for 3 weeks. The complementary mutants for 4 F. oxysporum f. sp. apii race 2 isolates and the F. oxysporum f. sp. apii race 1 isolate were paired with 2 to 4 nits from each of the 11 other fermae speciales of F. oxysporum. glossificatioo of mutants, The terminology used for the nit mutations described herein are based on those of Correll et al. (1987) (Table 1). Mutants with mutations in the nitrate reductase structural locus were designated as nitl mutants. Those with a mutation in any of the genes coding for the molybdenum cofactor were called NitM mutants, and any with a mutation in a pathway-specific regulatory locus were termed nit3 mutants. 30 Table 1. Classification of nitrate-nonutiliting mutants based on growth on different nitrogen sources. Growth on nitrogen sources1 Mutant class2 Mutation Nitrate Nitrite Hypoxanthine Uric acid Wild-type - - - +3 + + + Nitl Nitrate reductase - + + + structural locus Nit3 Pathway-specific - - + + regulatory locus NitM Molybdenum cofactor - + - + loci 1 Based on growth on a minimal medium supplemented with 0.2 M sodium nitrate, 0.03 M sodium nitrite, 0.7 M hypoxanthine (Cove 1976), or 0.0012 M uric acid (Correll et al. 1987). 2 Based on designations of Correll (Correll et al. 1987). 3 Nonutilizing of nitrogen source (-) was distinguished by lack of growth or thin expansive growth. Utilizing of nitrogen source (+) was determined by dense, wild-type growth. 31 In order to determine which enzymes were nonfunctional test isolates were grown on MM with 0.7 M hypoxanthine (Cove 1976), MM with 0.03 M NaNOz (Cove 1976), and MM with 1.2 mM uric acid (Correll et a1. 1987) in place of NaNO3. The colonies were examined for nonutilization of the nitrogen source (distinguished by the lack of growth or thin expansive growth) or dense wild type growth after 1 to 3 days. Nits unable to utilize hypoxanthine would be NitM mutants since the molybdenum cofactor required for nitrate reductase is also required for xanthine dehydrogenase which reduces hypoxanthine to xanthine (Figure 1) (Pateman et a1. 1964). Nits unable to grow on NaN02 would contain a mutation in the locus which regulates both nitrate and nitrite reductase (nit3 mutants) (Figure 1) (Cove 1976, Rlittich et a1. 1986). Remaining nits would fall into the nitl category. Since all nit mutants are able to grow on uric acid, it was included to determine if any other mutation had occurred which might prevent growth on MM. IV. POLYACRYLAMIDE GEL ELECTROPHORESIS Qultutes, Because culture medium ingredients affect the number and placement of bands on polyacrylamide gels (Glynn and Reid 1969), isolates for polyacrylamide gel electrophoresis were grown in a defined medium, MM broth, to reduce this source of variability. Flasks (250 ml) containing 100 ml MM broth were inoculated with one 13-mm-diameter plug from a PCA or MM agar culture. In preliminary experiments, the broth cultures were kept on a rotary shaker at 100 rpm for 5, 8 or 10 days in the dark. The S-day-old cultures contained the highest concentration of 32 soluble proteins and gave the most consistent results with electrophoresis. They were used in all subsequent experiments. Etotoio_1§olot1ont The mycelium from S-day-old MM broth cultures was collected by vacuum filtration and washed with 150-200 ml distilled water. Mycelium from 3 cultures for each isolate were combined and homogenized in 5 ml of 0.02 M Tris-HCl (pH 7.4) in a glass vessel with a motor driven Potter-Elvejhem pestle set at 500 rpm. The homogenate was centrifuged in a Sorvall refrigerated centrifuge at 2000 rpm for 5 min, and the supernatant extracted and stored frozen (-l4° C). Elootroohoresis oroceduto, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a discontinuous buffer system (Laemmli 1970) with a Sturdier Slab Gel Electrophoresis Unit SE400 (Hoefer Scientific Instruments, San Francisco, California). Resolving gels were 14 cm wide X 12 cm high X 1.5 mm thick. Stacking gels were 4 cm high X 12 cm wide X 1.5 mm thick. In preliminary experiments, resolving gels contained either (1) 8% (w/v) acrylamide and 0.21% (w/v) bisacrylamide, (2) 12% acrylamide and 0.32% bisacrylamide, or (3) 15% acrylamide and 0.40% bisacrylamide. The protein bands spread throughout the 12% acrylamide, but clustered together at the bottom and top of the 8% and 15% gels, respectively. Consequently, 12% acrylamide/0.32% bisacrylamide gels were used in all experiments reported in this paper. The resolving gel also contained 0.375 M Tris-HCl (pH 8.8), 0.1% (w/v) SDS, 0.5 M Urea, and was polymerized with 0.03% (w/v) ammonium persulfate and 0.1% (v/v) N,N,N'N'-tetramethylethylenediamine (TEMED). 33 The stacking gel consisted of 3% acrylamide, 0.08% bisacrylamide, 0.125 M Tris-H01 (pH 6.8), 0.1% SDS, and was polymerized with 0.05% ammonium persulfate and 0.2% TEMED. The optimal protein concentration in preliminary gels was 400 ug protein/electrophoresis well, giving distinct, darkly stained bands. This protein concentration was used in all gels described in this report. The protein concentration of each sample was determined by the manual assay of Markwell et a1. (1981), using bovine serum albumin as the standard. Samples were mixed with sample buffer and placed in a boiling water bath for 5 min. The sample buffer contained 0.125 M Tris- HCl (pH 6.8), 4% (w/v) SDS, 20% (v/v) glycerol, 0.1 M DL-dithiothreitol and 0.002% bromophenol blue (Toth 1988). Molecular weight standards included lysozyme (approximate molecular weight of 14,300), B-lactoglobulin (18,400), trypsinogen (24,000), pepsin (34,700), egg albumin (45,000), and bovine albumin (66,000) (Dalton Mark VI”, Sigma Chemical, St. Louis). Pepsin migrates anomalously in the Laemmli system used (Sigma Technical Bulletin # MWS- 887) and so was excluded from the regression analysis. Plots of log molecular weight vs RF were linear (r - .99) (P < 0.01) for all gels presented in this report. Gels were run for 1 hr at 25 mA and then for 4 hr at 30 mA. Gels were fixed overnight in a solution of 50% (v/v) methanol, 40% acetic acid, and 10% water; stained with 0.25% (w/v) Coomassie brilliant blue R250; destained in a solution of 5% (v/v) methanol, 7.5% acetic acid, and 87.5% water; and stored in 7% acetic acid. All procedures were carried out at room temperature (approximately 21-24“ C). 34 V. GREENHOUSE PATHOGENICITY TESTS Test isolates were grown for 3 to 4 weeks on PCA. One half of a 10- cm Petri dish culture was mixed into a 10-cm-diameter pot containing Baccto' professional planting mix (Michigan Peat Company, Houston, Texas) and two 4-week-old celery plants were transplanted into each pot. Uninoculated PCA and cultures of a F. oxysporum f. sp. apii race 2 isolate were the controls. The plants were kept in the greenhouse under sodium vapor lights with a 16 hr photoperiod for 9 weeks. Then the plants were uprooted and their crowns and tap roots cut open to reveal any vascular discoloration. Isolates which caused vascular discoloration in any inoculated plants were identified as F. oxysporum f. sp. apii race 2. VI. ISOLATION AND IDENTIFICATION OF UNKNOWN FUNGAL ISOLATES Soil samples from muck fields containing infected celery plants were passed through a sieve with a 2 mm diameter pore size. Thirty grams of each sample were air-dried for 2 days. Five grams of air-dried soil were stirred into 500 ml of a 1% (w/v) solution of carboxymethyl cellulose for 30 min. One ml of the soil suspension was diluted with 9 ml sterile distilled water. A 5 m1 aliquot from the dilution was added to 50 ml molten (50° C) Romada's medium (Romada 1975) and immediately poured into 5 Petri dishes (10 X 1.5 cm). Three samples were taken from each soil suspension. All cultures were kept in an incubator at 25-28° C under a 12 hr photoperiod. After 7 days, a few aerial hyphal fragments from colonies which had white aerial mycelium with pink, purple, or orange pigment on Romada's 35 medium were transferred to PCA. Colonies on PCA identified as F. oxysporum based on microscopic examination (Nelson et a1. 1983) were transferred to RPS for auxotrophic mutant selection. One to two nit mutants from an unknown isolate were paired on minimal medium with complementary nits from two different known F. oxysporum f. sp. apii race 2 isolates. F. oxysporum colonies were also transferred to minimal medium broth for SDS-PAGE, and to PCA for greenhouse pathogenicity tests . 36 RESULTS I. VEGETATIVE COMPATIBILITY TEST om ementa o e b we 8 orum f s a " race 2 and otoet {ozoae soecialeo of E, oxyoootom, All 14 F. oxysporum f. sp. apii race 2 isolates, the F. oxysporum f. sp. apii race 1 isolate, and the 11 other formae speciales of F. oxysporum tested produced several nit mutants, and all produced at least one set of complementary nit mutants. The nit mutants were stable, and did not revert back to wild-type prototrophic growth during storage on MM for several weeks. At least one nit mutant from F. oxysporum f. sp. apii race 2 isolates Fba 2, Fba 6, Fba 11, and Fee P13 formed a nitrate-utilizing heterokaryon with at least one nit mutant from every other F. oxysporum f. sp. apii race 2 isolate (Table 2). The nits from the remaining 10 F. oxysporum f. sp. apii race 2 isolates formed nitrate utilizing heterokaryons only with some of the other isolates (Table 2). The nit mutants from Fee 2, Fba 6, Fee 11, and Foa P13 which formed nitrate- utilizing heterokaryons with all other isolates were the NitM phenotype (Table l) (Correll et al. 1987). The remaining 10 F. oxysporum f. sp. apii race 2 isolates produced only nit1 and nit3 mutants (Correll et a1. 1987) (Table 1). F. oxysporum f. sp. apii race 2 isolates Fee 2, Fee 6, Fee 11, and Fee P13 were designated as tester isolates and their nit mutants used in further studies reported below. 37 Table 2. Heterokaryon formation between nitrate-nonutilizing mutants from 14 Fusarium oxysporum f. sp. apii (Foa) race 2 isolates and one F. oxysporum f. sp. apii race 1 isolate. Fba isolate1 Foa isolate 2 3 5 6 7 8 9 ll 12 18 A1 A2 P13 NR1 2 +3 + + + + + + + + + + + + + 3 + - + + - - + - + + + + - 5 + - + - - - + - - - + + - 6 + + + + + + + + + + + + + 7 + + - + + - + + + - - + - 8 + - - + + - + + - - + + - 9 + - - + - - + + + + + + - 11 + + + + + + + + + + + + + 12 + - - + + + + + + - - + - 18 + + - + + - + + + - + + - A1 + + - + - — + + - - + + + A2 + + + + - + + + - + + + + P13 + + + + + + + + + + + + + A8 - - - - - - - - - - - - - - 1 Fee A8 was a F. oxysporum f. sp. apii race 1 isolate from France. The rest were F. oxysporum f. sp. apii race 2 isolates from the United States. 2 Presence (+) or absence (-) of a nitrate utilizing heterokaryon between nit mutants of the test isolates when grown together on medium with nitrate as sole nitrogen source. 38 The NitM mutants for Fba 2, Fba 6, Fee 11, and Foa P13, and a nit1 from Fba 9 incited the same severity of disease on susceptible celery in the greenhouse as their respective wild-type parents (Table 3). Isolates Fee 9, Fba 11, and Fba P13 had lost virulence during long term culturing, and this was also observed in their corresponding nit mutants. Retention of pathogenicity by nit mutants has also been reported for other isolates of F. oxysporum (Ratan and Ratan 1988). None of the nit mutants from the 14 F. oxysporum f. sp. apii race 2 isolates formed a nitrate-utilizing heterokaryon with the nit mutants from the F. oxysporum f. sp. apii race 1 strain (Foa A8) from France (Table 2). F. oxysporum f. sp. apii race 1 was unable to cause disease in the greenhouse on green celery cultivars Tall Utah 52-70 R and Tall Utah 52-70 HR which were highly susceptible and moderately resistant to F. oxysporum f. sp. apii race 2, respectively (Table 4). Likewise, none of the nits from the 4 F. oxysporum f. sp. apii race 2 tester isolates or the F. oxysporum f. sp. apii race 1 strain formed nitrate-utilizing heterokaryons with nit mutants from 11 other formae speciales of F. oxysporum (Table 5). No Fusarium yellows disease symptoms were observed on Tall Utah 52-70 R and Tall Utah 52-70 HR plants inoculated with any of the 11 other formae speciales of F. oxysporum in greenhouse tests (Table 4). Ioentification of unkoogo isolotoo wtth vegetative comolementatioo, Not all unknown F. oxysporum cultures isolated by soil dilution produced nit mutants on RPS. Some isolates were naturally tolerant of chlorate and readily grew on RPS. The growth of other isolates remained restricted, not producing any chlorate-tolerant sectors within 5 weeks. 39 Table 3. Comparison of disease severity caused by wild-type and nitrate- nonutilizing (nit) mutants for 5 isolates of Fusarium oxysporum f. sp. apii race 2. Mean Corresponding Mean Isolate1 disease rating2 Nit disease rating Fee 2 1.383 2-16 1.57 Foa 6 2.63 6-3 2.38 Fba 9 1.25 9-5 1.00 Fba 11 1.00 11-3 1.00 Fba P13 1.25 P13-3 1.00 1 Fee 2, Fee 6, Fee 9, and Fee 11 were F. oxysporum f. sp. apii race 2 isolates from Michigan. Foa P13 was an isolate from California. 2 Plants were rated on a 1 to 5 class scale: 1 - no vascular discoloration in root or crown area; 2 - trace of vascular discoloration in root or crown area; 3 - less than 50% crown area discolored; 4 - more than 50% crown area discolored; 5 - dead or nearly dead plant. 3 Disease ratings for celery cultivar Tall Utah 52-70 R which was highly susceptible to F. oxysporum f. sp. apii race 2. Mean of 8 plants/ isolate. 40 Table 4. Comparison of disease severity on celery by different formae speciales of Fusarium oxysporum. Mean disease rating1 Formae speciales Tall Utah 52-70 HR2 Tall Utah 52-70 R apii race 1 1.03 1.0 apii race 2 1.5 4.5 cepae 1.0 1.0 chrysanthemi 1.0 1.0 congiutinans 1.0 1.0 dianthi 1.0 1.0 gladioii 1.0 1.0 glycine 1.0 1.0 lycopersici 1.0 1.0 medicaginis 1.0 1.0 meionis race 2 1.0 1.0 redolans 1.0 1.0 tuberosi 1.0 1.0 Uninoculated 1.0 1.0 PCA 1.0 1.0 1 Plants were rated on a 1 to 5 class scale: 1 - no vascular discoloration in root or crown area; 2 - trace of vascular discoloration in root or crown area; 3 - less than 50% crown area discolored; 4 - more than 50% crown area discolored; 5 - dead or nearly dead plant. 