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Rizal...‘ a 02.3.3.3 I . ‘ 3.11. -0 ¥¥¥¥¥¥¥ 4‘49 UBRARY Michigan State University This is to certify that the dissertation entitled The use of dimethomorph in the control of potato late blight (Phytophthora infestans): Sensitivity survey, insensitivity generation, and field optimization. presented by Jeffrey Michael Stein has been accepted towards fulfillment of the requirements for PhJ)- degree in Writ Pathology MAMM Major professor Date jut? 10!}50A M5 U is an Affirmative Action/Equal Opportunity Institution 0-12771 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 c;/CIRC/DaleDue.p65-p.15 THE USE OF DIMETHOMORPH IN THE CONTROL OF POTATO LATE BLIGHT (PHYTOPHTHORA INFESTANS): SENSITIVITY SURVEY, INSENSITIVITY GENERATION, AND FIELD OPTIMIZATION. BY Jeffrey Michael Stein 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 2002 5"! bis - "- \rlbu -... ~§‘§ .l...‘ m I) I7. ABSTRACT THE USE OF DIMETHOMORPH IN THE CONTROL OF POTATO LATE BLIGHT (PHYTOPHTHORA INFESTANS): SENSITIVITY SURVEY, INSENSITIVITY GENERATION, AND FIELD OPTIMIZATION. BY Jeffrey Michael Stein The sensitivities of 11 isolates of Phytophthora infestans to dimethomorph at all stages of the asexual life cycle and when inoculated onto potato leaf disks were examined. Once the sensitivities were determined, the generation of dimethomorph insensitive strains of It infestans was attempted using ethidium bromide and UV light mutagenesis and repeated culturing on amended media. The results from these, and previously reported trials, were used to develop and examine several aspects concerning the field use of dimethomorph for the control of foliar and tuber potato late blight. Zoospore encystment and cystospore germination were highly inhibited at leaf disks was most effective at inhibiting symptom development when applied before inoculation. Dimethomorph inhibited sporulation from leaf disks at concentrations >10.0 ug mlfl, regardless of application timing. The generation of nmderately CUnethomorph insensitive strains of P. infestans using both ethidium bromide and UV mutagenesis and repeated. culturing' on sub—lethal amended media was successful. Ethidium bromide and UV mutagenesis created strains with significantly higher Ergo values of in vitro growth on dimethomorph amended media than sub-lethal culturing, but with lower fitness on un—amended media. Strains created. by sub-lethal culturing occasionally' had reduced virulence on both leaf disks and in whole tubers. Rate reduction. of the jpre—mixed. commercial mancozeb 1' week.’1 was as and dimethomorph fungicide to 1.34 kg ha" effective: at controlling foliar late Iblight. as the full rate program, 1.74 kg ha"1 week%. When examined as components of a season—long chlorothalonil program, none of the alternative dimethomorph mixture partner fungicides was significantly more effective at controlling foliar or tuber late blight than the others. Reduced rate dimethomorph and pyraclostrobin, and pyraclostrobin alone, programs offered effective foliar and tuber blight control. A process was developed that produced late blight usage recommendations for‘ dimethomorph. using isolate sensitivity, resistance development, and field evaluations. Dedicated to: My parents, who have always supported, encouraged, and respected my choices and to my wife Kimberly, from whom I have learned so much. iv a .5“ u “a on.“ g ACKNOWLEDGEMENTS I would like to express my extreme gratitude to the members of my committee: Drs. Willie Kirk, Ray Hammerschmidt, Andy Jarosz, and Joe vargas, my fellow lab mates, especially Robert Schafer, Brandon Dunlap, and Sarah Shaw for countless hours of assistance, and BASF (was American Cyanamid), who fully funded my research. Other people that deserve appreciation are the staff at the MSU Muck Farm, especially Ron Gnagy, the folks involved in the Michigan Potato Industry, who have been extremely supportive of our program, and my fellow graduate students in the department, with whom I was able discuss my research and de—stress. To :my family' and friends, while they' may' not have understood what I kept doing in school, were always willing to learn about potatoes and talk about what was next. Finally, I would like to express my appreciation to Michigan State University and the Department of Botany and Plant Pathology for allowing me to have such a wonderful academic experience, especially all of the Professors and Teaching Assistants who taught me so much and guided my education. "Vv‘ I. v... ‘-\‘l J‘.‘ TABLE OF CONTENTS LIST OF TABLES ........................................ LIST OF FIGURES ....................................... KEY TO SYMBOLS AND ABREVIATIONS ....................... CHAPTER ONE LITERATURE REVIEW ..................................... Introduction ..................................... The Host .................................... The Pathogen ................................ The Disease ................................. Fungicidal Control of Phytophthora infestans ..... Definitions and Fungicide Groups ............ Fungicide Development for Commercial Release ..................................... Contemporary Fungicide Usage for Potato Late Blight Control .............................. Non—systemic Fungicides Currently In Use.... Systemic Fungicides Currently In Use ........ Future Fungicides ........................... Research Objectives .............................. CHAPTER TWO DIMETHOMORPH SENSITIVITY IN PHYTOPHTHORA INFESTANS.... Abstract ......................................... Introduction ..................................... Materials and Methods ............................ Preparation of Fungicide Stock Solutions and Amended Media ............................... Production of Viable Sporangia .............. Inhibition of Hyphal Growth and Sporulation in Vitro .................................... Inhibition of Direct and Indirect Germination ................................. Isolate Sensitivity Ranking and Correlations of the Asexual Life Cycle Stages ............ Protectant, Curative, and Antisporulation Activity of Dimethomorph in vivo ............ Results .......................................... Discussion ....................................... vi viii xii LOKOONLAJl-‘HH 15 17 22 27 36 37 38 38 39 41 41 42 42 45 46 47 48 7O ,“va '5 v b...“ ”Q." ,. u goo-J W O... 7""— , H ,- gola 0-- CHAPTER THREE THE GENERATION, QUANTIFICATION, AND CHARACTERIZATION OF DIMETHOMORPH INSENSITIVITY IN PHYTOPHTHORA INFESTANS AND OTHER PHYTOPHTHORA SPECIES .............. Abstract ......................................... Introduction ..................................... Materials and Methods ............................ Media Preparation ........................... Generation of Insensitivity: UV Mutagenesis. Generation of Insensitivity: Sub-Lethal Culturing ................................... Dimethomorph Sensitivity Assays ............. Virulence Assays ............................ Results .......................................... Discussion ....................................... CHAPTER FOUR FIELD OPTIMIZATION OF DIMETHOMORPH FOR THE CONTROL OF PHYTOPHTHORA INFESTANS ON POTATO: APPLICATION RATE, INTERVAL, AND MIXTURE PARTNERS ........................ Abstract ......................................... Introduction ..................................... Materials and Methods ............................ Experimental Design and Agronomic Management .................................. Inoculation ................................. Data Collection and Metric Generation ....... Data Analysis ............................... Fungicide Trials ............................ Results .......................................... Dimethomorph/Mancozeb Pun}; Manipulation (DMR) Trial ................................. Dimethomorph/Mancozeb Rate and Interval Manipulation (DMRI) Trial ................... Dimethomorph Mixture Partner and Rate Manipulation (DMP) Trial .................... Dimethomorph/Pyraclostrobin Rate Manipulation (DSR) Trial .................... Discussion ....................................... CHAPTER FIVE SUMMARY AND CONCLUSIONS ............................... LITERATURE CITED ...................................... vii 75 75 76 78 78 78 79 81 82 84 100 106 106 107 110 110 111 112 113 114 115 115 118 122 126 131 134 139 q d .5 .3 r . 3. .mu. 5.. ul VXV. ”in .,I\ ant“ .... H D a a; 1. AV 0. wFU . g F“ .3 a .7. T v i 1,. . .2 s . u . 1nd 2:. Y; .Q l . «a .. ¢ 0.. Dr» W. T . . .I P w . C C Cc . . a C. .D E H . as .. I In no a D. E n .Q .Q . O E t. S G. i\ 51 :l D. C . L "wt NM. H \ . ad. 9 C LIST OF TABLES Table 1. P. infestans Isolate ID, mating type, genotype, and State in which isolated (U.S.A.) ........ Table 2. The effective concentration of dimethomorph (pg ml'l) required for a 50% reduction of in vitro hyphal growth (colony diameter) and Sporulation for P. infestans isolates .................................... Table 3. The effective concentration of dimethomorph (pg ml’l) required for a 50% reduction of zoospore encystment, cystospore germination, and direct sporangia germination for P. infestans isolates ....... Table 4. Median rank of isolate sensitivity when EC“) values were ranked within hyphal growth, Sporulation (in Vitro), direct sporangia germination, zoospore encystment, and cystospore germination and compared with an analysis of variance on ranks ................. Table 5. Correlation of the stages of the asexual life cycle of P. infestans examined and the P-value for each, excluding zoosporogenesis, analyzed with Pearson's Product Moment Correlation .................. Table 6. Isolate identification code, E&wtophthora species, mating type (if applicable), P. infestans genotype (if applicable), and State in which isolated (U.S.A.) .............................................. Table 7. Calculated Ergo for in vitro hyphal growth (diameter) and resistance factors for all strains of P. infestans isolates examined. Statistical comparisons shown within each isolate using Fischer’s LDS (0,: 0.05) ....................................... Table 8. Calculated Ergo for .hi Vitro hyphal growth (diameter) and resistance factor, for all strains of P. capcisi (Pcap), P. cactorum (Pcac), and It erythroseptica (Pe) isolates examined. Statistical comparisons shown within each isolate using Fischer's LDS (a,= 0.05) ....................................... viii 43 53 58 60 61 80 93 96 n... a f L .. O F. A .v C . I a :. nal‘ “Vs o. h» ...... mi. .2 a My; ur‘ ct. « Hus ..‘ av a FL Table 9. Percent of infection by P. infestans isolates and strains, expressed as percent incidence, when inoculated onto both excised leaf disks and into whole tubers (>4.0 cm, <6.5 cm). Statistical comparisons shown within each isolate using Fischer’s LSD (a = 0.05) ....................................... Table 10 (a-b). The effect of increasing, decreasing and maintaining a constant rate of active ingredient of mancozeb / dimethomorph fungicide programs on (a) final foliar late blight, disease progress, yield, (b) infected marketable tubers plantd, marketable tubers plant'l, and mean mass (g tuber'l) for 1999 and 2001 combined. Statistical comparisons performed using Fischer’s LSD (a = 0.05) ............................. Table 11 (a-b). The effect of increasing, decreasing and maintaining a constant rate of active ingredient of mancozeb / dimethomorph fungicide programs with a five or seven day application interval on (a) final foliar late blight, disease progress, yield, (b) infected marketable tubers plantd, marketable tubers plant'l, and mean mass (9 tuber'l) for 2000 and 2001 combined. Statistical comparisons performed using Fischer’s LSD (a = 0.05) ............................. Table 12 (a-b). The effect of dimethomorph at two rates when tank~mixed with chlorothalonil, mancozeb, or metiram and incorporated into a season—long chlorothalonil program on (a) final foliar late blight, disease progress, yield, (b) infected marketable tubers plant4, marketable tubers plantJ; and mean mass (g tuberfl) for 2000 and 2001 combined. Statistical comparisons performed using Fischer's LSD (a = 0.05) ........................................... Table 13 (a-b). The effect of dimethomorph and pyraclostrobin at varying rates when incorporated into a season-long chlorothalonil program on (a) final foliar late blight, disease progress, yield, (b) infected marketable tubers plantd, marketable tubers plant’l, and mean mass (g tuber”) for 2001 . Statistical comparisons performed using Fischer’s LSD (a = 0.05) ........................................... ix 98 116 119 123 127 LIST OF FIGURES Figure 1 (a-k). The percent inhibition, relative to the untreated. control, of in Vitro Zhyphal growth, Sporulation, and direct sporangia germination, for P. infes tans vs . dimethomorph concentration (0 . 0 , 0 . 01 , 0 . 1 , 1 . 0 , and 10. 