2 Tall Utah 52-70 HR and Tall Utah 52-70 R are celery cultivars moderately resistant and highly susceptible to F. oxysporum f. sp. apii race 2, respectively. 3 Mean for 8 plants/cultivar/formae speciales. 41 Table 5. Complementation between nitrate-nonutilizing mutants (nit) of FUsarium oxysporum f. sp. apii race 2 or F. oxysporum f. sp. apii race 1 isolates with 11 other formae speciales of F. oxysporum. F. egzsootoo f, so, aoii strain formae speciales Fee 21 Foa 6 Fba 11 Foa P13 Foa A8 cepae - - - - - chrysanthemi - - - - - conglutinans - - - - - dianthi - - - - - gladioli - - - - - glycine - - - - - 1ycopersici - - - - - medicaginis - - - - - melonis race 2 - - - - - redolans - - - - - tuberosi - - - - - 1 Fba 2, Fee 6, and Fee 11 were F. oxysporum f. sp. apii race 2 isolates from Michigan, Foa P13 was a F. oxysporum f. sp. apii race 2 isolate from California, and Fba A8 was a F. oxysporum f. sp. apii race 1 isolate from France. 2 Presence (+) or absence (-) of a nitrate utilizing heterokaryon between nit mutants of the test isolates when grown together on medium with nitrate as sole nitrogen source. 42 Still others produced chlorate-tolerant sectors which were able to utilize nitrate, which has been noted for chlorate-tolerant sectors from other fungi (Correll et a1. 1987, Cove 1976, Rlittich and Leslie 1987). The two tester nits used to identify unknown isolates were complementary NitM mutants from different F. oxysporum f. sp. apii race 2 tester isolates. Two different NitM mutants were used to decrease the chances of false negative identifications which would occur if the same gene was inoperative in both tester nit and the unknown nit. A total of 128 unknown F. oxysporum isolates were identified by the VCG test and greenhouse pathogenicity test (Table 6). Of these, 27 were identified as F. oxysporum f. sp. apii race 2, and 96 as not F. oxysporum f. sp. apii race 2 by both procedures (Table 6). Only 4 isolates were identified as F. oxysporum f. sp. apii race 2 by vegetative compatibility test which were apparently not F. oxysporum f. sp. apii race 2 by greenhouse pathogenicity tests. These four may be isolates with very low virulence which the greenhouse pathogenicity test fails to identify. One isolate was identified as F. oxysporum f. sp. apii race 2 by pathogenicity tests but not by the vegetative compatibility test. This isolate might have been a self-incompatible isolate which would be unable to anastomose with itself or any other isolate (Bosland and Williams 1987, Correll et al. 1988, Jacobson and Gordon 1988). 43 Table 6. Identification by vegetative compatibility and greenhouse pathogenicity tests of F. oxysporum f. sp. apii race 2 cultures isolated from muck soil. Procedure Number of Vegetative Greenhouse cultures1 compatibility pathogenicity 27 +2 + 96 - - 4 + - 1 - + 1 F. oxysporum cultures isolated from muck soil collected from celery fields. 2 to be F. oxysporum f. sp. apii race 2 (-). Identified as F. oxysporum f. sp. apii race 2 (+), or determined not 44 II. ELECTROPHORESIS ace and other fo,zm_a_o_o1,2oo_i_,a__l_Ls:§_l-:_,__oxy§oo,1_'om_L A 100% homology in number and placement of bands occurred between F. oxysporum f. sp. apii race 2 isolates Foa 20, Foa W31, Foa Al, and Fba P13 (Figure 3). Isolate Fee 2 appeared to be missing 3 bands present in all other F. oxysporum f. sp. apii race 2 isolates (Figure 3). Since Fba 2 did not have as high a concentration of proteins within the mol. wts. examined as did the other isolates (evidenced by lightly stained bands), it is possible that the bands were present but the concentration of protein was too low to detect with Coomassie blue. Four prominent bands in the F. oxysporum f. sp. apii race 2 isolates were either missing or faintly stained in F. oxysporum f. sp. apii race 1 (Fee A8) (Figure 3). These bands corresponded to proteins at the approximate weights of 67,300, 45,000, 38,600 and 19,600 based on the linear regression equation developed from the molecular weight standards. A 90 to 100% homology in number and placement of bands was obtained between F. oxysporum f. sp. apii race 2 isolates and 10 other formae speciales of F. oxysporum (Figures 4,5,6). Foa A8 had a band at an approximate molecular weight of 61,000 which was absent in the banding patterns of Foa P13, F. oxysporum f. sp. redolans, F. oxysporum f. sp. glycine, F. oxysporum f. sp. gladioli, and F. oxysporum f. sp. conglutinans (Figure 4). F. oxysporum f. sp. melonis race 2 and F. oxysporum f. sp. medicaginis contained a band corresponding to a molecular weight of 31,800 which was absent in all other isolates on 45 k!) 1 66.0 f _. l as o 1 ' 3427 i ~ 24.0 18.4 i 14.3‘ L Figure 3. Sodium dodecylsulfate polyacrylamide gel electrophoresis of soluble proteins from Fusarium oxysporum f. sp. apii isolates and molecular weight standards in 12% polyacrylamide gels. Samples were: (lane 1) molecular weight standards (from top to bottom) bovine albumin, egg albumin, pepsin, trypsinogen, B lactoglobulin, and lysozyme; (lanes 2-6) F. oxysporum f. sp. apii race 2 isolates (left to right) Fee 20, Fee 2, Foe W31, Foa P13, Foe Al; and (lane 7) F. oxysporum f. sp. apii race 1 (Fee A8). Arrows on right mark differences in protein bands between race 1 and race 2. 46 66.0 , 4 .0 32.7 . 24.0 § 18.4 14.3 ....D Figure 4. Sodium dodecylsulfate polyacrylamide gel electrophoresis of soluble proteins from F. oxysporum isolates and molecular weight standards in 12% polyacrylamide gels. Samples were: (lane 1) molecular weight standards (from top to bottom) bovine albumin, egg albumin, pepsin, trypsinogen, B lactoglobulin, and lysozyme; (lane 2) F. oxysporum f. sp. redolans; (lane 3) F. oxysporum f. sp. glycine; (lane 4) F. oxysporum f. sp. gladioli; (lane 5) F. oxysporum f. sp. apii race 1 (Fee A8); (lane 6) F. oxysporum f. sp. conglutinans; and (lane 7) F. oxysporum f. sp. apii race 2 (Fee P13). Arrows on right mark differences in protein banding patterns between lanes. 24.01 18.4 14.3 Figure 5. Sodium dodecylsulfate polyacrylamide gel electrophoresis of soluble proteins from F. oxysporum isolates and molecular weight standards in 12% polyacrylamide gels. Samples were: (lane 1) molecular weight standards (from top to bottom) bovine albumin, egg albumin, pepsin, trypsinogen, B lactoglobulin, and lysozyme; (lane 2) F. oxysporum f. sp. apii race 2 (Fee 20); (lane 3) F. oxysporum f. sp. dianthi; (lane 4) F. oxysporum f. sp. apii race 2 (Foe 2); (lane 5) F. oxysporum f. sp. apii race 2 (Fee W31); (lane 6) F. oxysporum f. sp. melonis race 2; and (lane 7) F. oxysporum f. sp. medicaginis. Arrows on right mark differences in protein banding patterns between lanes. 48 24.0 14.3 Figure 6. Sodium dodecylsulfate polyacrylamide gel electrophoresis of soluble proteins from F. oxysporum isolates and molecular weight standards in 12% polyacrylamide gels. Samples were: (lane 1) molecular weight standards (from top to bottom) bovine albumin, egg albumin, pepsin, trypsinogen, B lactoglobulin, and lysozyme; (lane 2) F. oxysporum f. sp. apii race 2 (Fee P13); (lane 3) F. oxysporum f. sp. Chrysanthemi; (lane 4) F. oxysporum f. sp. apii race 2 (Fee Al); (lane 5) F. oxysporum f. sp. tuberosi; (lane 6) F. oxysporum f. sp. cepae; (lane 7) F. solani f. sp. phaesoli; and (lane 8) F. solani f. sp. pisi. Arrows on right mark differences in protein banding patterns between lanes. 49 that gel, including Fee 2, 20 and W31 (Figure 5). F. oxysporum f. sp. melonis race 2 was missing a band at molecular weight 48,000 which was present in all other isolates on the gel (Figure 5) and the band corresponding to a molecular weight of 41,000 was less prominent in F. oxysporum f. sp. melonis and F. oxysporum f. sp. medicaginis than the other lanes (Figure 5). No differences in number or placement of bands was observed between banding patterns for Fba 20, Foa W31, and F. oxysporum f. sp. dianthi (Figure 5). Fee P13, Foa A1 and F. oxysporum f. sp. cepae were missing a band corresponding to a molecular weight of roughly 44,500 which was present in the other form species on the same gel (Figure 6). F. oxysporum f. sp. chrysanthemi and F. solani f. sp. pisi had a double band at molecular weight of 65,600 which was only a single band, corresponding to the smaller of the double bands, in the other lanes (Figure 6). F. solani f. sp. phaesoli had a banding pattern more similar to F. oxysporum isolates than did F. solani f. sp. pisi (Figure 6). Ioentificatioo of ooknogog with 525-2565, The protein banding patterns for 4 of the 12 unknown isolates were indistinguishable from the banding patterns for F. oxysporum f. sp. apii race 2 isolates (Table 7). These 4 isolates were also identified as being F. oxysporum f. sp. apii race 2 isolates by the nit-mutant procedure and pathogenicity tests (Table 7). Seven of the unknown isolates had banding patterns different from F. oxysporum f. sp. apii race 2, and were also identified as not F. oxysporum f. sp. apii race 2 with the other two procedures (Table 7). One isolate had a banding pattern similar to that of F. oxysporum f. sp. apii race 2 isolates, but it was missing one of the 50 prominent bands, and so was labeled as not F. oxysporum f. sp. apii race 2 using SDS-PAGE. This isolate was identified as being F. oxysporum f. sp. apii race 2 by the nit-mutant and pathogenicity methods (Table 7). Table 7. Identification by SDS-PAGE, vegetative compatibility, and greenhouse pathogenicity tests of F. oxysporum f. sp. apii race 2 cultures isolated from muck soil . Procedure Number of Vegetative Greenhouse cultures1 SDS-PAGE compatibility pathogenicity 4 +2 + + 7 - - - 1 - + + 1 F. oxysporum cultures isolated from muck soil collected from celery fields. 2 Identified as F. oxysporum f. sp. apii race 2 (+), or determined not to be F. oxysporum f. sp. apii race 2 (-). For SDS-PAGE, + - protein banding pattern not different from that of F. oxysporum f. sp. apii race 2, and - - banding pattern very different from that of F. oxysporum f. sp. apii race 2. 51 DISCUSSION A successful identification technique relies on a characteristic which is unique to the strain being identified. Pathogenicity tests achieve this since individual isolates of F. oxysporum show a high degree of host specificity (Snyder and Hansen 1940). Vegetative compatibility tests and gel electrophoresis also capitalize on unique gene products. Complementation between nit mutants of vegetatively compatible isolates is dependent on the type of mutation present in the nits being paired. NitM mutants readily complement nit1, nit3, and some other NitM mutants from vegetatively compatible isolates (Correll et al. 1987). Nitl mutants infrequently complemented each other, and nit3 mutants never complemented other nit3 mutants. Complementation between nit1 and nit3 mutants is slow and weak, and sometimes no complementation is observed between nit1 and nit3 mutants from the same fungal isolate (Correll et al. 1987). It is highly probable that the 10 F. oxysporum f. sp. apii race 2 isolates which were rated as negative for heterokaryon production (Table 2) would have formed nitrate-utilizing heterokaryons with nits from every other F. oxysporum f. sp. apii race 2 isolate if they has produced NitM mutants. The F. oxysporum f. sp. apii race 2 isolates from Michigan, California, and New York examined in this report appeared to all belong to a single vegetative compatibility group, and no other form species or 52 saprophytic strain of F. oxysporum tested was placed within that VCG. Puhalla (1985) and Correll et al. (1986a) also reported that the F. oxysporum f. sp. apii race 2 isolates they studied belonged to one unique vegetative compatibility group. Thus, F. oxysporum f. sp. apii race 2 contains at least one unique allele for vegetative compatibility. Although there were a few differences between banding patterns for F. oxysporum f. sp. apii race 2 isolates and some other form species of F. oxysporum, there was no particular banding pattern unique to F. oxysporum f. sp. apii race 2 by which to distinguish this pathogen from all other F. oxysporum isolates. Comparing isozyme patterns might have given more distinctive results since different proteins can have the same electrophoretic mobility (Micales et al. 1986). Isozyme patterns have been used to distinguish different formae speciales within F. oxysporum (Biles and Martyn 1988, Reddy and Stachmann 1972, Scala et a1. 1981). Likewise, esterase isozyme patterns distinguished isolates of F. oxysporum f. sp. spinaciae which caused spinach decline from those responsible for spinach wilt (Madhosingh 1980). The results of vegetative compatibility test and SDS-PAGE banding patterns provides some information on the biology of F. oxysporum f. sp. apii race 2. The data suggest that F. oxysporum f. sp. apii race 2 arose from a single mutant, which probably originated in California and quickly spread to the other states. How it spread to other states is not known, but it probably spread within Michigan in infested muck soil (Elmer 1985). Whether F. oxysporum f. sp. apii race 2 originated from F. oxysporum f. sp. apii race 1 cannot be determined. Since no isolates of F. 53 oxysporum f. sp. apii race 1 from the United States can be found (P. Hart, personal communication), and researchers in the United States were unable to isolate F. oxysporum f. sp. apii race 1, even from fields in continual celery production for more than 50 years (Elmer 1985), direct comparisons cannot be made. Isolates taxonomically placed within a particular form species of F. oxysporum yet separated into races based on host specificity are usually vegetatively incompatible from one another (Bosland and Williams 1987, Ratan and Ratan 1988, Jacobson and Gordon 1988, Larkin et al. 1988). Like host specificity, vegetative incompatibility may be a mechanism for adaptation (Bosland and Williams 1987). Host specificity allows a new genotype to proliferate in an environment with reduced competition from possibly more fit sibling species. Likewise, a genetic change leading to vegetative incompatibility would also reduce the chance of genetic recombination between a wild-type isolate and a mutant, decreasing the chance of genetic recombination with the resulting loss of fitness that many new genotypes would have. Different VCG's would also be observed within a species, form species, or race if pathogenic genotypes arose independently yet sympatrically, and so would remained genetically isolated from each other due to vegetative incompatibility. The vegetative compatibility test accurately identified 127/128 of the unknown F. oxysporum cultures (either + or - for being F. oxysporum f. sp. apii race 2). The greenhouse pathogenicity procedure only identified 124/128 accurately, since isolates of low virulence rarely cause noticeable disease in the greenhouse. With gel electrophoresis, 11/12 of the unknowns tested were identified the same as with vegetative 54 compatibility and pathogenicity methods, but a larger number of unidentified F. oxysporum isolates would need to by examined to adequately test the accuracy of SDS-PAGE in identifying F. oxysporum f. sp. apii race 2 isolates. Gel electrophoresis, if reliable, would identify F. oxysporum f. sp. apii race 2 cultures isolated from soil samples or plant tissue faster than the vegetative compatibility test or the greenhouse pathogenicity procedure. SDS-PAGE could identify cultures 10 to 12 days after transfer of colonies from Romada's medium. With the nit mutant procedure, isolates could be identified within a minimum of 20 days after transfer of cultures from Romada's medium, whereas the greenhouse pathogenicity method would take at least 12 weeks. Although slower than gel electrophoresis, the vegetative compatibility test proved to be a better procedure for identifying F. oxysporum f. sp. apii race 2 isolates in the laboratory. The nit-mutant results were clear-cut (either a heterokaryon formed or it did not). Analyzing the gels left more room for subjective interpretations. The nit test also is less labor intensive than gel electrophoresis. Several more person-hours were needed per isolate to homogenize the MM broth cultures and prepare the samples for electrophoresis than were needed to transfer cultures to RPS, to identify nits, and to pair mutants with the tester nits. 55 LITERATURE CITED Alexopoulus C. J. and Mims, C. W. 1979. Introductory Mycology. John Wiley and Sons, Inc. New York. 632 pp. Anagnostakis, S. L. 1982. Genetic analyses of Endothia parasitica: Linkage data for four single genes and three vegetative compatibility types. Genetics 102:25-28. Armstrong, G. M. and Armstrong, J. R. 1968. 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Heterokaryosis and the role of cytoplasmic inheritance in dark resting structure formation in Verticillium spp. Molec. Gen. Genet. 146:17-26. CHAPTER 3 SOMACLONAL VARIATION AS A MEANS FOR INCREASING RESISTANCE IN CELERY TO FUSARIUM OXYSPORUM F. SP. APII RACE 2 60 61 INTRODUCTION Fusarium oxysporum f. sp. apii race 2 (R. Nels. & Sherb.) Snyd. and Hans., the causal agent of Fusarium yellows of celery (Apium gravealens L. var dulce (Mill.) Pers.), has become the limiting factor in celery production throughout much of the United States (Awuah et a1. 1986, Elmer and Lacy 1984, Hart and Endo 1978, Martyn 1987). Infection of celery by F. oxysporum f. sp. apii race 2 leads to vascular discoloration, chlorosis, stunting, and wilt in susceptible plants, eventually progressing to necrosis and rot in roots and crowns (Hart and Endo 1978). Host resistance is the only feasible means for controlling F. oxysporum f. sp. apii race 2, but to date there are few cultivars available which have the high level of resistance necessary for growing celery in Fusarium infested fields. Potential sources of resistance to F. oxysporum f. sp. apii race 2 include celeriac (A. gravealens var. rapaceum (Mill.) DC) (Awuah et a1. 1986, Elmer et a1. 1986, Opgenorth and Endo 1979, 1985, Quiros et al. 1988), parsley (Petraselinum cripsum (Hoffm.) Nym.) (Elmer et al. 1986), smallage (A. gravealens var. secalinum) (Opgenorth and Endo 1985), and Apium species from other countries (Opgenorth and Endo 1979, 1985). When these plants are crossed with domestic celery cultivars, many undesirable phenotypic characteristics may be inherited along with resistance (Honma and Lacy 1980, Orton et a1. 1984, Opgenorth and Endo 62 1985). Several rounds of backcrossing and selection are necessary to produce a highly resistant cultivar which is horticulturally acceptable to celery growers. Although celery is a biennial, it can be forced to flower the first year by a vernalization treatment (Honma 1959). Even so, it still takes 12 to 14 months to go from seed to seed, and developing a new celery cultivar takes a number of years. Unfortunately, celery growers that have Fusarium-infested fields cannot wait the number of years required for highly resistant cultivars to be produced by conventional breeding methods. Tissue culture techniques offer an alternative way to induce heritable changes in existing cultivars. Tissue culture techniques were first used as a means of asexual propagation to produce clones (genetically identical organisms) for research purposes and for planting stock (Chaleff 1983, Larkin and Scowcroft 1981). It was quickly observed that phenotypic variants were often found in clonal populations (Chaleff 1983, Larkin and Scowcroft 1981). Researchers have utilized this variability to produce new genotypes of plants, including resistant genotypes from susceptible hosts (Brettell and Ingram 1979, Daub 1986, Evans et al. 1984, Larkin and Scowcroft 1981). The term "plant tissue culture" refers to the in vitra cultivation of plant parts under aseptic conditions (Biondi and Thorpe 1981). Most tissue culture techniques involve the induction of undifferentiated cell cultures from differentiated plant tissues. The initial explant may come from any plant organ or cell type, including, but not limited to, roots, leaves, buds, embryos and protoplasts. Undifferentiated cell 63 cultures grow in the form of callus, which is a mass of undifferentiated cells on a solid medium, or cell suspensions in a liquid medium. Auxins and cytokinins are usually necessary for initiation and maintenence of undifferentiated cell cultures (Ammirato 1983, Flick et a1. 1983, Gamborg and Schluk 1981). Usually the most difficult step in a tissue culture cycle is the regeneration of plants from cultured cells, and cell cultures which lack the ability to regenerate plants have limited application (Flick et al. 1983). Regeneration often involves the manipulation of culturing conditions to induce or select for differentiating cells (Ammirato 1983, Flick et al. 1983, Evans et al. 1981). Plants are regenerated from undifferentiated cultured cells via in vitra organogenesis and embryogenesis (Ammirato 1983, Evans et a1. 1981, Flick et a1. 1983). In vitra organogenesis is the formation of organs (such as roots, shoots, and flowers) de novo by cultured plant tissues (Thorpe 1980). The first reports of organogenesis were shoot formation on callus of Nicatiana species and root formation from carrot callus (Thorpe 1980). Regeneration of plants via in vitra organogenesis has been reported for a variety of plant species from different families (Flick et a1. 1983). Successful organogenesis involves the selection of suitable explant material and cell culture media and conditions, which can be different for various plant species and even cultivars within a species (Thorpe 1980). The initiation and maturation of embryos from somatic cells (termed embryoids) in culture were first observed with carrot cell cultures (Ammirato 1983). Since then, somatic embryogenesis has been reported 64 for a number of plant species (Ammirato 1983, Evans et a1. 1981). Somatic embryoids appear to go through the same development stages as zygotic embryos, becoming polarized with cotyledon initials at one end and radical at the other (Ammirato 1983). Auxins, and in a few cases cytokinins, are vital for embryoid induction, but the hormone levels need to be reduced or eliminated for embryoid maturation and plantlet development (Ammirato 1983, Evans et al. 1981). Somaclone is the general term given to a plant regenerated from any type of cell culture. Larkin and Scrowcroft (1981) defined somalconal variation as variation detected in plants derived from any form of cell culture, but Evans et a1. (1984) narrowed the definition to include only cell cultures which originated from somatic tissues. Gametoclones and gametoclonal variation were terms for plants regenerated from cell cultures originating from gametic tissues (Evans et al. 1984). Protoclones were used to describe plants regenerated from protoplasts (Shepard et a1. 1980). Much of the variability observed in regenerated plants was of genetic origin and was transmitted to self-fertilized progeny in sexually propagated crops (Evans et al. 1984, Larkin and Scowcroft 1981). Somaclonal variation appeared to result from both preexisting genetic mosaics in the explant donor tissue and from genetic changes induced by the cell culture environment (Evans et al. 1984). Rinetin and the synthetic auxin 2,4-dichlorophenoxy acetic acid (2,4-D), the two plant hormones most commonly used in tissue culture media, can cause chromosome aberrations and alterations in chromosome number at the concentration used for tissue culture (Rallak and Vapper 1985). Single 65 gene mutations, chromosome and gene rearrangements, transposable elements, gene amplification and deletion, and cryptic virus elimination are other mechanisms proposed for somaclonal variation (Evans et a1. 1984, Larkin and Scowcroft 1981, Maliga 1980). Genetic changes in mitochondrial genes of regenerants were also reported (Dixon et a1. 1982, Gengenbach et al. 1981). Aneuploidy and polyploidy were also observed in cultured cells and, less frequently, in regenerated somaclonal variants (Maliga 1980). In some cases, selective culturing conditions favoring the growth of a specific mutant type have successfully produced desired phenotypes in regenerated plants (Chaleff 1983, Daub 1986, Maliga 1980). Mutants resistant to herbicides were regenerated from cells or callus grown in the presence of the herbicide (Chaleff and Reil 1981, Chaleff and Parsons 1978, Thomas and Pratt 1982). Host-specific and host- nonspecific toxins and elicitors from different pathogens were also used at the cellular level to select for desired genotypes (Behnke 1979, Behnke 1980, Buiatti et a1. 1987, Carlson 1983, Daub 1986, Rines and Luke 1985, Shahin and Spivey 1986, Witsenboer et al. 1988). Somaclones were also regenerated with no selective pressure and then screened for desired phenotypes (Daub 1986). Somaclones were then screened for increased resistance to a particular pathogen while still in culture conditions, using either the pathogen (Dunbar and Stevens 1988, Heath-Pagiluso et a1. 1988, Ostry and Skilling 1988) or toxins (Daub 1986); or after transfer to the greenhouse (Miller at al. 1985) or field (Taylor et al. 1988) with pathogens. These procedures have proven 66 to be just as effective in providing pathogen-resistant variants as when a selective agent is used at the cellular level (Daub 1986). Regeneration of plants from callus or cell suspension cultures was first initiated for vegetative propagation of celery for breeding purposes (Chen 1976, Williams and Collin 1976a, 1976b). Somatic embryoids were observed in callus and cell suspension cultures from celery (Al-Abta and Collin 1978a, 1978b, 1979, Chen 1976, Dunstan et al. 1982, Williams and Collin 1976a, Wright 1985), leading researchers to propose using embryoids as planting stock (Orton 1985). Although much variability in chromosome number and structure was observed in cultured celery cells (Browers and Orton 1982a, 1982b, Murata and Orton 1983), cells with gross chromosomal changes rarely regenerated into plants (Browers and Orton 1982-H). Several kinds of somaclonal variants were observed in celery (Orton 1983, Orton 1985), including variants with increased resistance to F. oxysporum f. sp. apii race 2 (Heath-Pagliuso et al. 1988, Wright 1985, Wright and Lacy 1988), and to Septaria apiicala, Cercospara apii, and Pseudamanas cicharii (Wright 1985, Wright and Lacy 1988). Tissue culture techniques have provided novel sources of resistance for a variety of other plant species. Six of 370 somaclones regenerated from a tomato cultivar fully susceptible to tobacco mosaic virus were resistant to the virus (Barden et a1. 1986). The resistance was passed on to the progeny of the six somaclones. Somaclone-derived resistance in tomatoes to tomato mosaic virus involved multiple nuclear genes and maternally inherited factors (Smith and Murakishi 1987, 1988). Somaclonal variants with a single dominant gene for resistance to F. 67 oxysporum f. sp. lycapersici race 2 were regenerated from susceptible tomato lines (Miller et al. 1985, Shahin and Spivey 1986). An increased phytoalexin response was observed in somaclones of tomato when inoculated with F. oxysporum f. sp. lycapersici and Phytaphthara infestans (Buiatti et a1. 