0 ug ml‘l) . Error bars represent Fischer’s LSD (a = 0 05) ............................. Figure 2 HrJU.. The percent inhibition, relative to the untreated control, of in Vitro zoosporogenesis, zoospore encystment, and cystospore germination for P. infestans vs. dimethomorph concentration (0.0, 0.01, 0.1, 1.0, and 10.0 1.1g ml'l). Error bars represent Fischer’s LSD (a = 0.05) ............................. Figure 3 (a—k). The percent inhibition, relative to the untreated control, of the incidence of leaf disk symptom development vs. dimethomorph concentration (0.0, 1.0, 10.0, 100.0, and 1000.0 0g mi‘l). Dimethomorph was applied as a commercial formulated product (Acrobat 50WP, BASF Corporation) at 48 or 24 hours before inoculation (HBI) or 24 or 48 hours after inoculation (HAI) by a sporangia / zoospore suspension of P. infestans. Error bars represent Fischer’s LSD (on = 0.05) ........................................... Figure 4 (a—k). The percent inhibition, relative to the untreated control, for P. infestans Sporulation from inoculated leaf disks vs. dimethomorph concentration (0.0, JHCL 10.0, 100.0, and 1000.0 pg ml”). Dimethomorph was applied as a commercial formulated product (Acrobat 50WP, BASF Corporation) at 48 or 24 hours before inoculation (HBI) or 24 or 48 hours after inoculation (HAI) by a sporangia / zoospore suspension of P. infestans. Error bars represent Fischer’s LSD (a = 0.05) ................... 49 55 63 67 m D. a S .m C A: .W I ’v \ “ $1 a 4.. r. :. ”Ms A, AU P Figure 5. Representative colony growth, relative to the untreated control, in diameter (mm) day‘l vs. cycle number for isolate Pi213 when grown on rye B media amended with 0.0 or 1.0 ug ml‘1 dimethomorph. Error bars represent one standard deviation for replicate plates ...................................... Figure 6 (a-k). Final colony diameter of P. infestans isolates for the wild—type strains (0), repeated culturing control strains (D), repeated culturing on 1.0 pg mflfl dimethomorph amended media strains (A), and UV / ethidium bromide generated strains (0) at 11 days after inoculation. Isolates were grown on rye B media amended with 0.0, 0.1, 1.0, and 10.0 [lg ml"1 dimethomorph. Error bars represent Fischer’s LSD (a = 0.05) ............................................... Figure 7 (a-e). Final colony diameter at 5 days after inoculation. of the ‘wild—type strains (0), repeated culturing control strains (D), repeated culturing on 1.0 ug m1"l dimethomorph amended media strains (A), and fast growing sector of P. erythroseptica that spontaneously appeared (O) for Pu capcisi (Pcap), P. cactorum (Pcac), or P. erythroseptica (Pe). Isolates were grown on rye B media amended with 0.0, 0.1, 1.0, and 10.0 ug m1“l dimethomorph. Error bars represent Fischer’s LSD (a = 0.05) ............................. Figure 8. Diagram of the general steps required from the public release of a novel fungicide to the optimization. of time commercial product ‘under field conditions, and the parameters examined at each step.. xi 85 87 91 135 ‘1‘ “i H-- W.‘ .- ~ ‘2‘ ANOVA CT DAI DAP diHZO DNA EBDC ECso FCD FFLB HBI LSD PASS RAUDPC RF SL USD WT KEY TO SYMBOLS AND ABBREVIATIONS Active Ingredient(s) Analysis Of Variance Control Days After Inoculation Days After Planting De—ionized Hg) Deoxyribonucleic Acid EthyleneBis(DithioCarbamates) Effective Concentration for a 50% reduction Final Colony Diameter Final Foliar Late Blight Hours After Inoculation Hours Before Inoculation Least Significant Difference Pivot—Attached Spray System Relative Area Under the Disease Progress Curve Resistance Factor Ribonucleic Acid Sub-Lethal U.S. Dollars Ultraviolet light Wild—type xii T“)!— y. 4“ a- V?!" UV 6"— q an“. () C) 01’ " U PL." ““- fiv.‘ DMR FR HF RD RI RP UC DMRI DMP CHL CHLDl CHLD2 CTL MADl MAD2 MEDl MED2 Dimethomorph / Mancozeb Rate trial Full Rate, season-long Half of Full rate, season-long Rate Decreasing through season Rate Increasing through season Rate Peaking at mid-season Untreated Control Dimethomorph / Mancozeb Rate and Interval trial untreated Control rate Decreasing through season Full Rate season—long Lowest Rate season-long rate increasing (Up) through season Dimethomorph Mixture Partner trial Chlorothalonil, season—long Chlorothalonil / Dimethomorph partial rate Chlorothalonil / Dimethomorph partial rate untreated Control Mancozeb / Dimethomorph partial rate Mancozeb / Dimethomorph full rate Metiram / Dimethomorph partial rate Metiram / Dimethomorph full rate xiii DSR P25 P50 P75 P100 PD25 PDSO PD75 PDlOO Dimethomorph / Pyraclostrobin Pyraclostrobin Pyraclostrobin Pyraclostrobin Pyraclostrobin Pyraclostrobin Pyraclostrobin Pyraclostrobin Pyraclostrobin Rate trial 25% of full rate 50% of full rate 75% of full rate at full rate / Dimethomorph 25% of full rate / Dimethomorph 50% of full rate / Dimethomorph 75% of full rate / Dimethomorph at full rate xiv The H05‘ 1""... 5 s4! ‘ . fififiqy. ' 1 ‘gnLL/V‘L. . . crop L: as £000 i?“ ‘77\ “"1“. I I 0". ww- . LV‘A¢-“E 7r -- I . .0», I: J.( #2 «vim ' “h 18 QQH ~u 0 n CHAPTER ONE LITERATURE REVIEW Introduction The Host The potato (Solanum tuberosum L.) is the most important dicotyledonous and fourth most important food crop in the world (127). Potatoes are consumed by humans as food, fermented for alcohol, processed for starch, used as animal fodder, have a relatively high nutritional value (32,127), and grown in both developed and developing countries. In 2001, world production of potatoes was over 300 million metric tons (48). In 2000, Michigan growers cultivated approximately 20,000 hectares of which about 70% was used for chipping, representing a $100 (USD) million annual market (88). Potato cultivation, both commercial and for home gardens, occurs in a greater variety of environments and locations than any other food crop except maize (74). The cultivated potato is part of a complex consisting of diploid, triploid and tetraploid forms. The tetraploid form is separated into two subspecies (69): S. tuberosum ssp. andigena for those cultivated. in the .Andes and S. tuberosum ssp. tuberosum for those cultivated elsewhere in ".‘ the world (108). Potatoes are believed to have been introduced into Europe from the region now known as Chile and spread. to the rest of the *world from. Europe (44). Potatoes have been adapted to a variety of climates and selected or bred to flower and produce tubers regardless of day length (day neutral) or according to photoperiod and during long day conditions. The cultivation of potatoes in regions with weather extremes necessitates the ability to store fresh market and processing potatoes for up to one year at 7-10°C and 90% relative humidity (106). Crops are regenerated from “seed” stored between seasons near 3°C, which inhibits sprout development. Seed pieces are small intact tubers or larger tubers that are sectioned mechanically to multiply the amount of planting material with eyes (sprouts). Following cutting, seed pieces are generally stored at 7—100C prior to planting to allow for suberization of the cut surface and may be treated with powdered tree bark and/or fungicides to ahd in healing and reduce the risk of pathogen infection. The conditions and practices employed to store seed tubers enhance survival of tuber—borne pathogens between growing seasons and promote spread to previously uninfected seed pieces during cutting and after planting (83,92,93,113). The Path< . .‘ f-Q bu. h” e t an". .n6 RIP "n filogva ”a“‘ “P \— U ‘ A ya 88.; G OCIO :n '0 the A 54. FY 3- “Gen ,Qy. V» The Pathogen The causal agent of potato late blight, Phytophthora infestans (Mont.) de Bary, infects both foliar and tuber tissues of the potato. P. infestans will also infect a few related species within the family Solanaceae, such as tomato (Lycopersion esculentum Mill.) and pear melon (Solanum muricatum Ait). The parasitic nature of P. infestans ‘was not easily' defined. because the jpathogenic characteristics closely matched those of an obligate biotroph (46) including: Sporulation from asymptomatic green tissue as well as lesions, a narrow host range, and intercellular hyphae and haustoria-like structures. In contrast, saprophytic growth occurs on relatively simple defined media, a characteristic of facultative biotrophic pathogens. Compared txD other Phytophthora species, the saprophytic ability of P. infestans was minimal (46). The type of parasitism of P. infestans was considered to be of the hemibiotroph type in which the initial interaction between the pathogen and the host results in a delay in cell death until a large degree of infection has occurred (25). The asexual lifecycle begins with production of multinucleate sporangia borne upon branched sporangiophores that emerge from stomata, lenticels, or cracks in the 1"?" V‘ook Li--. r. , .. C) m '1 C) 1-1 I. I‘ ‘9 epidermis or periderm (46). Sporangia are disseminated in air currents or water and can germinate and infect directly at 15-25°C (58). However, the optimal temperature range for infection is from 12-18°C because in this range and under conditions of free water, sporangia differentiate into 3—8 biflagellate motile zoospores (9,107,136). Zoospores can remain motile for many hours, demonstrate negative geotaxis and. positive chemotaxis towards roots exudates and biological compounds such as amino acids and ethanol (82). Zoospores encyst on contact with a hard surface or agitated. Cysts germinate and penetrate the host directly or through stomata (25). Following penetration, P. infestans grows intercellularly and produces haustoria-like structures between the cell walls and plasma membranes within the cells it contacts (25). Optimal temperatures for growth of mycelium in Vitro is 20°C (46) and 15°C for sexual and asexual Sporulation (101). When both mating types are present (A1 with A2) and isolates are compatible, oogonia and amphigynous antheridia are produced and mating occurs, resulting in oospores. Oospore production in the field has only been recently noted in temperate regions (5,61). Excluding oospores, P. infestans lacks over- wintering structures. The minimal temperature for growth of P. infestans is approximately 3°C (46) and temperatures U‘ m V‘fi’" “‘y'.‘ .gcy- '. I - be. h‘ :- H \rd‘ u.‘ sgv ‘ ‘5‘- (I) ’4. 0‘4 _() {U ’(1 (I) below OWC are tolerated only for short durations of time by non—sexual tissues (118). Because of the lack of over— wintering structures and poor saprophytic ability, P. infestans requires a plant host, such as potato tubers, to over-winter in areas with extended periods of freezing. The genus Phytophthora and its relatives are occasionally termed “water molds” or “pseudofungi" because they superficially resemble true fungi in certain aspects of gross morphology. Pseudofungi are in the kingdom Chromista and are unrelated to true fungi in the kingdom Mycota (20,46). Oomycetes have B1,3-glucan and cellulose based cell walls instead of chitin, primitive ribosomes and mitochondria. compared tx> other eukaryotic organisms, substantially different nutritional requirements (71) including a necessity for exogenous sterols and thiamine, and numerous other basic biological activities. The differences between the two groups complicates chemical control as many of the basic biological activities and structures differ significantly' or are lacking completely ir1 Phytophthora (143). Fungicides tflun: target processes specific to true fungi, SUChL as demethylation inhibitors of sterol biosynthesis, typically' have little effect on pseudofungi and are not used in control programs (115). Multi—site fungicides that affect multiple and non- specific sites within the pathogen generally have an effect on both true fungi and pseudofungi. The Disease Historical Importance Potatoes were cultivated. throughout Europe for over 200 years before the introduction and spread of It infestans during the 1840’s (108). The agrarian population of Ireland had become heavily dependent on potatoes as a primary food source because of the high productivity of the crop and nutritional value of the tuber (32). Changes in the structure of Irish society included a population explosion and increased cost of grain and milk resulting in an increased dependence on Emmatoes (39). Other European countries were less dependent on the potato and thus less affected by late blight epidemics than Ireland (121). The first noted loss to the potato crop from P. infestans in Ireland. was in the latter portion of the 1845 growing season when an estimated 25% reduction in total yield occurred due to tuber blight and the subsequent rotting (44). In 1846 the epidemic occurred early in the growing season and severely limited tuber production, causing an almost complete loss of the crop. Because of the dependence on potatoes as a base of the diet and a lack of a . - y...~ ‘ CAWVN‘ , . V191 ‘ -“" .u I ‘F .