1987). Cells and protoplasts of tobacco screened for resistance to methionine sulfoximine, a structural analog of the toxin produced by Pseudomanas syringae pv. tabaci, regenerated plants which were resistant to methionine sulfoximine and to P. syringae pv. tabaci toxin (Carlson 1973). Protoplast-derived callus screened for resistance to toxins of P. syringae pv. tabaci and Alternaria alternate regenerated tobacco plants resistant to both pathogens (Thanutong et al. 1983). The resistance was stable and transmitted to the sexual progeny. Likewise, tobacco somaclones transmitted their increased resistance to Phytaphthora nicotianae var. nicotianae to their sexual progeny (Daub and Jenns 1988). Soybean somaclones and their progeny expressed resistance to races of Phytaphthara megasperma f. sp. glycinea to which the parental cultivars were susceptible (Olah and Schmitthenner 1988). Somaclones regenerated from Texas male-sterile cytoplasm corn were resistant to Drechslera maydis (- Helminthosparum maydis) race T and were male fertile (Brettell and Ingram 1979, Brettell et al. 1980, Gengenbach et al. 1977). Regenerants from potatoes were resistant to P. infestans (Behnke, 1979, Behnke 1980, Shepard et a1. 1980), A. solani (Shepard et a1. 1980), and Erwinia caratovara subsp. caratavara (Taylor et a1. 1988). 68 Tissue culture has provided resistance to Fiji disease (Rrish. and Tlaskal 1974, Nickell 1977), to H. sacchari (Nickell 1977, Larkin and Scowcroft 1981), Scleraspora sacchari (Nickell 1977), and to Ustilaga scitaminea (Liu 1981, Liu et a1. 1983) in sugarcane. Increased resistance was reported in somaclones of oats to victorin toxin from H. victoriae (Rines and Luke 1985); in somaclones of alfalfa to Verticillium albo-atrum (Latunde-Dada and Lucas 1983) and to F. oxysporum f. sp. medicaginis (Hartman et a1. 1984); and in somaclones of rice to Rhizoctania salani (Xie et al. 1987). Tissue culture was also used to obtain Papulus species and Larix species resistant to Septaria musiva and Grenmeniella abietina, respectively (Ostry and Skilling 1988). Variability in several other characteristics was observed in plants regenerated from tissue cultures. Somaclones are frequently male- sterile (Edallo et al. 1981, Evans and Sharp 1983, Prat 1983), female- sterile (Pratt 1983), or self-sterile (Daub and Jenns 1988). Regenerants with altered amino acid composition of enzymes (Brettell et a1. 1986), deficiency in ribosomal RNA genes (Landsmann and Uhrig 1985), or containing unique polypeptides (Dixon et al. 1982) and distinct mtDNA organization (Gengenbach et a1. 1981) were also reported. Variability in morphology, pigmentation, yield, and other traits under simple and quantitative genetic control (Buiatti et al. 1985, Engler and Grogan 1984, Evans and Sharp 1983, Larkin et a1. 1984, Larkin and Scowcroft 1981, Liu 1981, Orton 1980, Shepard et al. 1980, Taylor et al. 1988), and even loss of resistance to a pathogen (Olah and Schmitthenner 1988) were all observed in regenerants of a variety of plant species. 69 This study was undertaken to determine whether somaclones highly resistant to F. oxysporum f. sp. apii race 2 could be regenerated from suspensions of celery cells started from callus cultures of a moderately resistant cultivar of celery. After regeneration of whole plants, resistant somaclones were forced to flower, were self-fertilized, and their progeny analyzed for resistance to F. oxysporum f. sp. apii race 2 to determine if genetically stable resistance was induced. Somaclone progeny were also put through a primary screen in the greenhouse for resistance to the foliar pathogens Septaria apiicola Speg., the causal agent of late blight, and Pseudamanas cichorii (Swing.)Stapp, the bacterial blight pathogen. 70 MATERIALS AND METHODS I. GENETIC TERMINOLOGY The term somaclone is applied here to plants regenerated from cell cultures which originated from somatic tissues (Evans et al. 1984). Somaclonal variation was defined as variation detected in plants derived from cell culture (Larkin and Scowcroft 1981). The progeny from self- fertilization of somaclones were referred to as the R1 generation (Chaleff 1981); subsequent generations produced by self-fertilization are termed R2, R3, and so on. II. PLANT GROWTH CONDITIONS Unless otherwise noted, plants were grown in Baccto° professional planting mix (Michigan Peat Company, Houston, Texas) in the greenhouse. The fertilizer used was 20-20-20 (N-P-R) (Peterso soluble fertilizer). Plants were kept in the greenhouse under sodium vapor or fluorescent lights set for a 16 hr photoperiod. Hoagland's solution was made by combining 5% (v/v) stock solution A, 8% stock solution B, 0.1% A-Z solution and 86.9% distilled water. Stock A contained 1.928 M Ca(NO3)2-4H20. Stock B consisted of 1 M RN03, 0.5 M RH2P04, l M MgSOa-7H20, and 2.5 M NaCl. Both solutions were autoclaved and then stored at 4 C. The A-Z solution was made by autoclaving separately a solution of 0.037 M FeCl3'6H20 and a solution containing 71 0-091 H H3303. 0-47 m“ CuClz-zuzo, 0.44 mM znc12, and 3.94 mM MnC12°4H20. These two solutions were combined after autoclaving and stored at 4° C. III. TISSUE CULTURE MEDIA The media and procedures used followed those of Wright and Lacy (1988) with the following modifications. The duration between establishing cell suspensions and regenerating plants from the suspensions was 7 weeks longer than previously allowed, and the somaclones reported herein were grown to a height 4-7 cm taller under culture conditions than previous somaclones, before being transferred to the greenhouse (Wright and Lacy 1988). Murashige and Skoog medium (MS) (1962) was used in these studies. The mineral stock solution contained 0.206 M NH4NO3, 0.188 M RN03, 0.03 M CaC12-2H20, 0.015 M MgSOa°7H20, 12.5 mM RH2P04, 1.00 mM H3BO3, 1.12 mM MnSOa-HZO, 0.369 mM ZnSOa-7H20, and 0.05 mM RI in glass distilled H20. The MS iron stock solution contained 2.00 mM FeSOA-7H20 and 2.21 mM NazEDTA in glass distilled H20. These solutions were stored at 4° C. The MS-Orton stock solutions consisted of 0.270 mM glycine, 5.55 mM myo-inositol, 41 uM nicotinic acid, 24.3 uM pyridoxine HCl, and 2.97 uM thiamine HCl in glass distilled water. The MS-Orton stock solution for callus initiation also contained 45.3 uM 2,4-dichlorophenoxy acetic acid (2,4-D) and 89 HM 6-benzyladenine, and that for callus maintenance contained 22.6 uM 2,4-D and 4.65 MM kinetin. A11 MS-Orton stock solutions were stored in 10 ml lots at -l8° C. 72 Culture medium used to initiate callus tissue (CI) contained 0.5% (v/v) MS stock iron solution, 10% MS stock minerals, 1% MS-Orton stock solution for callus initiation, 0.88 M sucrose, and 88.5% glass distilled water. The pH was adjusted to 5.8 with NaOH or HCl, and 9.5 g DifcoO agar (Difco Laboratories, Detroit, Michigan) per liter of medium added prior to autoclaving. Callus maintenance medium (CM) was prepared by mixing 0.5% (v/v) MS stock iron solution, 10% MS stock minerals, 1% MS-Orton stock solution for callus maintenance, 0.88 M sucrose, and 88.5% glass distilled water. The pH was adjusted to 5.8. For agar media, 9.5 g agar per liter of medium was added. Celery regeneration medium (CRM) was made with 0.5% (v/v) MS stock iron, 10% MS stock minerals, 1% MS-Orton stock solution without hormones, 0.058 M sucrose, 88.5% glass distilled water, and 0.5 g activated charcoal per liter of medium. Again the pH was adjusted to 5.8, and 9.5 g agar per liter was added before autoclaving. IV. CALLUS INITIATION Celery cultivar Tall Utah 52-70 HR, moderately resistant to F. oxysporum f. sp. apii race 2 (Elmer et a1. 1986), was grown in the greenhouse for 11 weeks. The axillary buds from the plants were excised, surface sterilized with 10% commercial chlorine bleach for 10 min followed by two rinses with sterile distilled water, placed on CI agar, and kept at 22° C. After 9 days, buds with callus formation were transferred to CM agar. Six weeks later the callus was transferred to 73 250 m1 flasks containing 50 m1 CM broth, and these cultures were incubated on a rotary shaker at 100 rpm in the dark. After 2 months and then monthly, the cell suspension cultures were supplied with fresh CM broth. Cell suspensions were diluted 1:1 (v/v) with fresh CM broth, and 50 ml of the diluted cell suspension were poured into a sterile 250 m1 flask and placed back on the shaker. V. SOMACLONE REGENERATION Fifteen to 20 weeks after the callus was transferred to CM broth, 2 m1 aliquots from cell suspensions containing cell aggregates were plated onto CRM. Two weeks later green plantlets were observed on the CRM plates. The plantlets (somaclones) were transferred every 2 to 4 weeks to new CRM in either Petri plates (100 X 15 mm) or culturing boxes (7.5 X 7.5 X 10 cm ) (GA7 vessels, Magenta Corporation, Chicago, Illinois). Somaclones were planted into potting mix when they were 8 to 10 cm tall and had a set of secondary leaflets (4 to 8 months after regeneration). The somaclones were planted individually in Styrofoam cups containing potting mix which was previously autoclaved for 30 min. The transplants were heavily watered and quickly placed in plastic bags to minimize moisture stress. The somaclones were kept in the dark for 2 days, then placed under fluorescent lights (16 hr photoperiod) in the laboratory for 1 to 3 weeks, and then transferred to a mist chamber in the greenhouse. The plastic bags were removed and the plants kept under intermittent mist (15 min on, 15 min off) for 5 days. By this time, the somaclones could be placed on a greenhouse bench under fluorescent lights without wilting. 74 VI. SCREENING SOMACLONES FOR RESISTANCE TO F. OXYSPORUM F. SP. APII RACE 2. Somaclones were transplanted into muck soil naturally infested with F. oxysporum f. sp. apii race 2 either in the greenhouse or in the field 1 to 2 months after being planted in the potting mix. Somaclones screened in the greenhouse were kept under sodium vapor lamps for 15 to 18 weeks and then rated for disease severity. Somaclones screened for resistance in the field were rated for disease severity after 11 to 13 weeks. Somaclones screened in the greenhouse, and which had no vascular discoloration in the root or crown area, were replanted, placed back into the greenhouse for 3.5 to 4 months, and then vernalized. Somaclones screened for resistance in the field were uprooted with a shovel and their crowns cut in half. Somaclones with no, or a trace of, vascular discoloration were placed in plastic bags and taken back to the greenhouse. These somaclones were trimmed back to just a few inner petioles, replanted into 25 cm pots and kept under a greenhouse bench for 5 days to reduce tranplanting shock. The somaclones were fertilized and placed under sodium vapor or fluorescent lights in the greenhouse for 5 to 6 weeks before vernalization. VII. VERNALIZATION AND SELF POLLINATION Somaclones were vernalized at 4° to 6° C under a 12 hr photoperiod for 8 weeks in a large controlled-temperature chamber, then placed back into the greenhouse under a 16 hr photoperiod to induce flowering (Honma and Lacy 1980). During and after vernalization, the somaclones were 75 sprayed 3 times per week with approximately 5 ml of a solution of 0.13 M CaC12 to prevent black heart disease (Lacy and Grafius 1980). Vernalized somaclones were fertilized weekly, and were also given 100 ml of Hoagland's solution every two weeks. When a somaclone started to flower (at least 2 months after vernalization), the foliar parts were placed under a cage of cheese cloth suspended from a wire ring to prevent insect cross-pollination, and shaken daily to release pollen for self-pollination. The seed was harvested when the pedicel had turned yellow and the seed was brown. The seed was cleaned of debris and stored at 4° C. VIII. SCREENING SOMACLONE PROGENY FOR RESISTANCE E. oxysoarum f, so, ooi; toce 2, To hasten germination, seeds collected from somaclones were soaked in aerated water for 7 days on a laboratory bench prior to sowing. Seeds were then germinated in potting mix and transplanted into flats 3 to 4 weeks later. Thirty 8-week old R1 plants from individual somaclones were transplanted 15 cm apart into a 37.5 cm row in a muck field naturally infested with F. oxysporum f. sp. apii race 2. Transplants of Tall Utah 52-70 HR grown from commercial seed were the controls. After 76 days of growth in the field, 6 to 7 plants per line were uprooted, the crowns cut open, and the plants rated for disease severity. Ratings were on a l to 5 scale as follows: 1 - no vascular discoloration in root or crown area; 2 - trace of vascular discoloration in root or crown area; 3 - less than 50% 76 crown area discolored; 4 - more than 50% crown area discolored; 5 - dead or nearly dead plant. Plants which had no vascular discoloration were saved for seed production. When the plants were brought back into the greenhouse their roots were washed in a dilute commercial chlorine bleach solution in an attempt to control the secondary rotting organisms, and rooting hormone (RooTone’, Pratt-Gabriel, Hanover, Pennsylvania) was sprinkled on the cut area prior to planting in the potting mix. The plants were vernalized and cared for as described above. Foliar oathogeoo. The following procedure was used to determine whether somaclones resistant to F. oxysporum f. sp. apii race 2 might also have resistance to two important foliar pathogens. Side shoots from R1 plants saved for seed production were removed, dipped in rooting hormone, and planted in the greenhouse. The shoots were kept under a greenhouse bench for 2 weeks and then placed under fluorescent or sodium vapor lights. The cuttings were allowed to grow for several months, then cut back and new petioles (1 to 2 weeks old) were inoculated with Septaria apiicala or Pseudamonas cicbarii in the greenhouse. A Tall Utah 52-70 R plant grown from seed and a rooted cutting inoculated with distilled water were the controls. Dried celery leaves containing pycnidia of S. apiicola were soaked in distilled water for 30 min and the resulting spore suspension diluted to 5 x 105 spores/ml (Wright 1985, Wright and Lacy 1988). Three ml of the spare suspension was sprayed onto leaflets on one petiole per plant. The inoculated plants were kept under intermittent mist for 2 days, then placed back on a greenhouse bench under fluorescent lights. The plants 77 were rated for disease severity after 3 weeks using a l to 5 rating scale: 1 - no disease; 2 - 1 to 5% of leaflet surface had necrotic lesions with chlorosis extending beyond the lesions; 3 - 6 to 25% leaf surface diseased; 4 - 26 to 50% leaf surface diseased; 5 - > 50% leaf surface had symptoms (Wright 1985, Wright and Lacy 1988). P. cichorii was isolated from symptomatic celery leaves collected in the field. Leaves with symptomatic lesions were surface sterilized in 10% commercial chlorine bleach for 4 min followed by 2 rinses with sterile distilled water. The lesion and surrounding green leaf tissue were excised and chopped up in l to 2 drops of sterile distilled water which was then streaked onto Difco' Pseudomonas agar F (PsF) (Difco laboratories, Detroit, Michigan). The cultures were incubated at 25° C for 4 days. Colonies on PsF which were fluorescent under UV light were streaked onto fresh PsF and Nutrient broth-yeast extract agar (NBY). Colonies on NBY which were oxidase positive (Schaad 1980), and tested positive to the ROH test (- gram negative) (Schaad 1980) (Rreig and Holt 1984) were used to inoculate celery plants in the greenhouse to determine pathogenicity. A drop of concentrated solution of bacteria was put on a leaflet, and 4 holes pricked in the leaflet under the drop with a sterile needle (Wright and Lacy 1988). The plant was kept under intermittent mist for 4 days, then kept in the greenhouse until symptoms developed. One isolate which was pathogenic on inoculated plants above was used to inoculate R1 cuttings. One drop of a bacterial suspension (2 x 103 cells/m1) was placed on each of two leaflets on the same petiole, and 4 holes immediately pricked in the leaf surface under the drop. The 78 plants were left in the mist chamber for 4 days, then kept in the greenhouse under fluorescent lights until rated for symptoms 3 weeks later. The plants were rated based on a l to 5 scale: 1 - no visible necrosis around the inoculated wounds; 2 - necrosis surrounding less than 50% of inoculated wounds; 3 - more than 50% wounds with necrosis; 4 - necrosis and chlorosis spreading from less than 50% of wounds; 5 - necrosis and chlorosis spreading from more than 50% of the inoculations (Wright 1985). 79 RESULTS 1. SOMACLONE REGENERATION AND SCREENING FOR RESISTANCE TO F. OXYSPORUM F. SP. APII RACE 2. The results of previous research from our laboratory showed that the higher the level of resistance present in a cultivar, the higher the frequency of increased resistance in somaclones regenerated from that cultivar (Wright 1985, Wright and Lacy 1988). For this reason, celery cultivar Tall Utah 52-70 HR, which was moderately resistant to F. oxysporum f. sp. apii race 2 (Ireland et al. 1987), was chosen as the cell donor for the tissue culture procedure. Callus produced from buds of Tall Utah 52-70 HR plants was very friable and easily broken up in broth culture. Plants were regenerated from 5 of a total of 9 cell suspensions established from individual callus cultures. The rest of the suspensions either became contaminated or did not produce cell aggregates indicative of embryoid production. Somaclones from 3 of the cell suspensions expressed a high level of resistance to F. oxysporum f. sp. apii race 2. When activated charcoal was deleted from the regeneration medium, the embryoids either stopped developing or regenerated only shoots. On regeneration medium with activated charcoal, root growth was observed prior to shoot development. Activated charcoal is necessary for somatic 80 embryoid development in a number of species, probably because it absorbs a number of compounds, including hormones, which inhibit embryogenesis or embroyid maturation (Ammirato 1983). The first group of somaclones, which was transplanted when 3 to 5 cm in height, all died during the hardening-off process in the greenhouse. It appeared that they were not large enough to survive the shock of transplant. Wright (1985) transplanted celery somclones which were only 3 to 4 cm in height, but had only a 47% survival rate during greenhouse hardening. In contrast, by allowing the somaclones regenerated in this study to grow longer under axenic culture conditions, survivability was increased to 64%. Much variability was observed in the somaclones. Some were stunted and did not grow past 1 to 2 cm tall. Others had very abnormal morphology and many of these died within a few weeks. A few even began to form callus on the regeneration medium. Many of the somaclones had very different leaf morphologies or growth habits in the greenhouse than parental Tall Utah 52-70 HR plants from commercial seed. Four percent of the somaclones screened for resistance in the field had a complete loss of resistance to F. oxysporum f. sp. apii race 2. The somaclones were given a letter and number designation based on where they were screened for resistance. Somaclones with prefix of C were screened for resistance in the greenhouse. Those with prefixes of H were screened in a field trial in near Hudsonville, Michigan, and those with a D were screened in the field at Decatur, Michigan. A total of 544 somaclones were transplanted into potting mix. Of the 350 which survived the transfer from culture to the greenhouse, 160 81 were screened for resistance to F. oxysporum f. sp. apii race 2 in the greenhouse, and 59 of them had no vascular discoloration after 15 to 18 weeks. Twenty three of these somaclones had an open growth habit which more resembled celeraic than celery, and were discarded prior to vernalization. One hundred ninety somaclones were screened for resistance to F. oxysporum f. sp. apii race 2 in the field. Twenty two had no disease or just a trace of discoloration when rated and were replanted in the greenhouse. Two somaclones developed soft rot before or during vernalization and were discarded. A total of 35 somaclones set enough seed for a progeny screen. Some somaclones either did not flower or did not produce enough viable seed to screen. Five of the 35 flowered and set seed too late for the subsequent summer's field trial. Unique variants were observed in the R1 progeny from two somaclones. Four of seventeen seedlings from one line were dwarf mutants. In the second line, 7 of 56 seedlings were albino mutants. The albino seedlings were completely white, and did not grow past the cotyledon stage in the greenhouse. Chlorophyll deficient and dwarf mutants were also reported in somaclonal progeny for other plant species (Buiatti et al. 1985, Evans et al. 1983, Prat 1983). II. SCREENING SOMACLONE PROGENY FOR RESISTANCE. E. QXZSEOIEE i, so, ooii tace 2, Most of the R1 lines screened for resistance to F. oxysporum f. sp. apii race 2 were uniform in height and appearance in the field, and resembled parental Tall Utah 52-70 HR 82 plants in gross appearance and growth. A few lines had plants of variable heights, but all these lines had uniform disease ratings, so the stunting of some plants was not the result of increased vascular disease. Plants from line D 157 had abnormally thin petioles, and line M 50 had a very different leaf morphology than Tall Utah 52-70 HR plants. A total of 29 plants from 11 lines (Table 1) were saved for vernalization and seed set. Nine of these R1 lines, D 44, G 33(2), G 128, H 37, G 26, G 16, H 33, G 108, and D 128, had mean disease ratings significantly lower than the 2.2 rating for Tall Utah 52-70 HR plants from commercial seed (Table l) (P - 0.05). Lines G 26 and H 33 were the two most vigorous lines, and were uniform in field appearance. Lines G 108, D 128, and M 37 were also fairly uniform in field appearance and disease reaction, but G 16 contained plants of variable sizes. The other two lines, G 129(2) and G 134, had disease ratings not significantly different from Tall Utah 52-70 plants (Table 1). All 11 lines selected for seed production had disease ratings which were less variable than Tall Utah 52-70 HR plants from commercial seed. Plants from the 11 somaclonal lines all fell within the disease rating classes of 1 or 2. Tall Utah 52-70 HR control plants fell into disease rating classes of l, 2 or 3. Since celery cultivars are open- pollinated, and the somaclones were self-pollinated, less variability in some characteristics might be expected. All the R1 plants brought back to the greenhouse survived, and their R2 progeny will also be screened for resistance to F. oxysporum f. sp. apii race 2 in the field. 83 Table 1. Mean disease ratings for somaclone R1 progenies screened for resistance to F. oxysporum f. sp. apii race 2 in the field. R1 progeny1 Disease rating2 R1 progeny Disease rating H 54 4.0 G 150 3.0 G 112 3.0 G 34 2.8 H 19 2.6 G 13 2.5 H 55 2.5 G 28 2.4 G 26(2) 2.4 G 56 2.3 Tall Utah 52-70 HR3 2.2 H 13 2.2 G 120 2.2 G 151 2.0 G 130 2.0 G 39 2.0 G 129(2) ** 2.0 H 50 2.0 D 12 2.0 G 8 2.0 D 157 2.0 G 134 ** 1.9 D 44 ** 1.8 * G 33(2) ** 1.8 * G 128 ** 1.8 * H 37 ** 1.7 * G 26 ** 1.6 * G 16 ** 1.5 * H 33 ** 1.5 * G 108 ** 1.4 * D 128 ** 1.3 * 3 Letter prefix of somaclone lines designates where somaclone parent was screened for resistance to F. oxysporum f. sp. apii race 2. Lines with G or HR prefixes were screened in the greenhouse, and those with H and D prefixes were screened in the field near Hudsonville and Decatur, Michigan, respectively. 2 Disease rating was based on a l to 5 scale: 1 - no vascular discoloration; 2 - trace of vascular discoloration in root or crown area; 3 - less than 50% crown area discolored; 4 - more than 50% crown area discolored; 5 - dead or nearly dead plant. Data presented is the mean of 6 to 7 plants/line. 3 Parental cultivar somaclones were derived from. Seeds of Tall Utah 52-70 HR were from a commercial source. ** Lines from which plants were saved for seed production. * Mean disease ratings significantly lower than that for Tall Utah 52-70 HR, the cultivar from which the somaclones were produced; LSD - .36 (P - 0.05). 84 £211§1_2§;h2ggn§‘ Most of the side shoots from the R1 plants excised and treated with rooting hormone produced roots and grew well in the greenhouse. Disease reactions ranging from 2 to 5 were observed in the somaclonal lines in response to the S. apiicola inoculations (Table 2). Individual plants within somaclonal lines G 33, G 16, and H 33 were uniform in disease expression, but plants from lines lines H 37, G 108, and D 128 had a variety of disease reactions (Table 2). The low disease rating in the Tall Utah 52-70 R plant was unexpected since most commercial varieties are considered susceptible to late blight. Since this was the first plant inoculated, it may not have received an adequate level of inoculum. The somaclonal lines which had disease ratings of 2 will be screened for resistance to late blight in the field. No conclusions regarding resistance to late blight can be made until after the field screening. All of the plants inoculated in the greenhouse with P. cichorii had disease ratings 5 3 (Table 2), and no secondary spread of chlorosis or necrosis was observed. A bacterial dilution from ground up surface- sterilized leaflets from one inoculated plant showed that the inoculum was of a single bacterial type (a fluorescent Pseudomonad). Possibly it was an isolate with low virulence. Most of the somaclone progeny had bacterial blight ratings less than that of the Tall Utah 52-70 R check plant (Table 2), but since only one plant per genotype was inoculated, statistical analysis could not be performed to determine if the differences were significant. No conclusions on possible resistance in the somaclonal lines to bacterial blight can be determined from these data. Table Line (plant number)2 2. 85 Disease ratings for somaclonal R1 progeny screened for resistance to Septoria apiicola and Pseudomonas cichorii in the greenhouse. Disease rating1 S. apiicola P. cichorii 44 128 37 37 26 16 16 16 33 33 33 108 108 108 108 128 128 128 128 128 UUUUUQOQQEEEOQQQEIQOQUOO Tall Utah 52-70 R Uninoculated check 129(2) (2) 134 (l) (1) 33(2) (1) 33(2) (2) (1) (1) (2) (1) (2) (4) (5) (1) (2) (3) (1) (2) (3) (4) (1) (2) (3) (4) (5) Hmmwwbmwkmwmmmmbbwbmwmbmub OOOOOOOOOOOOOOOOOOOOOOOOOO HWP—‘I—‘NHHwNHHMNHHHHHHNwNHHt-‘N OOOU‘IOOOOU‘IOOOOOOUIOOUIOOmU‘OOO 1 Ratings for both diseases were on a l to 5 scale. For S. apiicola: l - no disease; 2 - 1 to 5% of leaflet surface had necrotic lesions with 86 Table 2 continued. chlorosis extending beyond the lesions; 3 - 6 to 25% leaf surface diseased; 4 - 26 to 50% leaf surface diseased; S - > 50% leaf surface had symptoms. One petiole/ plant was inoculated, and a visual rating was given to the whole petiole. For P. cichorii: l - no visible necrosis around the inoculated wounds; 2 - necrosis surrounding less than 50% of inoculated wounds; 3 - more than 50% wounds with necrosis; 4 - necrosis and chlorosis spreading from less than 50% of wounds; 5 - necrosis and chlorosis spreading from more than 50% of the inoculations. Each of two leaflets on one petiole were inoculated, rated separately, and the mean recorded. 2 Line refers to the parental somaclone and plant number in parenthesis refers to individual R1 plants. 