‘. Ia ‘( sufficient social support structure, the majority of the population. was ‘without adequate :nutrition. and starvation occurred resulting in a famine that lasted until 1849 (39). During the period of the famine, malnutrition was common and resulted in epidemics of human diseases and death. The combination of a massive number of deaths due to starvation and an emigration of 1.5 million people caused the population of Ireland to be reduced almost in half in the span of five years (44). Integrated Potato Crop Management Following the Irish Potato Famine, breeding for resistance to P. infestans was initiated in Europe and resulted i1) the development CHE resistant varieties. Breeding for high levels of stable resistance has not been fully successful (42) because P. infestans has readily overcome vertical resistance (10) and horizontal resistance is incomplete. Current. strategies for the control of potato late blight include the use of certified seed potatoes, less susceptible varieties that pass a late blight field tolerance test, removal of cull piles and volunteer potatoes, crop scouting, weather-based disease modeling, and chemical control programs (51,116). Cultural management of the potato crop includes irrigation q Infifi‘l’lim ‘AIUbulwu ‘ growma 1:001:11; ' .: in.ec: P2)": op .in t- “\r management and hilling of rows to reduce tuber contact with free water, planting and harvesting timing to reduce tuber damage, optimal nutrition. management, and. desiccation of foliage prior to harvest to eliminate foliar-borne inoculum. Epidemiology P. infestans is tflua most economically important pathogen of potatoes (75). In temperate regions the disease cycle begins each spring with an infected seed tuber, volunteer plant, cull pile, or non-potato host Solanum sp. that harbored a PR infestans infection between growing seasons. These sources serve as the primary inoculmn and the sporangia produced from lesions can then infect neighboring plants and initiate aui epidemic. Phytophthora infestans grows, reproduces asexually and infects best under cool and humid conditions (9). Under optimal conditions the asexual portion of the life cycle results in a polycyclic epidemic (144) that can completely defoliate the canopy of a susceptible variety through lesion expansion and re—infection. Yield loss occurs indirectly through photosynthetic capacity reduction due to the loss of foliage and directly through tuber infection and the subsequent invasion of the tuber by secondary it t mi e the Def u: it . t :. e .C .u .0 a X T. -1 t a Y :3 e 5.. 4. . t C. ... C S . t T. v... T C S 0 0 «G 4 . e S -l O O -l -l «1 p . n 1; ‘C ‘ I u a i C C. 0. e a E 0.. a C. a rd. nu n . "V p t he. a no h .. P: «x. S u .1“ e 0 e :1 r u r e .1 has PE. 96 0.. d "a ~—r)U p‘ ~ AU pathogens (108,125). Losses from.an1 epidemic (EH1 include the entire crop and the severity can be independent of the foliar epidemic duration as late season infection can result in complete crop loss (76). Fungicidal Control of Rhytqphthora infestans Definitions and Fungicide Groups Fungicidal vs. Fungistatic The common nomenclature used for the compounds employed to control fungal and pseudofungal plant pathogens is confused in that all are typically called ‘fungicides'. Fungicides are compounds that kill fungal organisms. Fungistatic compounds reversibly inhibit growth and/or development. Many of the compounds used in the control of potato late blight can be fungistatic or fungicidal depending on concentration (14,15,26,90,ll4). For simplicity, the term ‘fungicide’ will be used in its broadest sense and. will cover both true fungicides and fungistats that affect both true fungi and. pseudofungal organisms. Protective vs. Curative Fungicides can be used to both protect uninfected plant tissue from infection with a fungal plant pathogen or .— ' "-v ‘U . R‘ ’1. “~‘ F v.4 --..a.. -.v _, p'r" fill (I) ’05“ v ...--v-" " ...w‘ w-‘ ‘.-v - i . .w‘ u 2 F ' - _--u--oou v - . - .~ A Var veg .yd‘Q- -u - -' t A n‘ A _ F an» 0..“ - . .. . " ‘qnb . ~ogu..L. C v r . . _ 0”,”. A. N‘- ‘ — ~m.u.v-“‘ v Q ’. l Fae. g. gnfi. -.¢~--\.\v 5‘. In“; .- .....,~ 0‘ . . :6": "V‘w- "‘ v54“: . .... .2 -¢\. . ".5; h? ‘ U» . ‘AIA Lu“: “VA” = f“ t’edgebs ~~ j-u . "-Ug‘;‘ 5"! Prn,..._w . 3sufib. . to cure or inhibit an already established infection. Biologically, the prevention of a polycyclic epidemic, such as that of late blight of potatoes, is more feasible than attempting to control or halt a previously initiated one. Eliminating or limiting the number of initial infections will reduce the total spore production, and therefore reduce the re—infection capacity and fungicide dose required to limit the rate of epidemic development (142). The majority of fungicides used to control potato late blight are used as protectants. Curative usage of fungicides to control potato late blight is generally ineffective using the currently available fungicides as none of the chemistries can completely halt epidemic development (124). Biological vs. Physical Mode of Action The asexual life cycle of P. infestans on potatoes can be broken down into two general stages: A) the infection process consisting of zoosporogenesis, release and motility, encystment, germination and penetration, and B) growth inside and Sporulation from infected tissue. The effects of fungicides on specific points of the disease cycle are often referred to as the “physical mode(s) of action” (130). Various classes of fungicides can have 10 u RV. 5 “‘~ \, ‘ 1 :1 S be‘u‘C 9 ' . 5.0 kl 0n tne effects on one or both stages, and may only affect a single point within the cycle, e.g. zoospore release. The “biological mode of action” is the specific metabolic process or processes being disrupted by the fungicide, e.g. the disruption of cell wall formation. NOn-systemic Fungicides The two basic groups of fungicides are defined by the degree of plant uptake that occurs: non—systemic and systemic. Non—systemic, including both residual and contact compounds are not taken up by the plant, are at a higher risk for washing off, and must be present on the surface for activity. Once P. infestans penetrates the host, mycelium grows within the leaf or tuber tissue and is not in contact. with. these compounds and. therefore unaffected. Non—systemic fungicide usage can be divided into contact and residual (73). Contact fungicides are used to kill fungal colonies and/or propagules that are present on the plant as well as in the local environment, such as the soil and/or plant debris. Residual fungicides are applied to the plant surface and affect the pathogen on arrival of inoculum in a protectant fashion. In current potato agricultural systems, most non—systemic usage is residual to reduce the .cost and environmental impact by 11 ‘- v 0. \y aCCIH arm" 'f ‘ gv-tn-ooa germina‘ pro contac rt“ D‘ae“'b ; (ild I Fv .n~ meta ‘ 71““: Chow‘s .e H applying the fungicide to the plant only. Residual fungicides typically interrupt a large number of cm ea few extremely important metabolic processes and. have a ‘wide spectrum of biological activity, including toxicity to plants if infiltrated. into tissues. This general disruption of metabolic activity typically causes the inhibition of both direct and indirect sporangia germination, zoospore molitity, and/or cystospore germination and growth (115) resulting in the inhibition of infection. Residual fungicides are generally applied with .a protectant strategy, are subject t1) environmental degradation and therefore have a limited duration of contact with the fungus resulting in a low level of resistance selective pressure (135). To become resistant to a multi-site fungicide, either multiple drastic changes in metabolism or the development of a detoxification or efflux mechanism would be required (134). The combination of a comparatively low resistance selective pressure and the complexity of development of fungal resistance to the fungicide correspond to a small resistance development risk for multi—site fungicides. 12 C rtam and cu: previous u . I 1 TFQ?‘ I *1‘ob-a- Systemic Fungicides Systemic fungicides are highly' resistant to ‘washing off after absorption by the plant tissue, and remain biologically active until diluted within developing and expanding plant tissue, actively degraded or metabolized. Certain systemic fungicides can be used in both protective and curative schemes an; some compounds will affect previously established infections in addition to inhibiting initial infection. Fungicides that limit the growth and Sporulation of successful infections can reduce the amount of photosynthetic area lost by limiting lesion expansion, and may reduce resporulation and therefore the re-infection potential, thus altering the rate of epidemic development. In studies of the potato late blight pathosystem, none of the commercially available fungicides completely prevented infection under conducive conditions (51,124). Systemic fungicides generally have a specific biological mode of action, interrupting one or a few required. metabolic processes or specific enzymes. Such targeted biological activity means that often only small groups of related organisms are affected. .Activity of systemic fungicides on P. infestans may include the disruption of in planta growth and resporulation as well as the inhibition of sporangia or cystospore germination 13 ’5 a 9" “’ 5 en v a“: nf‘kflf'fl" “av..-vv.¢ . J s;:.p-e Cf. “V A a Y'A .3: 08. c ’V R . A /‘ 31:09:30.3: ccmpex «5"»: ‘h: ,. Oteoa‘u‘ s, r ‘HA ' EAO: C‘j’toc h‘ I, .- .me 145 ~.- Mn Au Spe vs‘ «Want 0 SVM . I .Vse‘ulp typical cflf non-systemic fungicides. Systemic fungicides that affect in planta growth or Sporulation of the fungal pathogen can be used.ir1ea curative fashion and can limit, but typically do not eliminate, previously established infections. The resistance risk for systemic fungicides is higher than that cf rxmrsystemic fungicides because their specific biological activity may be overcome by relatively simple changes in target enzyme gene sequence, resulting in the development of resistance. In the true fungus Mycosphaerella fijiensis, resistance tx> the cytochrome tel complex inhibitors such as the strobilurins (Qo inhibitors), results from a change in the DNA sequence of the cytochrome 1) gene that translates into EH1 alanine at the 143 position in the peptide, instead of glycine (119). The specific activity of many systemic compounds, occasional curative usage by the grower, and prolonged duration of contact between the fungus and fungicide inside the plant results in an increased level of selective pressure applied and.a1 higher resistance development risk in comparison to a multi-site fungicide (134). Systemic fungicides range in systemicity depending on the type of movement (102). Compounds that move within the plant cells, or symplast, are generally termed “fully systemic” because application of the compound to any 14 v-v A 0' 3‘” GUrle.3 GRCLIE 1C \ ‘7 Q‘_ :u‘...j U“ . n" Y”! 0:. OS VA PF 7‘ .UD»: (\A. . R‘fifil ab rith-ab v. '1 ”Tin #- Vp¢-\' 3,. .0e maxi aCIODECa] portion of the plant generally provides protection for the entire pflant, including leaves, stems, roots, and tubers. Fully systemic compounds have the potential to move both towards the shoot (acropetal movement) and towards the roots (basipetal movement) (26,115). Fungicides that move only within the intercellular spaces of the plant, or apoplast, can move locally or acropetally. Minimal apoplastic systemicity is termed translaminar, in which application of the compound to one surface of the foliage results in the infiltration. and. movement throughout the entire leaf and protects both leaf surfaces from infection. The maximum degree of apoplastic systemicity is complete acropetal movement, in which application of the compound to the root system confers protectbmn to all aerial portions of the plant. Fungicide Development for Commercial Release Fungicide development in the current agrochemical industry is a. mmlti-step process typically involving initial discovery, early' assessment. of activity, initial field trials, public release, additional examination of activity and resistance risk assessment, refinement of field usage, and registration for commercial use. Fungicide discovery typically involves the mass screening 15 CLSCOVEfi .q COBCIOé" ‘ Ef sl liab. {+u~- ‘bdv. ghtom O. L fika' conduc crop 0:0 Er‘ .*Q "\ ..-a-S a a r‘ he. of compounds with unknown properties on representative species from various groups of plant pathogens, e.g. Basidiomycetes, Ascomycotes, Oomycetes, etc. Following discovery, compounds are assessed both in vitro and under controlled environment conditions for activity. This assessment often examines both the biological and physical mode(s) of action of the fungicide, but typically uses a limited. number of isolates and species. Initial field trials are always conducted at industry research facilities, and may be continued and expanded at external institutions, such as Luaversities. Public release generally occurs at an international plant pathology’ or crop protection meeting, such as with dimethomorph at the Brighton Crop Protection Conference in 1988 (1). Following public release, additional research on the fungicide is conducted under in vitro, in vivo, and field conditions and the specifics of activity and resistance risk assessments on the target organisms are determined, e.g. dimethomorph efficacy on downy mildew of grapes (137) and dimethomorph resistance in P. parasitica Dast. (21). The refinement of field activity occurs in various regions and climates, often on multiple crops and is followed by the registration process. Following registration, the fungicide is available for commercial use and additional research often 16 Cont emPC 15:82 3 r e s r y . nond (c 1a S A: a» H: w“ r. e «nu .n:“ u at 7M .v RD .ato Rn.