87 DISCUSSION A successful tissue culture program involves the establishment and proliferation of essentially undifferentiated cells, followed by the regeneration of whole plants, and the selection of desired phenotypic variants which are capable of passing on the genetic changes to their sexual progeny (Evans et al. 1984, Larkin and Scowcroft 1981). We were successful with celery in regenerating somaclones which expressed a higher level of resistance to F. oxysporum f. sp. apii race 2, and which transmitted that resistance to their progeny through self-fertilization. Resistance in Tall Utah 52-70 HR is coded for by at least two loci, one dominant gene with a strong effect and at least one gene with a supplemental effect on resistance (Quiros et al. 1988). Somaclonal variation could affect this resistance in a number of ways. The resistance could be increased by amplification of either gene, or by a mutation adding another gene to further supplement the two pre-existing genes. Likewise, one or both of the genes could be deleteriously affected by the tissue culture procedure, resulting in somaclones with a decreased or total lack of resistance. This was in fact observed in 4 percent of the somaclones screened for resistance in the field. Genetic and cytological studies would be needed to determine the nature of the somaclonal variation observed. One benefit of somaclonal variation is its use in the creation of useful genetic variation without hybridization, thus allowing 88 maintenance of the desirable characteristics of a cultivar while one characteristic (for example disease resistance) is added. Celery somaclones produced herein were also selected for gross morphology and growth habits resembling the parental cultivar, Tall Utah 52-70 HR. Most R1 plants from the somalconal lines resembled Tall Utah 52-70 HR plants in the field, but future generations will have to be examined in more detail for variabiltiy in other characteristics, such as petiole width, height, or tendency to form side shoots, which would affect yield. Tissue culture techniques do, however, have some draw-backs. A researcher has little or no control over the level of variability induced. We observed a number of different morphological variants in somaclonal populations, and even had variants with an increased level of susceptibility to F. oxysporum f. sp. apii race 2. Tissue culture, especially without any selection pressure at the cellular level, is similar to other types of mutation breeding in that the desired genotypes may not be produced at all, or a large number of plants may have to be screened in order to find one of the desired type (Daub 1986). We were unable to select for resistance to F. oxysporum f. sp. apii race 2 at the cellular level. No host-specific toxin has been implicated in the infection of celery by F. oxysporum f. sp. apii race 2. Heath-Pagliuso et al. (1988) screened somaclones in culture by growing them on a mat of F. oxysporum f. sp. apii race 2, but all the somaclones eventually died from the disease. Wright (1985) was able to find resistant somaclones when regenerants were screened in the 89 greenhouse. Likewise, by screening regenerants in the field and greenhouse, we were able to identify lines with a high level of resistance to F. oxysporum f. sp. apii race 2. Somaclonal lines described here could provide a source of increased resistance to F. oxysporum f. sp. apii race 2 for celery breeding programs. Lines which are horticulturally similar or superior to Tall Utah 52-70 HR could be developed into new celery cultivars. Somclonal variation could be taken further. Resistance might be increased further by placing first or second generation progeny from highly resistant somaclonal lines into a tissue culture cycle, and regenerating whole plants from them. Tissue culture would provide a means of increasing resistance to F. oxysporum f. sp. apii race 2 and other diseases in cultivars favored by celery growers, and likewise, also has potential for changing other characteristics of celery plants, even quantitative characteristics to increase yield. 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Additional sources of resistance to race 2 of Fusarium oxysporum f. sp. apii. Plant Dis. 69:882-884. Orton, T. J., Hulbert, S. H., Durgan, M. E., and Quiros, C. F. 1984. UCl, Fusarium yellows-resistant celery breeding line. HortScience 19:594. Orton, T. J. 1980. Chromosomal variability in tissue cultures and regenerated plants of Hordeum. Theor. Appl. Genet. 56:101-112. Orton, T. J. 1983. Spontaneous electrophoretic and chromosomal variability in callus cultures and regenerated plants of celery. Theor. Appl. Genet. 67:17-24. 95 Orton, T. J. 1985. Genetic instability during embryogenic cloning of celery. Plant Cell Tissue Organ Cult. 4:159-169. Ostry, M. E. and Skilling, D. D. 1988. Use of tissue culture and in Vitro bioassays for the development and selection of disease-resistant trees. (Abstr.) Phytopathology 78:1608. Prat, D. 1983. Genetic variability induced in Nicotiana sylvestris by protoplast culture. Theor. Appl. Genet. 64:223-230. Quiros, C. F., D'Antonio, V., and Ochoa, O. 1988. Breeding celery for disease resistance and improved quality. Annual Report, California Celery Research Advisory Board,, Dinuba. CA. 2:1-3. Rines, H. W. and Luke, H. H. 1985. Selection and regeneration of toxin-insensitive plants from tissue cultures of oats (Avena sativa) susceptible to Helmintbosporium victoriae. Theor. Appl. Genet. 71:16- 21. Schaad, N. W. 1980. Laboratory Guide for Identification of Plant Pathogenic Bacteria. The American Phytopathological Society, St. Paul, Minnesota. 72 pp. Shahin, E. A., and Spivey, R. 1986. A single dominant gene for Fusarium wilt resistance in protoplast-derived tomato plants. Theor. Appl. Genet. 73:164-169. Shepard, J. F., Bidney, D., and Shahin, E. 1980. Potato protoplasts in crop improvement. Science 208:17-24. Smith, S. S. and Murakishi, H. H. 1987. Genetic characteristics of somaclonal resistance to tomato mosaic virus. (Abstr.) Phytopathology 77:1705. Smith, S. S. and Murakishi, H. H. 1988. Nature of resistance of tomato somaclones to tomato mosaic virus. (Abstr.) Phytopathology 78:1536. Taylor, R. J., Ruby, C. L., and Secor. G. A. 1988. Assessment of field performance and soft rot resistance in a population of protoplast- derived potato clones. (Abstr.) Phytopathology 78:1595. Thanutong, P., Furusawa, I., and Yamamoto, M. 1983. Resistant tobacco plants from protoplast-derived calluses selected for their resistance to Pseudomonas and Alternaria toxins. Theor. Appl. Genet. 66:209-215. Thomas, B. R. and Pratt, D. 1982. Isolation of paraquat-tolerant mutants from tomato cell cultures. Theor. Appl. Genet. 63:169-176. 96 Thorpe, T. A. 1980. Organogenesis in Vitro: structural, physiological, and biochemical aspects. pgs. 71-111. In: International Review of Cytology, Supplement 11A: Perspectives in Plant Cell and Tissue Culture. I. R. Vasil (ed.). Academic Press, New York. 253 pp. Williams, L. and Collin H. A. 1976. Embryogenesis and plantlet formation in tissue cultures of celery. Ann. Bot. 40:325-332. Williams, L. and Collin H. A. 1976. Growth and cytology of celery plants derived from tissue cultures. Ann. Bot. 40:333-338. Wright, J. C. 1985. Somaclonal Variation Occurring in Disease Response of Regenerated Celery Plants from Cell Suspension and Callus Cultures. M. S. Thesis. Michigan State University. 90 pp. Wright, J. C. and Lacy M. L. 1988. Increase of disease resistance in celery cultivars by regeneration of whole plants from cell suspension cultures. Plant Dis. 72:256-259. Xie, Q. J., Rush, M. C., Massaquoi, R., and Cao, J. 1987. Potential for rice improvement through somaclonal variation for disease resistance. (Abstr.) Phytopathology 77:1723. CHAPTER 4 EVALUATION OF CELERY GERM PLASM FOR RESISTANCE TO FUSARIUM OXYSPORUM F. SP. APII RACE 2, AND THE EFFECT OF HIGH SOIL TEMPERATURES ON THE EXPRESSION OF THE RESISTANCE. 97 98 INTRODUCTION Fusarium oxysporum f. sp. apii (R. Nels. & Sherb.) Snyd. and Hans., the causal agent of Fusarium yellows of celery (Apium gravealens L. var dulce (Mill.) Pers.), was the limiting factor in production of yellow and self- blanching celery cultivars grown during the first half of this century (Nelson et al. 1937, Ryker 1935), but the disease vanished after the introduction of highly resistant green celery cultivars in the 1950's (Opgenorth and Endo 1985). In 1978, Fusarium yellows of celery reappeared in California due to a new race of the fungus (race 2) (Hart and Endo 1978). Race 2 attacked previously resistant green cultivars as well as yellow and self-blanching celery cultivars, whereas race 1 attacked only the yellow and self-blanching cultivars (Schneider and Norelli 1981). F. oxysporum f. sp. apii race 2 had spread to Michigan by 1981 (Elmer and Lacy 1984), and was identified in New York in 1982 (Awuah et al. 1986). Today, F. oxysporum f. sp. apii race 2 is also an important pathogen of celery in Ohio (R. Rowe, personal communication), Wisconsin (Elmer and Lacy 1984), and Texas (Martyn 1987). Fusarium yellows was observed on celery grown in soil from Florida (Toth unpublished), but has not caused important losses in that state. The most common diagnostic symptom of Fusarium yellows is a red to brown discoloration in the vascular tissue of the roots and crowns of infected plants (Awuah et al. 1986, Hart and Endo 1978, Lacy and Elmer 99 1985). The discoloration may extend into the petioles in severely infected plants, and be accompanied by rotting of the central crown area. As the disease progresses, plants become stunted and chlorotic, and severely infected plants wilt and die as the crown rots. Even slightly infected plants are of low quality because they are often stunted and bitter tasting (Awuah et al. 1986, Toth unpublished). I. HOST RESISTANCE Host resistance is the most practical method of controlling F. oxysporum f. sp. apii race 2 (Lorenz and Maynard 1988, Opgenorth and Endo 1985). Celery is generally monocropped, and since few vegetables are as profitable as celery to grow (Lorenz and Maynard 1988, Ryder 1979, Ware and McCollum 1975), commercial growers are reluctant to rotate fields out of celery. Soil fumigation is expensive, and has not yet proven to be a viable means of controlling F. oxysporum f. sp. apii race 2 (Awuah et al. 1986, Greathead 1988, Otto et a1. 1976). Likewise, no commercially available fungicides have controlled this pathogen (Awuah et a1. 1986). Field trials in California (Hart and Endo 1978, Opgenorth and Endo, 1979, 1985), Michigan (Elmer et al. 1986), and New York (Awuah et al. 1986) demonstrated that most yellow and green celery cultivars were susceptible to F. oxysporum f. sp. apii race 2. The green celery cultivars grown today were all derived from a single plant designated number 52-70 selected from the cultivar Crystal Jumbo for resistance to F. oxysporum f. sp. apii race 1 (Opgenorth and Endo 1985). This 100 narrowing of the genetic pool in celery might have been the reason for the nearly uniform susceptibility of celery cultivars to F. oxysporum f. sp. apii race 2. Cultivars with moderate resistance to F. oxysporum f. sp. apii race 2 were discovered (Awuah et al. 1986, Elmer et al. 1986, Hart and Endo 1978, Opgenorth and Endo, 1979, 1985). Tall Utah 52-70 HR and Deacon are two cultivars with moderate resistance to F. oxysporum f. sp. apii race 2 which are recommended for lightly infested fields in Michigan (Elmer et al. 1986). Unfortunately, neither of these cultivars possesses the high level of resistance necessary for growing celery in heavily infested fields. Potential sources of resistance to F. oxysporum f. sp. apii race 2 include celeriac (A. gravealens var. rapaceum) (Awuah et al. 1986, Elmer et al. 1986, Opgenorth and Endo 1979, 1985, Quiros et a1. 1988), parsley (Petroselinum cripsum) (Elmer et a1. 1986), smallage (A. gravealens var. secalinum) (Opgenorth and Endo 1985), and Apium species from other countries (Opgenorth and Endo 1979, 1985). When these plants are crossed with domestic celery cultivars, many undesirable phenotypic characteristics may be inherited along with resistance (Honma and Lacy 1980, Orton et a1. 1984, Opgenorth and Endo 1985). Several rounds of backcrossing and selection are necessary to produce a highly resistant cultivar which is horticulturally acceptable to celery growers. Tissue culture techniques offer an alternative way to induce heritable changes such as disease resistance into existing cultivars (Brettell and Ingram 1979, Daub 1986, Evans et al. 1984, Larkin and Scowcroft 1981, Thorpe 1981). Applying tissue culture to increase 101 disease resistance involves in Vitro cultivation of plant cells under aseptic conditions with the subsequent regeneration of plants (Thorpe 1981). With tissue culture techniques, the preferred characteristics of a cultivar may be maintained while a desired trait is added by mutation, prOtoplast fusion, or gene transfer (Evans et al. 1984, Larkin and Scowcroft 1981, Thorpe 1981). II. TEMPERATURE EFFECTS ON RESISTANCE TO FUSARIUM Environmental factors have a role in mediating the effectiveness of host resistance by placing stress on plants and limiting the energy available for resistance mechanisms (Bruhel 1987). In 1987, an increase in Fusarium yellows severity was observed in some moderately resistant celery cultivars and lines grown in field trials in Michigan. This breakdown in resistance occurred concurrently with higher temperatures during the growing season (Figure 1). Temperature can profoundly affect the expression of host resistance to pathogens, especially in cool weather crops like celery (Ware and McCollum 1975). Michigan celery growers often report a higher incidence of Fusarium yellows in celery harvested during July and August than in earlier and later plantings. In California, moderately resistant celery cultivars are more susceptible to Fusarium yellows when grown during the hotter summer months than during the winter months (Otto et al. 1976). Likewise, moderately resistant celery cultivars Tall Utah 52-70 HR and Tendercrisp produced marketable yields when temperatures were 3 15° C 102 36 -0- :54":- :52; so; 28- 26- 24- 22« 20- 18 ,I A V 1 Y : , 4., 4 , ' I .11- “F uln- ‘F -- di- q!- .1- qt— db 36 34 -- 32‘” \ /°\ \ E) / / Temperature (’0) L 30 - 28- 26 I 241* °\o/° o \ A 22 .. \ 2o; 18 1 1 1 1 1 1 1 1 1 1 1 1 1 I I I I I I I I I I I I j K 45”“ \ 23° / A R 1 / J V CD June July August Figure 1. Mean weekly temperatures for June, July and August during 1986 to 1988 for (A) Paw Paw - Decatur, Michigan, and (B) Hudsonville, Michigan. 103 despite Fusarium yellows infection, but when soil temperatures averaged above 15° C, no plants were harvestable from either cultivar (Endo et al. 1978). Fusarium wilts are generally warm soil diseases, being most destructive at soil temperatures near 28° C, which is the optimum temperature for F. oxysporum growth in culture (Bosland et al. 1988, Bruhel 1987, Clayton 1923, Tisdale 1923, Walker 1941). F. oxysporum f. sp. apii race 1 grew fastest in culture at 28° C (Ryker 1935), and was most destructive on susceptible self-blanching celery cultivars at 28° C (Nelson et a1. 1937, Ryker 1935). F. oxysporum f. sp. lycapersici incited the severest disease on tomatoes at 28° C, and no disease symptoms were observed below 20° C or above 35° C (Clayton 1923). Likewise, soil temperatures near 27° C were most favorable for wilt of watermelons by F. oxysporum f. sp. niveum (Walker 1941), and 24° to 28° C for wilt of flax by F. oxysporum f. sp. lini (Tisdale 1923). Resistance in cabbage to F. oxysporum f. sp. conglutinans race 2 was progressively less effective as soil temperatures increased from 14° to 24° C (Bosland et al. 1988). The amount of disease caused by F. oxysporum f. sp. chrysanthemi on Chrysanthemum cultivars steadily increased as temperatures increased from 24° to 35° C (Gardiner et al. 1987). In this investigation, commercial celery cultivars and experimental lines derived from highly resistant breeding lines, parsley X celery crosses, and somaclones were screened for resistance to F. oxysporum f. sp. apii race 2 in field trials in Michigan. Their Fusarium yellows disease reaction was compared with those of known susceptible and 104 moderately resistant cultivars. A greenhouse study was also undertaken to examine the effects of soil temperature on Fusarium yellows severity in susceptible and moderately resistant celery cultivars. 105 MATERIALS AND METHODS I. FIELD SCREENING Seven celery cultivars and 21 experimental lines were screened for resistance to F. oxysporum f. sp. apii race 2 in replicated field trails at two locations in Michigan from 1986 to 1988. Field trials were held on Willbrandt Farms near Decatur, Michigan in 1986-88, and on Gordon Martinie's farm near Hudsonville, Michigan in 1987-88. All plots were in fields naturally infested with F. oxysporum f. sp. apii race 2. Celery somaclones previously regenerated from cell cultures by Wright and Lacy (1988) were screened for resistance to F. oxysporum f. sp. apii race 2 in the greenhouse in 1986 (Chapter 3). Those somaclones which expressed a high level of resistance to F. oxysporum f. sp. apii race 2 were self-fertilized (Chapter 3) and their progeny screened for resistance in a nonreplicated field trial during 1987. In 1988, 28 breeding lines derived from parsley X celery crosses made by Honma and Lacy (1980) were screened for resistance to F. oxysporum f. sp. apii race 2 in a nonreplicated trial at Hudsonville. Seeds were germinated in potting mix (BacctoO professional planting mix, Michigan Peat Company, Houston, Texas), and then transplanted into flats when 3 to 4 weeks old. Seedlings were grown in the greenhouse under a 16 hr photOperiod and fertilized with 20-20-20 (N-P-R) (Peter'so soluble fertilizer). Plants were trimmed back to 15 cm the day prior to 106 transplanting into field plots to reduce the leaf area for transpiration so the plants would better survive the shock of transplant. Eight-week-old bare-root celery seedlings were transplanted into the field from June 1 to July 2 using equipment supplied by growers. Replicated plots were set up in a complete randomized block design with 4 replications. Each plot consisted of a 4.5 meter row containing 30 plants. Plants were cared for by the growers during the growing season, receiving the same fertilizer, calcium, and pesticide applications as celery grown for production. When plants were mature, 75 to 90 days after transplanting into the field, they were uprooted, crowns cut open, and rated for disease severity. Ten plants from each line and cultivar per replication were rated on a 1 to 5 scale (Figure 2): 1 - no vascular discoloration in root or crown area; 2 - trace of vascular discoloration in root or crown area; 3 - less than 50% crown area discolored; 4 - more than 50% crown area discolored; 5 - dead or nearly dead plant. Mean disease ratings were interpreted as follows: highly resistant - mean disease rating 5 1.9; moderately resistant - 2.0-2.5; moderately susceptible - 2.6-3.5; highly susceptible - mean disease rating > 3.5, Weights of plants from some of the more resistant lines and cultivars were measured to get an approximation of yield. The 10 plants rated for disease severity were trimmed for packing and weighed in the field. The weights were converted to tonnes/hectare (t/ha). Eight of the more promising lines from the 1986 field trial were also rated for some horticultural characteristics. Height from ground level to the tallest point of an average size plant in each replication 107 Figure 2. Fusarium yellows severity rating scale. From left to right: 1 - no vascular discoloration in root or crown area; 2 - trace of vascular discoloration in root or crown area; 3 - less than 50% crown area discolored; 4 - more than 50% crown area discolored; 5 - dead or nearly dead plant. 108 for the 8 lines was measured. The width at the top (first node) and base, and the length from base to first node of 2 petioles on each of 5 plants/line/replication were measured. The width of the widest point at the base of the 5 plants was also recorded. The plants were given a visual rating for the relative abundance of side shoots: l - few, 2 - medium amount, 3 - above average amount. These are characteristics used by other celery researchers to determine horticultural acceptability and superiority of different lines (B. Zangstra personal communication). II. FUNGAL POPULATION STUDY Soil samples were collected from areas in the fields at the Decatur and Hudsonville locations where the trials were planted. Each sample was forced through a sieve with a 2 mm diameter pore size. Two 30 gram portions from each soil sample were air-dried for 2 days. Five grams of air-dried soil samples were stirred into 500 m1 of a 0.5% (w/v) solution of carboxymethyl cellulose for 30 min. One ml aliquots from the soil suspensions were diluted with 9 ml sterile distilled water. A total of 11 l-ml samples were taken from the two soil suspensions from each field. A 5 ml aliquot from each dilution was added to 50 ml molten (50° C) Romada's medium (Romada 1975) and immediately poured into 5 Petri dishes (100 X 15 mm). Plates were examined for Fusarium colonies after 7 days. After 7 days, a few aerial hyphal fragments from colonies which had white aerial mycelia with pink, purple, or orange pigment on Romada's medium were transferred to potato carrot agar (PCA) (Chapter 2). Forty three to 100% of selected colonies on Romada's medium were transferred 109 to PCA, depending on the total number of colonies encountered. Colonies on PCA were examined microscopically to determine which were not F. oxysporum (Nelson et al. 1983). To distinguish F. oxysporum f. sp. apii race 2 colonies from nonpathogenic F. oxysporum colonies, nitrate nonutilizing (nit) mutants were induced for unknown isolates using a technique described by Puhalla (Puhalla 1985). Nit mutants were produced on a potato-sucrose medium containing 15 g RClO3/ liter of medium (Correll et al. 1987, Puhalla 1985). Nit mutants were identified by their thin, but normally expansive, growth and lack of aerial mycelium on a minimal medium containing sodium nitrate as sole nitrogen source (MM) (Puhalla 1985). One to 2 nit mutants from each isolate were paired for 3 weeks on MM with nit mutants from 2 known F. oxysporum f. sp. apii race 2 isolates which consistently pair with all other F. oxysporum f. sp. apii race 2 isolates. Isolates which formed a nitrate-utilizing heterokaryon with either of the F. oxysporum f. sp. apii race 2 nits were identified as F. oxysporum f. sp. apii race 2 (Correll et al. 1986, Ireland and Lacy 1986). Nitrate-utilizing heterokaryons were identified by the presence of wild-type growth with aerial mycelium and pigmentation where the mycelium from two nit mutants intergrew (Correll et al. 1987, Puhalla 1985). II. TEMPERATURE EFFECTS ON DISEASE RESISTANCE Eight week old plants of celery cultivars Tall Utah 52-70 R and Pilgrim (Peto) (susceptible to F. oxysporum f. sp. apii race 2), and Tall Utah 52-70 HR and Pilgrim (MSU) (moderately resistant to F. 110 oxysporum f. sp. apii race 2) were transplanted into containers 19 cm high and 21.5 cm in diameter. Coarse sand was put in the bottom 3 cm of containers which were then filled with muck soil naturally infested with F. oxysporum f. sp. apii race 2. Three 4-week- old seedlings of a single cultivar were transplanted into each container. Four containers (12 plants) were used for each cultivar and temperature. Containers filled with sterile muck were the controls. The plants were placed in temperature tanks at 20°, 25°, 30°, and 35° C under sodium vapor lights set for a 16 hr photoperiod. After 9 to 10 weeks in the temperature tanks, the plants were uprooted and rated for disease severity using the same scale as above. Shoot heights, in cm, of individual plants and total weight, in grams, of all plants were measured. The experiment was repeated once. 111 RESULTS I. FIELD TRIALS The field in which the trials were held near Decatur had a F. oxysporum f. sp. apii race 2 population of approximately 198 propagules per gram of soil (ppg). The field near Hudsonville had a lower population of 126 ppg. This could account for the higher disease ratings obtained at the Decatur site than at the Hudsonville site (Tables 1, 2). Both populations were high enough to cause serious disease in susceptible plants (Elmer and Lacy 1987). Average yields for celery are 60 t/ha, and good yields would be 77 to 79 t/ha (Lorenz and Maynard 1988). The yields in our plots varied from below average to well above average (Tables 3, 4). Since the weight of only 10 plants per replication was converted to yield (t/ha), discrepancies in trimming compared to commercial growers could incorrectly estimate commercial yield. Thus, yield data within the same year and plot can be compared, but care should be taken when comparing yields from different years or plots, or comparing our yield data with commercial celery crops. Celery cultivars Tall Utah 52-70 R and Florida 683 were included as race 2 susceptible checks (Elmer et al. 1986). Both cultivars had highly susceptible disease ratings in all trials (Tables 1, 2). Yields were not measured for these two cultivars since all of the plants were severely stunted or dead. Tall Utah 52-70 HR and Deacon were included 112 Table 1. Disease ratings of celery in F. oxysporum f. sp. apii race 2 field trials in Michigan during 1986 and 1987. Mean disease rating1 1986 198] Line Decatur Hudsonville Decatur MSU 71-288 5.0 a2 Florida 683 4.6 ab 4.0 a 3.9 b MSU 79-75 4.5 ab --- --- Pilgrim (Peto) --- 3.8 b 3.7 Tall Utah 52-70 R 4.4 abc 3.7 c 3.6 MSU 75-93 4.1 bc --- --- MSU 77-170 4.0 bc --- --- FM 1218 --- 3.8 bc 4.1 a Napoleon 3.7 cd --- --- MSU 79-27 3.0 de --- --- MSU 77-144 2.9 de --- --- Tall Utah 52-70 HR 2.9 de 2.5 e 2.9 f MSU 77-7 2.7 ef --- --- MSU 77-106 2.6 ef 3.0 d 3.3 MSU 63-69 2.6 ef 2.4 2.7 g Deacon 2.3 efg 2.5 e 3.0 e Companion 1.9 fg --- --- Pilgrim (MSU) 1.6 g --- --- FM 1217 1.6 2.9 d 2.8 fg MSU 74-70 1.5 1.9 g 2.4 h U0 1 --- 1.8 g 2.2 i 1 Disease ratings were based on a 1 to 5 scale: 1 - no vascular discoloration; 2 - trace of vascular discoloration in root or crown area; 3 - less than 50% crown area discolored; 4 - more than 50% crown area discolored, plant stunted; 5 - dead or nearly dead plant. 2 Mean of 10 plants from each of 4 replications. Within columns, all means followed by the same letter were not significantly different (P - 0.05) according to Duncan's multiple range test. ratings < 2.5 were considered resistant; those with ratings > 2.5 susceptible. Lines with disease 113 Table 2. Disease ratings of celery in F. oxysporum f. sp. apii race 2 field trials in Michigan during 1988. Mean disease rating1 Line Hudsonville Decatur Florida 683 4.1 a2 4.3 a Tall Utah 52-70 R --- 4.3 a Pilgrim (Peto) 3.4 b --- Deacon 2.7 c 3.1 b SS 7083 2.2 d 2.2 d Tall Utah 52-70 HR 2.2 d 3.1 b Pilgrim (MSU) 2.0 e --- Bosco 1.9 e 1.7 ef SS 7074 1.8 e 1.7 ef SS 7073 1.5 f 1.7 ef SS 7078 1.5 f 1.6 SS 7068 1.4 f 2.3 c MSU 74-70 1.4 f 1.8 e SS 7067 1.4 f 1.8 e SS 7076 1.4 f --- 1 Disease ratings were based on a 1 to 5 scale: 1 - no vascular discoloration; 2 - trace of vascular discoloration in root or crown area; 3 - less than 50% crown area discolored; 4 - more than 50% crown area discolored, plant stunted; 5 - dead or nearly dead plant. 