- UV . h _@81 yu.q‘ continues on various topics, e.g. dimethomorph activity on other Phytophthora species (97). Contemporary Fungicide Usage for Potato Late Blight Control Fungicide Programs Following the migration of phenylamide fungicide resistant populations of P. infestans from Mexico to Europe, the rest of the Americas, and other parts of the world (61), control recommendations for potato late blight changed. This :migration. necessitated. crop» jprotection schemes that relied primarily on non-systemic residual foliar fungicide applications and a transition in systemic fungicides away from phenylamide usage (115,116). In potato growing regions, three basic types of foliar fungicide programs exist: full-protectant, weather—based protectant and curative (124). Full—protectant foliar fungicide programs begin before the onset of potato late blight, consist primarily’ of residual fungicide applications that continue throughout the growing season at regular intervals, and may be initiated by weather-based potato late blight prediction models (77,95). Protectant fungicide programs that are fully weather-based are initiated like full—protectant programs, but the application interval and/or product rate(s) vary depending 17 protectaf . V n ~r.p"* -.uduv v “P “y or? a.‘ 6-5 a -SCOV a 5156 my” huh-V . M55 WJM d ,5...,.;A «Cum: A apt ‘ ‘ flank W Y bVe-L-O i crpl ca: . W ‘§ U . N US“ v V“ § sk- Cid 0"“ on meteorological events (87,126). Weather modeled protectant programs can potentially reduce fungicide usage without increased foliar disease (95). Failure occurs because many of the currently available potato late blight models in the United States were based on the displaced clonal USl/Al lineage (101), are generally region specific (63) and. may' not account for local climatic variations (77). Curative programs are initiated following the discovery or predicted onset of potato late blight (51). These can have the lowest level of fungicide input, but have the highest potential for failure. Disease detection aptitude 'varies and. current fungicide jproducts are less effective ‘with. curative usage (115,116,124). .Attempts at controlling potato late blight curatively following establishment and spread may result in extensive fungicide application of more expensive products (78), increased fungicide resistance selection pressures for systemic products (13,134), and potential yield loss. .Application Methodology Depending on the growing region, four types of foliar fungicide application methods are used: chemigation, pivot- aattached. sprayer systems (PASS), aerial and ground application. Chemigation through the existing irrigation 18 -0- gay lvva - V -I 3.4 e tnem: 61"?” ‘ y- 5.033031 EOWEVJE ‘ a .1 ‘ [7.6 A1 Z H A‘- a». “IY. «VEI‘S “Lom OI“. .4: . .. “y . ‘ "TLC F'HH‘ b; uuu- L- as holed! system requires minor modification of the equipment, and can deliver a more uniform fungicide distribution because of the larger water volume used in delivery (67). Chemigation with certain systemic products can result in acropetal redistribution of the fungicide via root absorption and protection of the entire plant tissue. However, the enormous volume of water used in this form of application may dilute and/or waste fungicide product and result in sub-active concentrations being distributed upon the leaf surface (56). PASS application requires additional modification of the irrigation equipment but does not dilute the fungicide to the same extent as Chemigation and offers adequate distribution (56,129). Aerial application systems use substantially less water and rely on rain or irrigation mediated redistribution of fungicides. Further advantages of aerial application include the minimization of physical crop disruption and soil compaction. In comparison to other forms of application, fungicide distribution is poor with aerial application; without water mediated redistribution and frequent applications the majority of the foliar surface remains inadequately protected (67). Ground application delivers appropriate coverage to the crop and is the most uniform in distribution (67). However, because of the 19 I.» v. "mm 2%. .ww a“ NC. CC V. 3.. r“ .3. 1.. «o r“ .G I. . a .C at. ‘3 : . C 3. . n C a .l- C. .. r. C .. . .. A c .Q T. .3 .C L .. . a a ... frequency of application required and the small area that can be treated with each sprayer pass, soil compaction and plant disruption is greatest with this method. Additionally, pathogens can be transferred between fields on equipment. Michigan growers use primarily ground and aerial applications, depending on region and farm size (88). Fungicides may also be applied as seed treatments prior to planting or in furrow at planting. Application Rates The rate of active ingredient (a.i.) applied depends on a variety of factors including: the bioactivity of the fungicide, the control program being followed, application methodology, maximum. product usage limit, meteorological conditions, the proximity of the nearest inoculum source, and the point within the growing season and/or crop maturity (11,18,23,51,52,70,78,105,133). As product labels dictate in the United States, a standard protectant late blight fungicide program using a residual non—systemic fungicide will typically consist of applications of 0.5 kg ha'1 to 1.8 kg ha’1 active ingredient with an application interval varying between five to 14 or more days between applications, depending on disease conditions and crop maturity. Incorporation of systemic fungicides into a late 20 , 1‘.‘L - p §.uy~; l -h..\ ((7 Eur. 1) (D m {4 If! blight control program permits a decrease in non-systemic fungicide rates (47,124), 51 possible lengthening of application interval without a loss of biological activity (84), and provides complimentary biological modes of action that may be synergistic (47,57). Rates for systemic fungicides vary between 0.1 kg ha"1 to 1.0 kg ha‘1 and require similar application intervals as non—systemic fungicides, depending on fungicide. Regional USage Fungicide programs employed by growers for the control of potato late blight in Michigan have not changed substantially since grower adaptation to the immigration of the phenylamide insensitive strains and the subsequent failure of phenylamide—based fungicide programs during the middle of the 1990’s. The current standard late blight control program consists of regular application of organic non-systemic protectant fungicides commencing before canopy closure and generally continuing until desiccation. In the absence of late blight, minimal systemic fungicide usage occurs and growers typically target such usage to correspond to row closure, tuber initiation, immediately pmior to desiccation, and/or during conducive weather 19eriods. In the 2000 growing season, both ground and 21 co Fw pm. A: aerial application methods were employed and the majority of growers applied nine to 15 applications (88). The average ground application carrier (water) volume ranged from 150 — 280 1 ha‘1 while aerial applications typically used 10 — 47 l ha'l. The average yearly costs for fungicide application during a late blight conducive season, such as 2000, were $100 — 250 (USD) ha’l, not including application costs (88). In locations with active late blight in the crop, a substantial number of systemic fungicide applications occurred as growers used combination applications of a protectant non-systemic and a systemic product in order to apply additional pressure to the late blight epidemic. Such programs are typically more expensive and are likely to only retard the epidemic rate (51,124) . Non-systemic Fungicides Currently In Use Inorganic Protectants - Copper Chemical control of potato late blight began with the use of copper compounds, such as the Bordeaux mixture (100), towards the end of the 19th Century. The Bordeaux mixture is considered the initiation of chemical control for crop pests (115) and was extremely successful because of its fungicidal efficacy, tenacity and distribution on 22 the leaf surface, and relative ease of application. Additionally, the Bordeaux mixture was produced from copper sulphate and lime, two relatively simple, inexpensive, and readily available compounds that could be applied to the crop as a dust or in water. The Bordeaux mixture can be phytotoxic and the development of other copper based fungicides, such as the copper oxides, resulted in the reduction. of phytotoxicity' (72). .All copper fungicides share the copper ion “hf' or Cu”W as the biologically active component of the compound and have a certain level of phytoxicity that can result in yield reduction when used in the absence of late blight (72). FUngitoxicity occurs because the copper ions interact with sulphhydryl, carboxylic acid and hydroxyl groups of proteins causing the denaturing of multiple enzymes, thus interfering with fungal metabolism and disrupting both direct and indirect sporangia. and. cystospore germination VK3). Copper—based fungicides gained widespread and extensive usage in many crop systems and were the dominant chemistry in potato late blight control until the development of the organic protectants (115). 23 Organic Protectants Dithiocarbamates With the discovery and development of the dithiocarbamate compounds (40) copper' was eventually displaced as the dominant fungicide for the control of potato late blight in the United States (73) and the organic fungicide era. began” The dithiocarbamates were less phytotoxic than copper compounds and easier to transport and apply. For the control of potato late blight, the) ethylenebis(dithiocarbamates), or‘ EBDCs, were found to be the most fungicidally active members of the group and are still used as a base for control programs (115,116). As with coppers, EBDCs are non-systemic, residual fungicides that function by disrupting spore germination and preventing penetration of the host by the pathogen (15). The active component is the dithiocarbamate anion which was thought to disrupt metal—containing enzymes or interact with thiol groups of enzymes (132). The EBDCs have: a. moderate level of 'non—target toxicity, are considered possible human carcinogens by the Environmental Protection Agency (EPA) and their use may eventually be restricted (62). 24 _ Li. «1 Phthalonitriles Chlorothalonil is the only commercially available product for late blight control from this group. Its biological activity is thought to be similar to that of the EBDCs because reaction with thiol groups resulted in the disruption of metabolism (31,54), thus preventing spore germination. and. penetration (128). Chlorothalonil is a non-systemic fungicide that must be present on the leaf surface for activity. In the 2000 growing season, Michigan growers used chlorothalonil as the primary late blight fungicide (88). Time application” distribution, and redistribution patterns of chlorothalonil for various application methodologies have been studied thoroughly (16,67). As with the EBDCs, chlorothalonil has a moderate non—target impact and is considered a possible human carcinogen by the EPA, thus usage may eventually be restricted (62). Organic Tins Two triphenyltin compounds represent the organic tin fungicides: triphenyltin hydroxide and triphenyltin acetate. Both compounds are primarily used in a protectant manner and share the triphenyltin group as the active component (31). Unlike.the other classes of protectant 25 fungicides, triphenyltin compounds have slight translaminar systemicity and an antisporulation activity (115). As with the copper fungicides, a low level of phytotoxicity occurs when applied to developing foliage. Triphenyltins are typically mixed with EBDC fungicides and applied in the latter portion of the season or when an active late blight epidemic is occurring. The biological mode of action is not completely understood but is thought to be the disruption of oxidative phosphorylation (31). The physical mode of action results in the inhibition of spore germination, zoospore motility, and vegetative growth (31). Non-host, and specifically mammalian (4,22) toxicity of triphenyltin fungicides is high and this group is at a high risk of usage restriction. Pyridinamines Fluazinam is the only commercially available product from this class for the control of potato late blight. The biological mode of action is not fully understood but an uncoupling of oxidative-phosphorylation has been reported (12,65) on both true fungi and pseudofungi (86). Fluazinam is biologically active in relatively small concentrations, inhibits spore germination and infection (3), and reduces the duration of zoospore motility (97). Fluazinam is non— 26 systemic and must be used on a regular schedule for disease control. Non-host toxicity is low compared to the other contact fungicides. Fluazinam. can be successfully incorporated into a control program using partial host resistance and varying fungicide rates and application intervals