2 Mean of 10 plants from each of 4 replications. Within a column, all means followed by the same letter were not significantly different (P - 0.05) according to Duncan's multiple range test. Lines with mean disease ratings less than 2.5 are considered resistant; those with ratings greater than 2.5 susceptible. 114 Table 3. Yields of celery lines in 1986 and 1987 Michigan celery field trials screening for resistance to F. oxysporum f. sp. apii race 2. Yield (t/ ha)1 1986 1987 Line Decatur Hudsonville Decatur Tall Utah 52-70 HR 51.52 bc 55.2 a 97.0 a Companion ' 47.8 c ---- ---- Pilgrim (MSU) 67.4 a ---- ---- Deacon 50.6 bc 47.0 b 91.3 a MSU 63-69 52.0 bc 40.8 be 83.9 a MSU 74-70 57.5 b 46.1 b 92.4 a FM 1217 59.4 b 34.9 c 85.4 a MSU 77-7 48.3 c ---- ---- UC-l ---- 55.7 a 105.2 a 1 Mean yield of 4 replications converted to tonnes per hectare. 2 Trimmed weights for 10 plants per replication. Within a column, all means followed by the same letter were not significantly different (P - 0.05) according to Duncan's multiple range test. 115’ Table 4. Yields of celery lines in 1988 Michigan celery field trials screening for resistance to F. oxysporum f. sp. apii race 2. Yield (c/ ha)1 Line Hudsonville Decatur Tall Utah 52-70 HR 54.4 2'bcd 28.5 c Deacon 54.0 bcd 51.7 a Pilgrim (MSU) 63.8 abc ---- MSU 74-70 63.8 abc 52.2 a Bosco 64.0 abc 50.6 a SS 7067 64.5 ab 46.6 ab SS 7068 69.9 a 48.9 ab SS 7073 57.2 be 46.2 ab SS 7074 56.2 bc 42.8 ab SS 7076 44.9 d ---- SS 7078 54.3 bcd 37.2 bc SS 7083 53.4 cd 43.3 ab 1 Mean yield of 4 replications converted to tonnes per hectare. 2 Yield of 10 plants/ replication. Within a column, all means followed by the same letter were not significantly different (P - 0.05) according to Duncan's multiple range test. 116 as moderately resistant controls (Elmer et al. 1986). The mean disease ratings for Deacon planted at Decatur increased from moderately resistant in 1986 to moderately susceptible for 1987 and 1988 (Tables 1, 2). Mean disease ratings for Tall Utah 52-70 HR planted in Decatur were consistently in the susceptible range from 1986 to 1988 (Tables 1, 2). Both cultivars had lower disease ratings in the Hudsonville trials than in the Decatur trials (Tables 1, 2). Deacon and Tall Utah 52-70 HR had significantly lower disease ratings than susceptible Florida 683 and Tall Utah 52-70 R in all trials (P - 0.05) (Tables 1, 2). Deacon and Tall Utah 52-70 HR had average to good yields compared to other lines within the same plots and years (Tables 3, 4). The low yield for Tall Utah 52-70 HR at Decatur in 1988 (Table 4) corresponded with a higher disease rating (Table 2). Since stunting is one symptom of Fusarium yellows, this would not be unexpected. Deacon and Tall Utah 52-70 HR were not as tall as some of the other lines tested, but they had wider petioles and less side shoot development (Table 5) Pilgrim (MSU) and Companion cultivars were developed by Honma et al. (1986) (Michigan State University) for slow flowering and resistance to Cercospora apii, respectively. Both cultivars contained celeriac parents in their pedigrees. In the 1986 Decatur trial Pilgrim (MSU) and Companion had disease ratings significantly lower than that for Tall Utah 52-70 HR, but not lower than Deacon (P - 0.05) (Table 1). Although Pilgrim (MSU) and Companion had similar ratings for horticultural characteristics (Table 5), Pilgrim (MSU) had a significantly higher yield than Companion (Table 3). Pilgrim (MSU) also had a better field appearance with a full heart and tall uniform petioles. 117 Table 5. Results of ratings for horticultural characteristics for celery cultivar trials in Decatur, Michigan in 1986. Plant1 ,Eg5121§_flidgh Petiole Base Shoot2 Line height Top Bottom length diameter rating Tall Utah 52-70 HR 65.83 2.0 3.3 23.3 6.5 1.3 Pilgrim 68.8 1.5 2.5 30.8 7.8 2.8 Companion 63.3 1.5 2.5 27.0 6.3 2.8 Deacon 68.8 1.8 2.8 24.5 6.5 2.0 Ferry Morse 1217 72.0 1.5 2.3 24.0 6.8 2.7 74-70 79.5 1.8 2.8 31.0 7.0 3.0 63-69 68.3 1.8 2.5 27.0 6.5 2.8 77-7 66.3 1.8 2.8 27.0 6.5 3.0 LSD (P - 0.05) 6.3 0.3 NS 3.3 0.8 0.7 1 Plant height, petiole width, petiole length, and base diameter are expressed in centimeters. Mean of 5 plants/line for each of 4 replications. 2Side shoot rating: 1- few, 2- medium amount, 3- above average amount. 118 Pilgrim (Peto) was a seed increase of Pilgrim (MSU) which was first available for the 1987 growing season. Pilgrim (Peto) had a highly susceptible disease rating in both trials in 1987 (Table 1). Pilgrim (Peto) was a mixture of horticultural types; plants had different leaflet shapes and growth habits not observed in Pilgrim (MSU). Both lines were included in the 1988 field trial at Hudsonville, and Pilgrim (MSU) had a significantly lower disease rating than Pilgrim (Peto) (Table 2). FM 1217 and FM 1218 were experimental lines developed by Ferry Morse Seed Company (San Juan Bautista, California). In 1986, FM 1217 was significantly more resistant than Tall Utah 52-70 HR, but in 1987, FM 1217 had a disease rating in Hudsonville which was significantly higher than for Tall Utah 52-70 HR and Deacon, and a rating in Decatur not different from Tall Utah 52-70 HR and Deacon (Table 1). FM 1218, which was released in 1988 under the name Hercules (Anon. 1988), was rated as highly susceptible to F. oxysporum f. sp. apii race 2 in the 1987 field trials (Table 1). Napoleon was released by Scattini Seeds (Salinas, California) for commercial production in 1986, and was highly susceptible disease in the 1986 field trial at Decatur (Table 1). Of the 11 experimental lines (MSU number lines in Table 1) developed by Honma and screened for resistance in 1986, only MSU 74-70 had a highly resistant disease rating (Table 1). The other 10 MSU lines had susceptible disease ratings and were segregating for disease resistance, height, and other horticultural characteristics. MSU 74-70 was also included in the 1987 and 1988 field trials, where it continued to have highly to moderately resistant disease ratings (Tables 1, 2). MSU 74-70 119 had average to good yields in each plot compared to other lines measured (Tables 3, 4). MSU 74-70 is a tall line with average petiole widths, but has a tendency towards numerous side shoots (Table 5). Line UC-l was a breeding line released by Orton et al. (1984) (University of California at Davis) because of its high level of resistance to F. oxysporum f. sp. apii race 2. The parentage of UC-l includes celeriac accessions, celery cultivars Tall Utah 52-70 R and Tendercrisp, and resistant introductions from China. UC-l had the lowest mean disease rating for all the lines tested at both locations (Table l) and the highest yields in both trials for 1987 (Table 3). UC- l was a tall vigorous line, but with thin, hollow petioles and numerous side shoots. Bosco, an experimental line from Brinker Oresetti Seed Company (Watsonville, California), had mean disease ratings of 1.92 and 1.70 in the 1988 Hudsonville and Decatur trials, respectively (Table 2). Bosco was a good line horticulturally, except for its tendency to flower early. Bosco produced one of the higher yields in Hudsonville and Decatur in 1988 (Table 4). The 88 lines screened in 1988 (Table 2) were experimental lines from SunSeeds (Hollister, California). Six of the 7 lines had moderately resistant disease ratings (Table 2). Although yields of 6 SS lines were not significantly different from the yields of Tall Utah 52-70 HR and Deacon in the Hudsonville plot, and Deacon in the Decatur trial (Table 4), they were horticulturally unacceptable lines. The plants were 120 taller than average, but with thin petioles which were hollow and brittle. The plants also had an open growth habit which looked wild and made them difficult to harvest. The celery X parsley crosses were an attempt to transfer resistance to late blight caused by Septoria apiicola from parsley into celery (Honma and Lacy 1980). Parsley is immune to S. apiicola and to F. oxysporum f. sp. apii race 2. Twenty of the 28 lines derived from celery X parsley crosses had highly susceptible disease ratings (Table 6). Only 3 lines (803, 808, and 835) had moderately resistant disease ratings (Table 6). Of these, line 808 most resembled celery in morphology and growth habit. Lines 803 and 835 resembled the parsley parentage more. These three lines will be examined again in future field trials. The somaclones whose progenies were screened for resistance were regenerated from cell cultures of Tall Utah 52-70 HR (Wright and Lacy 1988). None of the 15 lines screened in 1987 had mean disease ratings significantly less than the rating for Tall Utah 52-70 HR plants from commercial seed (Table 7). Lines J 4, J 7, J 8, J 9, J 12, J 20 and J 21 had individual plants which had no vascular discoloration (rating of 1.0). These plants were saved for seed production. None of the plants of Tall Utah 52-70 HR from commercial seed had disease ratings < 2.0. The progenies from two plants of the J 12 line were tested in a nonreplicated plot in 1988, but the lines were only moderately resistant. The progenies from the rest of the plants will be screened in a field trial during 1989. 121 Table 6. Disease ratings for lines derived from celery X parsley crosses and screened in a nonreplicated trial for resistance to F. oxysporum f. sp. apii race 2 at Hudsonville, Michigan in 1988. Line Disease rating1 Line Disease rating 801 4.5 802 3.3 803 2.3 804 4.0 805 4.5 806 4.0 808 2.3 809 3.4 810 2.6 811 2.5 813 4.0 814 5.0 815 5.0 816 5.0 817 4.5 818 5.0 820 5.0 821 5.0 822 5.0 824 5.0 825 5.0 826 5.0 827 5.0 828 4.5 829 5.0 834 3.3 835 2.0 837 5.0 1 Plants were rated on a l to 5 class scale: 1 - no vascular discoloration in root or crown area; 2 - trace of vascular discoloration in root or crown area; 3 - less than 50% crown area discolored; 4 - more than 50% crown area discolored; 5 - dead or nearly dead plant. Mean rating for 10 plants/line. 122 Table 7. Disease ratings for somaclonal progenies screened for resistance to F. oxysporum f. sp. apii race 2 at Decatur, Michigan in 1987. Line Disease rating1 Line Disease rating J 10 3.8 * J 1 3.4 * J 19 3.3 * J 14 3.3 J 22 3.3 J 13 3.1 J 21 ** 3.1 J 9 ** 3.0 J 17 2.9 J 8 ** 2.9 J 5 2.9 J 20 ** 2.8 J 4 ** 2.8 J 7 ** 2.7 J 12 ** 2.6 Tall Utah 52-70 BR 2.9 LSD (P - 0.05) 0.42 1 Plants were rated on a l to 5 class scale: 1 - no vascular discoloration in root or crown area; 2 - trace of vascular discoloration in root or crown area; 3 - less than 50% crown area discolored; 4 - more than 50% crown area discolored; 5 - dead or nearly dead plant. Mean rating of 20 plants/line. * Mean disease rating significantly different from Tall Utah 52-70 HR, the cultivar somaclone parents were derived from. ** Lines from which plants were saved for seed production. 123 II. TEMPERATURE EFFECTS ON RESISTANCE The greenhouse temperature study was an attempt to determine if the increased severity of disease observed in moderately resistant lines in the 1987 field trials were the result of higher temperatures experienced during 1987 compared to 1986 (Figure l). Fusarium yellows severity did not change significantly as temperatures increased from 20° to 35° C for all cultivars tested (P - 0.05) (Figures 3, 4). A trend towards increased disease ratings in Tall Utah 52-70 HR at 35° C and decreased disease ratings in Tall Utah 52-70 R at 30° and 35° C in the first study could not be repeated in the second study (Figure 3). Inoculated and uninoculated plants from celery cultivars Pilgrim (Peto), Tall Utah 52-70 HR, and Tall Utah 52-70 R grew tallest at 20° and 25° C, and growth significantly declined between 25° and 30° C (P - 0.05) (Figure 5). Although the slopes of the regression lines for all cultivars tested in the first test were significantly different from zero (P - 0.01), the relationships were nonlinear (P - 0.01). The weights for both inoculated and uninoculated plants of all three cultivars reflected the decrease in shoot height (Figure 6). The same trend in height and weight was observed in the second test, although data were not collected. Root growth corresponded to shoot growth for all cultivars tested. Plants grown at 20° C had roots which grew to the bottom of the containers (approximately 15 cm from the soil surface). Roots at 25° C had grown at least into the sand (within 3 cm of the container bottom). At 30° C some, but not all, roots were near the sand, and at 35° C the roots were only within the top 4 cm of the soil. The roots of plants 124 Figure 3. Mean Fusarium yellows severity ratings for moderately resistant celery cultivar Tall Utah 52-70 HR and susceptible cultivar Tall Utah 52-70 R grown at different soil temperatures. Plants were rated on a 1 to 5 class scale: 1 - no vascular discoloration in root or crown area; 2 - trace of vascular discoloration in root or crown area; 3 - less than 50% crown area discolored; 4 - more than 50% crown area discolored; 5 - dead or nearly dead plant. 125 WW .1 ®\\\\\.. \\\\\\\\\\\\\\\.- W \V\\\x. _ § mcsom mmomma :82 4o 25 30 35 Temperature (0C) 20 15 Figure 3. 126 [:1 Pilgrim El Pilgrim IXXI Pilgrim Peto second 8 MSU {Peta first stud III/[WM {udy second study ’A.‘.A’A‘ . O'OTOTO‘M .9 saw 9 O . ' . . 909.909.? C O 5 03 .E 44 .4.) O L (D .. m 3 O 8 '6 2‘1 c 0 <1) 1-- 2 O 15 Figure 4. class scale: I 20 81 Temperature (0C) Mean Fusarium yellows severity ratings for celery cultivars Pilgrim (Peto) (susceptible) and Pilgrim (MSU) (moderately resistant) grown at different soil temperatures. Plants were rated on a 1 to 5 l - no vascular discoloration in root or crown area; 2 - 4O trace of vascular discoloration in root or crown area; 3 - less than 50% crown area discolored; 4 - more than 50% crown area discolored; 5 - dead or nearly dead plant. 127 30 O ‘ O-O Pilgrim (Peta) >