in order to reduce the amount of season-long fungicide application (84). Systemic Fungicides Currently in Use First Generation Systemics The initial development of systemic fungicides for the control of true fungi occurred during the 19605 with the introduction of the benzimidazoles (133) and was followed later by additional groups. Systemic fungicides for the control of pseudofungi were not available until the almost simultaneous introduction of the cyanoacetamide—oximes, phosphonate, carbamates, and phenylamides in the late 19705 (115). Only tn“) fungicides: cymoxanil ha cyanoacetamide- oxime) and metalaxyl (a phenylamide) had initial widespread and intensive usage for the control of P. infestans. Cyanoacetamide—oximes As the single commercially available member of the cyanoacetamide—oxime fungicide group, cymoxanil is active 27 f 'U on certain members of the Peronosporales (27,115) and has both translaminar systemicity and a high level of acropetal transport \fiji root uptake (28). Breakdown.;n2 planta into glycine can occur within days (28) and is temperature dependent (98). When used for the control of potato late blight, cymoxanil is typically mixed with a non-systemic contact fungicide, providing a synergism of activities (47,57). The biological mode of action has not yet been elucidated but disruption of RNA synthesis was considered a possible target (38). Insensitivity' within the related Oomycete Plasmopara Viticola (Berk. & Curt.) Berl. & de Toni has developed in situ within certain populations (78) but no change in sensitivity has been detected to cymoxanil within P. infestans populations studied (66). Its primary physical mode of action is similar to the non—systemic contact fungicides in that zoospore release, germination and appressorium formation were inhibited (115). Cymoxanil also provided moderate post infection curative activity due to the partial inhibition of hyphal growth in planta (78). Phosphonate Compounds All of the compounds within this group breakdown in planta ‘which releases the jphosphonate anion, the active component of the fungicide (64). The alkyl phosphonates 28 may have additional fungicidal activity from their metal cation. Fungicidal activity (x1 foliar infections (HE P. infestans is sporadic and often very limited, resulting in little commercial use worldwide (64). The application of phosphonate fungicides to infected crops resulted in a reduction of tuber infection and demonstrated the full systemic movement (Hi the compound :hi planta (30). Phosphonates are more important for the control of Phytophthora diseases affecting tree roots than for P. infestans in potatoes (46). Carbamates Propamocarb and prothiocarb are both carbamate fungicides and highly specific to members of the Peronosporales. Propamocarb has received the most research and field use for the control of P. infestans. The biological mode of action has not been elucidated but a disruption of the cellular membrane and/or function has been observed (114). The biological activity of propamocarb is relatively low compared to other semi- systemic late blight fungicides in rate comparisons and large amounts must be applied for comparable activity (115). Propamocarb 'has local systemicity' movement only (110). The physical mode of action is proposed to be a 29 combination between a moderate effect on inhibition of sporangial germination and a greater effect on hyphal growth disruption (110). Post—infection activity is limited, therefore propamocarb is mast useful when applied in a protectant fashion (78,110). Phenylamides The phenylamide fungicide group is highly specific for members of the Peronosporales and is further divided into two groups: the acylalanines and the butyrolactones. Metalaxyl, a member of the former group, has been the most important systemic fungicide for time control of It infestans on potatoes in the United States. Discovery of the phenylamides occurred during the investigation of the structurally related chloroacetanilide herbicides (36). Metalaxyl has acropetal systemicity and application of the fungicide to roots or lower foliar portions of the plant often confers protection to the rest of the foliage, including new growth (26). Protection of tubers from foliar applications has also been reported (104,117). The biological mode of action is not completely understood but the inhibition of ribosomal RNA biosynthesis occurred via the putative disruption of the RNA polymerase I complex (36). Metalaxyl inhibited. Iboth. fungal growth. and 30 Sporulation (33,34,35). The development of resistance to metalaxyl in P. infestans can be induced with ultraviolet light irradiation (17), selected for in Vitro through repeated culturing (139) and is thought to occur from a DNA sequence mutation at the gene for the target site of the fungicide (36). Cross-resistance to other phenylamide fungicides occurred (21). Metalaxyl has high biological activity and unlike the non—systemic fungicides, does not affect zoosporogenesis or germination but instead inhibits fungal growth (14). The high level of systemicity and inhibition of hyphal growth allowed for effective post- infection usage of metalaxyl to control previously infected potato plants. Unfortunately, frequent usage, especially in a curative fashion results in high selective pressure placed on the pathogen, resulting in the development and spread of resistance. Second Generation Systemics Many potato-growing regions around the world became heavily dependent on phenylamide fungicides for control of P. infestans on potatoes and growers adopted fungicide application patterns that deviated from strict protectant programs, such as post-infection application programs in order to eradicate existing infections. The phenylamide 31 insensitive strains of P. infestans that migrated from Mexico to other potato growing regions in the early 1990's disrupted crop protection programs and resulted in severe crop losses (53). Additionally, these strains were often comparatively' more aggressive than. displaced strains (37,75), luui different physiologies resulting irl enhanced fitness (8), and often were of the complimentary mating type allowing for' possible sexual recombination and the putative generation of rumv genotypes (55). Following the migration. of these jphenylamide insensitive genotypes and subsequent crop losses, the development of existing and novel fungicides for the control of P. infestans was accelerated. Cytochrome bcl Complex Inhibitors Two fungicide groups, the strobilurins and oxazolidinediones share a common biological site of activity and are effective against both true fungi and pseudofungi. The only two currently commercially available strobilurin fungicides for time control of 1% infestans on potatoes within the United States are azoxystrobin and pyraclostrobin (F500), with trifloxystrobin and others under development for* possible future release. The strobilurin fungicides were derived from strobilurin A, 32 which is produced. by the Basidiomycete Strobilurus tenacellus (54). Famoxadone is an oxazolidinedione and is the only member of this group that is close to registration for use on potatoes in the United States. For both groups the biological mode of action is the inhibition of respiration through the binding of the cytochrome bcl complex (80,131). Because of the single site of activity, the development of resistance within the target organism is probable, and at least two mechanisms for insensitivity to these fungicides have been found (119). The first mechanism is the induction of the alternative oxidase pathway. With this type of insensitivity, the fungus is still able to respire, but at a reduced rate resulting in lowered fitness. The second type of insensitivity results from a change in the nucleic acid sequence of the target peptide gene and yields total resistance to the fungicide. The most common mutation resulting in the resistance of true fungi to fungicides of this group results in the replacement of glycine with alanine at the 143 amino acid position (119). The physical mode of action of both groups is identical in the disruption of zoosporogenesis, zoospore motility, and hyphal growth. Compounds within these groups are locally systemic and are applied in a protectant fashion with 33 limited applications per season to reduce the resistance development risk. Benzamides The only commercially available member of this group for the control of P. infestans on potatoes is zoxamide. Zoxamide binds B-tubulin and disrupts microtubule assembly, similar to the benzimidazole fungicides, resulting in the inhibition of mitosis (140). Attempts to generate P. infestans mutants resistant to zoxamide were unsuccessful and the resistance development risk was considered lower than that of the phenylamides (139,141). The physical mode of action for zoxamide is the disruption of mitosis and most stages of the life cycle were affected (140). The zoospore stages, including zoosporogenesis, encystment and germination were unaffected, as mitosis is not known to occur during these portions of the life cycle (25). Morpholines Dimethomorph is the only commercially available member of the morpholine fungicide group that is used to control late blight. It was derived from cinnamic acid, showed translaminar and acropetal systemicity, and was specific against the genus Phytophthora and certain members of the 34 Peronosporaceae (1). Dimethomorph was most effective when used as a protectant fungicide (29), however post-infection activity also occurred (2). Dimethomorph caused disruption of cell wall formation (1,90) and inhibition of sporangia formation irl P. infestans when applied. tx> normally developing lesions (2,29). The biological mode of action has not been elucidated, no activity on protoplasts was found, and dimethomorph was considered to have a single site of activity (2). Dimethomorph exhibited a lower resistance development risk than metalaxyl in P. parasitica (139) and in P. capsici and P. infestans (141) using single isolates. Dimethomorph negatively affected all stages of the P. infestans life cycle except zoosporogenesis and zoospore release as these stages do not involve cell wall biosynthesis (2,29,90). Dimethomorph was originally released for emergency use on potatoes in the United States as a pre—mixed wettable powder with mancozeb as its mixture partner (9% dimethomorph / 60% mancozeb by weight) to reduce the resistance development risk. The pmoduct label was later modified and. dimethomorph. was re—released for full non- emergency usage as a 50% dimethomorph single active ingredient product. Tank mixing dimethomorph with a protectant fungicide is mandatory according to the label 35 (“I (') (I) and allows growers more flexibility in mixture partner product choice. Rate and interval optimization and mixture partner assessment for dimethomorph within a Michigan potato system have not been performed. Future Fungicides Following the development and spread of phenylamide resistance in many of the Oomycete plant pathogens, the market for Oomycete fungicides has undergone a resurgence. Agrochemical companies are in the process of both active discovery of new compounds and further development and registratitml of existing' chemistries. Tlma cost of late blight control is currently one of the limiting factors in potato production (79,88) and none of the currently available late blight fungicides were as efficacious and had the same level of post-infection activity as metalaxyl did on sensitive strains (97). A search for suitable replacement products is a high priority in many industry research and development programs. The recent release of the strobilurins, fluazinam, and zoxamide for late blight management in the United States demonstrates the grower demand for suitable products with disease control comparable to currently used fungicides, but with reduced environmental impact. 36 Research Objectives The objectives of this study were to 1) the examine the sensitivity to dimethomorph of isolates of P. infestans from various genetic backgrounds at multiple stages within the asexual lifecycle and in Vivo, 2) attempt to generate insensitivity to dimethomorph within P. infestans and characterize the insensitivity if found, 3) optimize the field efficacy of dimethomorph within a potato late blight program by examining mixture partners, rate, and application intervals. 37 CHAPTER TWO DIMETHOMORPH SENSITIVITY IN PHYTOPHTHORA INFESTANS Abstract The sensitivities of 11 isolates of Phytophthora infestans to dimethomorph at all stages of the asexual life cycle and when inoculated onto potato leaf disks were examined. Zoospore encystment and cystospore germination were both highly sensitive to dimethomorph with Exgo values for' most isolates <0.20 pg lfllqfi while direct sporangia germination and in Vitro hyphal growth and Sporulation were less so. Zoosporogenesis was not significantly inhibited at the maximum dimethomorph concentration examined, 10.0 pg mlfiy ECw values between isolates were significantly different (Fischer’s LSD, (x = 0.05) for all stages of the asexual life cycle, except direct sporangia germination and zoosporogenesis. Application of 1000.0 pg mld'dimethomorph to potato leaf disks at 24 (n: 48 hours before inoculation completely inhibited symptom development for most isolates, while application after inoculation generally was not significantly different from the untreated control, regardless of concentration. Sporulation from leaf disks 38 treated with dimethomorph at 24 or 48 hours after inoculation was completely inhibited at 1000.0 pg ml"1. Introduction Dimethomorph, a cinnamic acid derivative, was one of the fungicides released in response to the migration (60) or spontaneous development (141) of phenylamide resistant strains of Phytophthora infestans. Initial studies with dimethomorph demonstrated a specificity of activity towards the genus Phytophthora and certain members of the Peronosporaceae (1). Dimethomorph was most effective when used as a protectant fungicide (29), however a degree of curative activity also occurred (1,29) . A moderate amount of translaminar and acropetal systemicity was also noted (1,29). One of the most interesting aspects of dimethomorph was the inhibition of P. infestans sporangia formation when applied to normally developing lesions under controlled conditions (2, 29) , also known as “antisporulation” activity. The specific biological mode of action of dimethomorph has not yet been elucidated but a disruption of cell wall formation, specifically the organization and not the synthesis of wall components, was noted (1,90). Dimethomorph disrupted all stages of the asexual life cycle 39 CHE P. infestans except zoosporogenesis, zoospore release, and. motility as these stages do not involve cell wall biosynthesis (2,29,90). The activity of dimethomorph on other Pfiytophthora species was examined (96) and found to be similar to that in P. infestans. However, differences in the effective concentration for a 50% reduction relative to the untreated. control (ECmfl for" mycelial growth. and cystospore germination were apparent between species. The majority of studies examining dimethomorph activity against Phytophthora species typically consisted of only one isolate per species. The single study that compared the sensitivity of several isolates of P. infestans to dimethomorph examined only the in vdvo activity and not the effects on the different stages of the asexual life cycle, nor antisporulation activity (29). Varying sensitivity to fungicides is present in other Oomycete plant pathogens (138) and it is possible that such variation is present in P. infestans to dimethomorph. The objective of this study was to examine the sensitivity of isolates of P. infestans from various genetic backgrounds to dimethomorph l) at multiple stages in the potato late blight disease cycle (asexual life cycle of P. infestans) and 2) protectant, curative, and 40 antisporulation activity of dimethomorph when inoculated onto potato leaves. MATERIALS AND METHODS Preparation of Fungicide Stock Solutions and Amended Media Assessment of the inhibition of in Vitro hyphal growth rates was performed on modified rye B agar (2,19) consisting of the filtrate of pre-rinsed rye (Secale cereale L.) seeds (100.0 g L'l) boiled for 1 hour, de- ionized (di) HflD added to a final volume of 1.0 L, glucose (8.0 g L‘l), B—sitosterol (0.05 g L'l) and agar (15.0 g 1:1). All plates for each replication of the experiment were prepared from the same batch of media in order to reduce variability. A dimethomorph stock solution. was prepared. by dissolving technical grade (95% pure) dimethomorph into 95% ethanol and performing serial dilutions as required. For solid media, the fungicide stock solutions were added to molten media at 10.0 md III when the temperature was 55%:. Sterility was obtained by filter sterilizing the fungicide solution through a 0.22 pm syringe driven filter (Millipore Corp., Bedford, MA, U.S.A.). 41 Production of Viable Sporangia To jproduce viable sporangia. of similar age, potato tubers (cv. Russet Burbank) were surface sterilized with 0.50% sodium hypochlorite in diH20 (10% commercial bleach solution) for 30 minutes, rinsed three times in sterile diHfi), and allowed to onyx Tubers were sliced into 7.0 mm1 sections and placed into sterile 150 mm plastic petri 2 agar sections previously colonized dishes on top of 1.0 cm by' P. .infestans. Plates ‘were sealed.‘with. Parafilml and incubated at 18°C / 15°C (12 hours light / 12 hours dark) until at least 50% of the tuber surface was covered by mycelia (typically five days). Sporangia were harvested by gently removing the mycelium with a plastic culture spreader, transferred into sterile macro-centrifuge tubes, and 1.0 ml sterile diHyD was added. Sporangia were counted with a hemacytometer and the concentration was adjusted to 1.0 x 104 sporangia mlqu Inhibition of Hyphal Growth and Sporulation in Vitro Previously characterized (Table 1) isolates of P. infestans that had been sub—cultured once after re- isolation from infected potato leaves were cultured on rye B agar for 21 days. Colonized plugs, 4.0 mm diameter, were transferred from the margin of the colony onto fungicide 42 Table 1. .P. infestans Isolate ID, mating type, genotype, and State in which isolated (U.S A.). Isolate ID Mating Type Genotype1 Origin Pi88 Al USl ND Pi95-5 Al USl MI P1671 Al USl4 WA Pi458 A2 U817 ID Pi670 A2 U87 OR Pi213 A2 U88 CO Pi94—4 A2 U88 MI Pi95-7 A2 US8 MI Pi97—2 A2 U88 MI Pi98-l A2 U88 MI Pi98—2 A2 US8 MI 1. Allozyme—based genotype”. 43 amended rye B media in 60 mm diameter plastic petri dishes and incubated an: ZTTL The fungicide concentrations used were 0.0, 0.01, 0.1, 1.0, 10.0 pg mld, with three replicate plates per concentration. Colony diameter was measured 11 days after inoculation (DAI). Percent inhibition of radial growth was calculated with respect to the mean colony diameter of the non-amended. plates within each isolate. Percent inhibition values were then transformed using probits, i.e. the inverse of the standard. normal distribution (89), and expressed (49) as a function of the lrxho of concentration. Curve equation parameters were then determined using linear regression (SigmaPlot, SPSS Inc., Chicago, IL, U.S.A.) and the ECw for hyphal growth (diameter) was calculated and reverse transformed for each isolate. 'Nme experiment was repeated three times and the IKgo values for each isolate were used as replicates for an analysis of variance (Proc (KAI - SAS/Stat, SAS Institute, Cary, NC, U.S.A.) at (1 == 0.05 by’ pair-wise comparisons using Fisher’s LSD. The effects of dimethomorph on sporangia production in Vitro were examined by using a modification of a previously described. method. of sporangia quantification (19). Ten colonized agar plugs, 0.1 mm diameter, were randomly excised from each replicate plate of the LU: Vitro 44 sensitivity assay, five plugs 2.0 mm from the colony margin, and five 2.0 mm from the initially transferred inoculum. The plugs were then placed into a 1.5 ml micro- centrifuge tube with 1.0 ml of sterile de—ionized Hg) (diHflD) and agitated to dislodge the sporangia. Sporangia were counted with a hemacytometer. Percent inhibition and ECso values were calculated and analyzed as described. Inhibition of Direct and Indirect Germination To assess the effects of dimethomorph on direct germination, aliquots of the previously prepared sporangial suspensions from inoculated tuber slices were transferred to 96 well polystyrene culture plates (Costar, Corning Incorporated) and dimethomorph stock solutions were added for a final concentration of 0.0, 0.01, 0.1, 1.0, and 10.0 pg mflfl dimethomorph, in each of three replicate wells per isolate. Sporangia/fungicide solutions were incubated for 72 hours at 21°C and the numbers of total and germinated sporangia were counted. To examine indirect germination and zoospore encystment, sporangia/fungicide solutions were incubated at 8°C for five hours to induce zoosporogenesis and the number of motile zoospores was counted. The solutions were then incubated at 21°C and the number of total cystospores was counted after 24 hours. 7R9 examine 45 cystospore germination, zoospore suspensions were prepared as described and incubated at 21°C for 24 hours to allow for zoospore encystment. Fungicide solutions were added, the solutions were incubated. at .2I%3 for“ 48 hours, and the _ number of total and germinated. cystospores was counted. Percent inhibition and ECg) values were calculated and analyzed as described. Isolate Sensitivity Ranking and Correlations of the Asexual Life Cycle Stages To compare the overall sensitivity of each isolate to dimethomorph, the isolates were ranked within each stage of the asexual life cycle using the least significant mean EC“) values generated from each analysis of variance. An analysis of variance on ranks (89) was performed from these values and a mean rank of sensitivity was generated for each isolate and compared. Zoosporogenesis was not included in this analysis because of the lack of sensitivity' of time; process tx> dimethomorph. Pearson’s Product. Moment Correlation. (89) was used. to examine the correlation. of time asexual life cycle stages of the P. infestans isolates used to detail any trends between stages. 46 Protectant, Curative, and Antisporulation Activity of Dimethomorph in vi vo The in Vivo activity of dimethomorph was assessed relative to the inoculation event by removing fully expanded leaflets of similar age from greenhouse grown potato plants (cv. Snowden) and surface sterilizing them with 0.50% sodium hypochlorite in diHZO (10% commercial bleach solution) for one minute. Leaflets were then rinsed three times in sterile diHZO, allowed to dry, and cut into 20 mm diameter leaf disks with a sterilized core borer. Leaf disks were placed onto water agar (15.0 g L’l) amended with rifamycin (37.5 mg 11), ampicillin (10 mg l"), and nystatin (37.5 mg 14) which was previously dissolved in 1.0 ml dimethylsulfoxide, stored frozen in the dark, and added to the molten media following sterilization. Leaf disks were temporarily removed from the agar for treatment and dimethomorph was applied until run-off using the formulated commercial product at 0.0, 1.0, 10.0, 100.0, and 1000.0 pg ml’1 at 24 or 48 hours before (HBI) or after (HAI) inoculation. Twelve leaf disks were inoculated per experiment, and the experiment was repeated three times. Following inoculation, leaf disks were incubated at 21°C light / 18°C dark (12 hour cycles) and assessed for infection by P. infestans at 96 hours after inoculation for 47 symptoms and signs of infection by P. infestans, such as necrosis and sporulation. To assay for the inhibition of Sporulation, time four leaf disks from each replicate were placed into a 15.0 ml centrifuge tube containing 4.0 ml of dino, agitated, and sporangia were counted with a hemacytometer. Percent inhibition and EC“) values were calculated and analyzed as described for both incidence of symptom development and the number of sporangia produced per leaf disk area (cmz). Results For all isolates, no significant difference (Fischer’s LSD, OL = 0.05) was observed in the percent inhibition of hyphal growth on media amended with 0.0 and 0.01 pg ml"1 dimethomorph (Figure 1). At 0.1 pg ml‘1 dimethomorph, all isolates exhibited less than 20% inhibition of hyphal growth except Pi94—4 and Pi95—5, which were 28% and 41% respectively. At 1.0 pg ml’l dimethomorph, more than 50% inhibition of hyphal growth occurred in all isolates, with Pi95—5 being completely inhibited, while 10.0 pg ml“l dimethomorph completely inhibited hyphal growth of all isolates. Most isolates had a sigmoidal shaped sensitivity curve when percent inhibition was plotted against the loglo 48 Figure 1 (aek). The percent inhibition, relative to the untreated control, of in Vitro hyphal growth, Sporulation, and. direct sporangia. germination, for P. .infestans ‘vs. dimethomorph concentration (0.0, 0.01, 0.1, 1.0, and 10.0 pg mlq). Error bars represent Fischer’s LSD (a = 0.05). 49 Percent Inhibition Relative to the Untreated Control 120 100: 80- 60- 40- 20i -4l—-Hyphal Growth -{}— Sporulation —4>— Direct Germination -20. 120 100« °' 80- 60- 40~ 204 -20. 120 100‘ e' 80- 60: 40: 20: 0. -204 0 0.01 0.1 l 10 0 0.01 0.1 l 10 Concentration Dimethomorph (pg mlq) 50 Percent Inhibition Relative to the Untreated Control 120 100~ 80« 607 40- 20. -20( 120 100 4 80 ~ 60 ~ 40 - 20 1 -20 . 120 100‘ j. Pi98-l k. Pi98-2 80: 60: 40« 20« 0. -20 . 0 0.01 0.1 l 10 0 0.01 0.1 1 10 Concentration Dimethomorph (pg ml-l) 51 of concentration. The calculated ECW values for inhibition of hyphal growth ranged from 0.131 to 0.800 pg ml"1 dimethomorph (Table 2). Five non—overlapping significance categories were present with the isolate Pi213 having a significantly higher ECW value than all others. Regardless of the concentration of dimethomorph in amended media, almost all isolates produced significantly less sporangia. per colony' area «3R5 than the ‘untreated control (Figure 1) and Pi95—5 exhibited a 35% inhibition at a concentration of only 0 . 01 pg ml‘1 dimethomorph. Sensitivity of in Vitro sporulation was more variable at 0.01 and 0.1 pg mflfl dimethomorph than was hyphal. Sensitivity curves tended to be more linear than sigmoidal, unlike those of hyphal growth. (Hue calculated Ergo values ranged from 0.036 to 0.437 pg mflfl dimethomorph (Table 2). The isolate Pi213 had a significantly higher'EKgo than Pi95- 5, Pi97—2, and Pi98—2. However, distinct trends in sensitivity and genetic background were not apparent. For all isolates except P1213, 0.01 pg ml"1 dimethomorph significantly inhibited direct sporangia germination in comparison to the untreated control (Figure 1). For most isolates, inhibition trends were similar to those of :U1 Vitro sporulation and sensitivity curves were 52 Table 2. The effective concentration of dimethomorph (pg mld) required for a 50% reduction of in Vitro hyphal growth (colony' diameter) and, sporulation for P. infestans isolates. . . -1 1 Isolate ID in Vitro ECso (pg m1 .) 3 Hyphal Growth Sporangia Production Pi88 0.425 c4 0.199 abc Pi95-5 0.131 e 0.036 c Pi671 0.625 b 0.320 ab Pi458 0.585 ‘b 0.203 abc Pi670 0.392 c 0.236 abc P1213 0.800 a 0.437 a Pi94—4 0.270 d 0.247 abc Pi95-7 0.533 b 0.171 abc Pi97-2 0.291 d 0.115 bc Pi98-1 0.419 c 0.246 abc Pi98-2 0.429 c 0.163 bc 1. Effective concentration for a 50% reduction, calculated using probit transformation (M5 the percent inhibition relative tx> the untreated control. 2. Calculated from colony diameter on rye B media amended with 0, 0.01, 0.1, 1.0, or 10 pg ml”1 dimethomorph. 3. Production of sporangia per cm? colony area on rye B media amended with 0, 0.01, 0.1, 1.0, or 10 pg ml'1 dimethomorph. 4. Means followed by the same letter are not significantly different using Fischer's LSD (a = 0.05). 53 primarily linear. No significant differences were calculated between isolates for the EC50 of inhibition of direct sporangia germination (data not shown). Ergo values ranged from 0.096 to 0.231, with a mean of 0.163 pg ml"l dimethomorph. Zoosporogenesis was not inhibited at any of the concentrations examined, 13) to 10.0 (mg ml"1 dimethomorph (Figure 2). No significant differences existed in EC50 values between isolates, and all exceeded 10.0 pg ml'31 dimethomorph (data not shown). Zoospore encystment and cystospore germination inhibition trends were similar and most isolates exhibited significant inhibition of both factors at 0.01 pg ml‘1 dimethomorph (Figure 2). For" both. factors, sensitivity trends 'were: generally' linear. lime isolate Pi213 had. a significantly higher EC“) value for zoospore encystment, than Pi88, Pi95—5, Pi94—4, and Pi97—2, while other distinct trends were not apparent (Table 3). For cystospore germination, the ECw values for Pi670 and Pi88 were significantly“ higher than five of the 11 isolates, but distinct trends were not apparent. No correlations were noted between EC“) value and isolate mating type, genotype, or location of isolation. 54 Figure 2 (a-k). The percent inhibition, relative to the untreated control, of in Vitro zoosporogenesis, zoospore encystment, and cystospore germination for P. infestans vs. dimethomorph concentration (0.0, 0.01, 0.1, 1.0, and 10.0 pg ml'l). Error bars represent Fischer’s LSD (at = 0.05). 55 Percent Inhibition Relative to the Untreated Control 120 100~ 80~ 60: 40: 20 -20. 120 100 —4r— Zoosporogenesis -{}— Zoospore Encystment -4¥- Cystospore Germination 80: 60* 40 20~ -20. 120 100 b. Pi95-5 C . J 80- 60- 40- 20 -20. e . O 0.01 0.1 1 10 0 0.01 0.1 l 10 Concentration Dimethomorph (pg mlfil 56 Percent Inhibition Relative to the Untreated Control 120 100~ 80: 60 40: 20: -20. 120 100 80: 60 40: 20: -20. 120 100 80 60: 40 20: -20 q f. g. Pi94-4 h. Pi95-7 i. Pi97-2 j. k. Pi98-2 *r 0.01 0.1 l 10 (l 0.01 0.1 l 10 Concentration Dimethomorph (pg mld) 57 Table 3. The effective concentratbmn of dimethomorph (pg ml‘l) required for a 50% reduction of zoospore encystment, cystospore germination, for P. infestans isolates. and direct sporangia germination in Vitro ECSO (pg ml'l)1 Isolate ID Zoospore2 Germination Encystment Cystospore3 Pi88 0.047 bc4 0.169 a Pi95-5 0.016 :bC 0.016 (3 P1671 0.067 abc 0.117 ab Pi458 0.084 abc 0.047 de P1670 0.036 bC 0.145 a P1213 0.136 a 0.114 abc Pi94-4 0.011 C 0.018 d Pi95-7 0.053 abc 0.042 de P197-2 0.033 bC 0.027 Cd Pi98-1 0.098 ab 0.071 abcd Pi98-2 0.063 abc 0.046 ‘de 1. Effective concentration for a 50% reduction, calculated using probit transformation of the percent inhibition relative to the untreated control. Encystment of zoospores in sterile diHZO amended with 0, 0.01, 0.1, 1.0, or 10 pg ml’l dimethomorph. . Germination of encysted zoospores in sterile dinD amended with 0, 0.01, 0.1, 1.0, or 10 pg ml’l dimethomorph. . Means followed by the same letter are not significantly different using Fischer’s LSD (a = 0.05). 58 The most sensitive isolate overall was Pi95-5, an Al/USl strain (Table 4). However, the sensitivity of time other Al/USl strain, Pi88, was near the average of the isolates examined. The isolate with the lowest overall sensitivity was Pi213. The isolates, Pi458, and Pi671 had significantly lower mean sensitivity than all other isolates, except Fd98—1. All stages of tflua asexual life cycle were positively and significantly correlated. with each other except cystospore germination” which. was not significantly correlated with inhibition of any other stage (Table 5). No significant difference between isolates was observed for the percent inhibition of the incidence of leaf disk symptom development at 0.0, 1.0, and 10.0 pg ml“1 dimethomorph, regardless of application timing (Figure 3). When dimethomorph was applied prior to inoculation, there were no significant differences in the inhibition of symptom incidence between application timings, at any concentration. Sensitivity curves for dimethomorph application prior to inoculation were sigmoidal and complete inhibition of symptom development occurred at 1000.0 pg mflfl dimethomorph. 1%) significant inhibition of symptom development occurred when dimethomorph was applied after inoculation, regardless of timing, although the 59 Table 4. Median rank of isolate sensitivity when ECSO values were ranked within hyphal growth, sporulation (in Vitro), direct sporangia germination, zoospore encystment, and cystospore germination and compared with an analysis of variance on ranks. Isolate ID maziggtgif I mediazeifiifit?°15te Pi88 Al/U81 52 b3 Pi95—5 Al/USl 1_ d Pi671 Al/USl4 9 a Pi458 A2/U817 9 a Pi670 A2/US7 7 jb Pi213 A2/U88 11’ a Pi94—4 A2/US8 2 cd Pi95—7 A2/US8 6 jbc Pi97-2 A2/US8 3 d Pi98-1 A2/U88 53 ab Pi98-2 A2/US8 5 jbc 1. Allozyme—based genotype” 2. Sensitivity ranking is from most sensitivity (lowest) to least sensitivity (highest). 3. Means followed by the same letter are not significantly different using Fischer’s LSD (a = 0.05). 60 Table 5. Correlation of the stages of the asexual life cycle cflf.P. infestans examined and the P—value for each, excluding zoosporogenesis, analyzed with Pearson’s Product Moment Correlation. 61 .xfluumfi ecu wo Hamo stuocm CM pwmemmfip no cemfiHMQEOUIMme wanmofiHQQMICOC 4 .m m mwumoaUCH Amo.0A wdfim>(mv om.ov ucmflufluumoo m wHflSB unmoflwflcmflm m mwumoflpcfl Amo.0A wzam>umv .coflumeMHoo w>fiummmc unmoaufismfim .mwanmflum> cwmzuwn cofiumeuHoo w>fluflmom Om.OA QM®£3 .mmuflwfloflwwwou COflUmHWMMOU US$502 UUSCOHQ COmewm .H 3mm . 8 3c a? 5c usosumhosu omm.o muonuoon Abvm.ov “moo.ov m\c m\c GoHuwnfifihow wam.o mam.o namnuhonm Avmo.ov Amfio.ov Awoo.ov m\s GOMuMHsuonm mmm.o wo>.o mw>.o m Ohuw> Ga Ahma.ov Aaoo.OAv Amoo.ov Amoo.ov #uBOHU mmv.o new.o m>>.o Haw.o Honmhm nodumnafiuow unmfiumhosu Goaumnwfihow :oflumasuomm whommoumho ouonmoon wamawnomm Ohuwb aw oHanHm> mosHu> 30m uo A05Hm>smv was ”Hsowuammmoo downwawunou .m magma 62 Figure 3 firJd. The percent inhibition, relative to the untreated control, of the incidence of leaf disk symptom development vs. dimethomorph concentration (0.0, 1.0, 10.0, 100.0, and 1000.0 pg ml'l). Dimethomorph was applied as a commercial formulated product (Acrobat 50WP, BASF Corporation) at 48 or 24 hours before inoculation (HBI) or 24 or 48 hours after inoculation (HAI) by a sporangia / zoospore suspension of P. infestans. Error bars represent Fischer’s LSD (a = 0.05). 63 120 100: (A H to (s m m o N) to (s m m o C) <3 0 :3 <3 <3 0 c: c: o 120 Relative to the Untreated Control Percent Inhibition of Symptom Development Incidence NibONCD OOOOO -{}— 48 HBI -<}— 24 HBI -—.—-24 HAI -4P— 48 HAI b. Pi95-5 100: 1 10 100 1000 0 l 10 1’ 100 1000 Concentration Dimethomorph (pg mld) 64 Percent Inhibition of Symptom Development Incidence Relative to the Untreated Control 120 100 - 80 : 40: 201 120 f. 100~ 80: 60: 20- 120 100- (I) O 60: 40~ 20( 0 J l 10 100 1000 0 l 10 100 1000 Concentration Dimethomorph (pg mld) 65 incidence of symptom development for most isolates was inhibited to approximately 20% at 1000.0 pg ml-l dimethomorph. Within each application timing, the ECw values for the incidence of symptom development were not significantly different among isolates (data not shown). Mean ECM,values for symptom (development ‘were :not significantly' different between application timings before inoculation; nor for application timings after inoculation. The mean.EIgo'values for the inhibition of the incidence of symptom development for dimethomorph application before and after inoculation were 36.18 and >1000.0 pg mflfl dimethomorph, respectively, with the latter being significantly larger. For all isolates, no significant difference in the percent inhibition of sporulation was present between application timings at each concentration of dimethomorph (Figure 4). No significant inhibition of sporulation occurred an: 1.0 or 10.0 pg mflfl dimethomorph, while 100.0 and 1000.0 pg ml'l dimethomorph significantly inhibited sporulation, relatively to the untreated control, to 70.7% and 98.4%, respectively. The lack of symptom development for leaf disks treated. with 1000.0 pg ml"l dimethomorph before inoculation did not allow for assessment of 66 Figure 4 (a—k). The percent inhibition, relative to the untreated control, for P. infestans sporulation from inoculated leaf disks vs. dimethomorph concentration (0.0, 1.0, 10.0, 100.0, and 1000.0 pg ml'l). Dimethomorph was applied as a commercial formulated product (Acrobat 50WP, BASF Corporation) at 48 or 24 hours before inoculation (HBI) or" 24 or 48 ihours after inoculation (HAI) by' a sporangia /’ zoospore suspension cflf P. infestans. Error bars represent Fischer’s LSD (a = 0.05). 67 Percent Inhibition of Sporulation Relative to the Untreated Control 120 100 ~ 80 60: 40: 20 120 100 . 80 60 40 20: 120 100 4 80 : 60 : 40 . 20: a. —C}-— 48 HBI —O— 24 HBI + 24 HAI + 48 HAI b. Pi95-5 C. d. e. l 10 100 1000 0 l 10 100 1000 Concentration Dimethomorph (pg ml—l) 68 i. k. 3 4|..- 2 .l P f O O O O O O O O O O O O O O O 2 O 8 6 4 2 2 O 8 6 4 2 2 1 l 1 1 1 Honusou poumoHuCD msu on w>flumamm cofluoaouomm mo QOHDHQHECH ucoouwm 10 100 1000 0 100 1000 10 Concentration Dimethomorph (pg ml”) 69 antisporulation. No significant differences between isolates, timings, or application relative to the inoculation event were present for the ECSO values of sporulation inhibition (data not shown). The mean ECgo value for the inhibition of sporulation was 49.44 pg ml’1 dimethomorph. Discussion The concentrations required for the inhibition of P. infestans in Vitro ihyphal growth, direct sporangia germination, zoospore encystment, cystospore germination, and inhibition of symptom development and sporulation in Vivo were similar to those previously reported (1,2,29,90,97). Zoosporogenesis was not sensitive to the concentrations of dimethomorph examined, while in Vitro hyphal growth and sporulation, and direct sporangia germination were moderately sensitive to inhibition by dimethomorph, with mean ECw values of 0.45, 0.22, and 0.19. In contrast, zoospore encystment and cystospore germination were both highly sensitive to dimethomorph with ECSO values for most isolates <0.10 pg ml"1 dimethomorph. The higher sensitivity of the zoospore encystment and cystospore stages of the asexual life cycle may be related to the physiology of PhytOphthora at those stages. 70 Specifically, disrupting the extension of hyphal tips or sporangiophore formation will not be immediately lethal to a colony and ir::hs possible that physiological changes in the cytoplasm could temporarily or partially off—set the disruption of cell wall formation. In contrast, disruption of the de novo synthesis of the cell wall, as in zoospore encystment, would be lethal irlaa much shorter time period as zoospores must actively offset osmotic pressure and have limited. energy' reserves (46). As ‘with zoospores, cystospores have relatively' limited. energy' reserves, saprophytic ability, and longevity [Coffey, 1991 #378]. Therefore, the inhibition of germ tube formation would be lethal to each cyst while a sporangium might be able to attempt multiple germinations. It is possible that direct sporangia germination was less susceptible because of the larger physical size and energy reserves of the sporangium in comparison to a zoospore or cystospore. Direct sporangia germination and in Vitro hyphal growth had the smallest ranges in sensitivity between isolates, with >4 and >6—fold differences of EC50 values between the most and least insensitive isolates, respectively. Ln? vdrzr> sporulation, zoospore encystment and cystospore germination all had larger ranges in sensitivity with >10—fold differences. The ranges of 71 dimethomorph sensitivity are much smaller than those reported for phenylamide insensitivity in P. infestans (128) and other Phytophthora species (49,103). However, these studies included isolates with field resistance to phenylamides, and exclusion of those isolates results in a similar sensitivity distribution. The sensitivity ranges determined from disease incidence for Plasmopara Viticola to the strobilurin fungicide azoxystrobin were similar to those presented here (138). Application of 1000.0 pg ml"1 dimethomorph at 24 or 48 hours before inoculation almost completely inhibited symptom development incidence in inoculated leaf disks, while application within 48 hours after inoculation failed to offer significant inhibition. When used to control P. infestans in the field, dimethomorph should be applied in a protectant fashion, reinforcing previously reported results (1,29,124). The ECSO values for the inhibition of in vivo symptom development were much larger than those at the specific stages in the asexual life cycle of P. infestans. While dimethomorph is considered to be slightly systemic, it is also known to have a high resistance to washing off with water following application (29). These results, and the fact that dimethomorph has low solubility in water, indicates that dimethomorph probably has a high affinity 72 for the cuticle of the leaf. Once applied, only a small percentage of dimethomorph is likely to dissolve into any free water on the leaf surface, therefore requiring larger concentrations of dimethomorph for in Vivo efficacy to offset the low solubility. Inhibition of sporulation of P. infestans occurred when dimethomorph was applied between 48 hours before and 48 hours after inoculation at 100.0 pg Inld' or higher. These results are similar to those previously reported (29). Outside of the examined time frame and under field conditions, dimethomorph may not have the same level of antisporulation activity. A study examining antisporulation under field conditions failed to demonstrate any inhibition of sporulation following dimethomorph, or other fungicides, applications (123). Application CHE fungicides under field conditions is unlikely to confer the same homogeneity of leaf coverage as application under controlled conditions. Thus, the lack of antisporulation activity 1J1 the field is chug to either 1) incomplete coverage leaving’ unprotected foliage, 2) sub— efficacious concentrations of dimethomorph on the leaf surface, or a combination of the two. Dimethomorph at low concentrations is highly inhibitory to most stages of the asexual portion of the IR 73 infestans life cycle. Frequent protectant applications of dimethomorph at concentrations exceeding 1000.0 pg mld' would likely inhibit infection by P. infestans in the field and possibly reduce sporulation from previously established infections, but not cure them. The P. infestans isolates examined all had similar sensitivity to dimethomorph in the assays performed. However, low levels of resistance to dimethomorph have been generated in Vitro for P. infestans (141) and other Phytophthora species (21,141), and therefore should be examined further in order to manage resistance development. 74 CHAPTER THREE THE GENERATION, QUANTIFICATION, AND CHARACTERIZATION OF DIMETHOMORPH INSENSITIVITY IN PHYTOPHTHORA INFESTANS AND OTHER PHYTOPHTHORA SPECIES Abstract The generation of dimethomorph insensitive strains of Phytophthora infestans was attempted using ethidium bromide / ultra—violet light mutagenesis and repeated culturing on amended media. Ethidium bromide / UV mutagenesis created two strains of P. infestans with resistance factors >20, i.e. the ratio of the Ergo of the mutant strain to that of the wild—type. For most P. infestans isolates, the rate of growth on dimethomorph amended media increased until the fourth sub-culture. Resistance factors for strains generated from repeated culturing were <8. For most isolates (HE P. infestans, dimethomorph insensitive strains had reduced growth rates on un—amended media, regardless of the level (ME insensitivity. Additionally; virulence on leaf disks and in whole tubers was significantly reduced in >20% of the isolates examined. Insensitivity generation of other Phytophthora species was attempted with repeated culturing on amended media and one strain of P. erythroseptica occurred through colony sectoring with a 75 resistance factor >7. Introduction Resistance to fungicides in fungal and Oomycete plant pathogens has become increasingly more common following the release of systemic fungicides in the 1960’s (45). Field resistance to every major systemic fungicide class has occurred in at least one species of plant pathogenic fungi or Oomycetes (109,122), leading to drastic shifts in fungicide programs for many pathosystems, such as potato late blight (61) and Phytophthora blight of bell pepper (103). The migration of phenylamide insensitive populations of Phytophthora infestans, the causal agent of potato and tomato late blight, from Mexico to the rest of North America (61), necessitated cultural control methods and crop jprotection strategies that relied. primarily’ on protectant foliar fungicide applications (116). Concurrently, the agrichemical industry developed and released systemic fungicides in.en1 attempt tx> replace the phenylamides. Dimethomorph, one of the fungicides released in response to phenylamide resistance, is a cinnamic acid derivative with a high specificity to the genus Phytophthora and certain members of the Peronosporaceae (1). Cells of P. infestans lacking cell walls, such as 76 zoospores and artificially generated protoplasts, were not affected by dimethomorph (2) and it was concluded that dimethomorph disrupted cell wall formation by interfering with the molecular arrangement of wall components and not the inhibition of component synthesis (90). Insensitivity to dimethomorph was generated with ultraviolet (UV) light mutagenesis in one isolate of P. parasitica (21) and with chemical mutagenesis in one isolate of P. capsici Leonian (141). In both cases, an approximate 20—fold decrease in sensitivity resulted. Virulence of dimethomorph insensitive P. parasitica strains was equal to, or less than, the wild-type. In addition, insensitivity in P. parasitica was stable in the absence of dimethomorph through several in Vitro sub-cultures for three months. Conversely, attempts to generate insensitivity using mycelial adaptation in P. infestans and P. capsici resulted in small (<2—fold) decreases in sensitivity to dimethomorph (141). The use of only one culture of P. infestans and P. capsici in the latter experiment may have limited the results as genetic variability was absent. The objectives of this study were to 1) generate strains of P. infestans insensitive to dimethomorph using ethidium bromide / UV mutagenesis or mycelial adaptation 77 and 2) determine the level of resistance generated and examine virulence of P. infestans insensitive strains on potato foliage and tubers. Materials and Methods Media Preparation Experiments involving hyphal growth were performed on modified (CHAPTER TWO) rye B agar (2,19) because of the relatively rapid growth compared to synthetic media. All plates for each sub—culture on dimethomorph amended media or run of the baseline sensitivity assay were prepared from the same batch of media in order to reduce variability within each run of an experiment. Fungicide amended media was prepared as described (CHAPTER TWO). Generation of Insensitivity: UV Mutagenesis Cultures of P. infestans for mutagenesis were grown on rye B agar at 21°C until complete colonization of the media. Plates were flooded with 5.0 ml of a 1 pg ml’1 ethidium bromide solution (in sterile diHZO), allowed to dry, and exposed to 254 nm ultraviolet (UV) light (2.03 x 103 ergs cm’2 5'1) for five minutes. From each isolate, four 4.0 mun diameter colonized agar plugs were transferred to rye B media amended with 0.0 or 10.0 pg ml"1 dimethomorph. Non- 78 exposed cultures were also transferred onto control and amended rye B media. Cultures were incubated in the dark and examined on seven—day intervals for colony diameter and rapid growth or sectoring on the fungicide amended media. The fastest growing sector of each isolate from the fungicide amended media was used for further assays, and hereafter labeled strain “UV”, e.g. Pi95—7W. Generation of Insensitivity: Sub-Lethal Culturing Using previously determined effective concentration for a 50% reduction in colony diameter (ECso) values for inhibition of in Vitro growth by dimethomorph (CHAPTER TWO), culturing of Phytophthora sp. on media amended with a highly inhibitory (1.0 pg ml’l), sub-lethal concentration of dimethomorph was performed to select for insensitivity in cultures. Initial wild—type isolates were previously characterized (Table 6), two sub—cultures from re-isolation from infected host tissue, and labeled strain “WT”, e.g. Pi95—7WT. Colonized agar plugs, 4.0 mm in diameter, from the margin of an actively growing colony were transferred mycelium-side down onto modified rye B media amended with 0.0 or 1.0 pg ml"1 dimethomorph and incubated at 21°C in the dark with three replicate plates per concentration. Colony diameter was measured after 11 days for P. infestans, and 79 Table 6. Isolate: identification. code, Phytophthora species, mating type (if applicable), P. infestans genotype (if applicable), and State in which isolated (U.S.A.). I 8°11; te Ph}; :3; 1:11:13 Mgr-$219 Genotype2 Source Pi88 P. infestans A1 U81 ND Pi95-5 P. infestans A1 U81 MI Pi671 P. infestans A1 U814 WA Pi458 P. infestans A2 U817 ID Pi670 P. infestans A2 U87 OR Pi213 P. infestans A2 U88 CO Pi94—4 P. infestans A2 U88 MI Pi95-7 P. infestans A2 U88 MI Pi97-2 P. infestans A2 U88 MI Pi98—1 P. infestans A2 U88 MI Pi98-2 P. infestans A2 US8 MI Pcap P. capcisi A2 n/a MI Pcac X P. cactorum PI n/a MI Pcac Y P. cactorum H n/a MI Pe96-2 P. erythroseptica II n/a MI Pe00-1 P. erythroseptica Ii n/a ID 1. Homothallic species designated by 2. Allozyme-based genotype”. 80 “H” for other Phytophthora species at 5 and 11 days for 0.0 and 1.0 pg mflfl, respectively; Isolates grown (n1 control (0.0 pg ml'l) and sub-lethal dimethomorph (1.0 pg ml‘l) amended media. were labeled strains “CT” and “SL”, respectively, e.g. Pi95#%n and Pi95-7SL. After the colony diameter was measured, one plate from each concentration was used to sub—culture the strain onto media with the same concentration of dimethomorph, for ten sub—cultures total. Portions of the colony were chosen for sub—culturing in an attempt to retain the wild—type colony phenotype of each isolate, while selecting sectors with higher growth rates. In the absence of accelerated growth on dimethomorph, plugs were chosen randomly from the margin of the colony. Following the tenth sub-culture, the cultures were examined for baseline sensitivity and pathogenicity. Dimethomorph Sensitivity Assays Prior to performing baseline sensitivity assays, all cultures were grown on non-amended media. Baseline sensitivity of in Vitro growth to dimethomorph was determined as previously described (CHAPTER TWO). Baseline sensitivity assays were conducted three times with three replication plates per run for each strain of every 81 isolate, e.g. Pi95-7WT, Pi95—7CT, Pi95—7SL, and Pi95-7W. EC50 values were calculated as previously described (CHAPTER TWO). Resistance factors were calculated for all strains using the following formula: _ ECsz ECsoWT RF where ECSOX is the ECSO value of the strain being examined, and ECSOWT is the EC50 value of the wild—type strain of that isolate. Analysis of variance was performed on the final colony diameter (FCD) measurement (mm) and the ECso values calculated (Proc GLM - SAS/Stat, SAS Institute, Cary, NC, U.S.A.) at a = 0.05 via pair—wise comparisons using Fisher’s LSD. Prior to the combined ANOVA, each run was analyzed separately and the homogeneity of variance was confirmed using the F Max test (68). Virulence Assays Leaf disks for inoculation, and sporangia/zoospore suspensions were prepared as pmeviously described (CHAPTER TWO) for a final concentration of 1.0 x 104 sporangia mld. The sporangia/zoospore suspension from each strain of every isolate was applied (50 pl) to four leaf disks per replicate, with three replicates. The experiment was repeated twice. Inoculated leaf disks were incubated at 82 21°C light / 18°C dark (12 hour cycles) and were examined with a dissecting microscope at 96 hours after inoculation for symptoms and signs of infection by P. infestans, such as sporulation. Pathogen identity was confirmed with a compound microscope. Leaf disk inoculations were not performed (n1 ethidiuml bromide / [A] generated. strains as sporulation was disrupted for most isolates. An analysis of variance on the number of leaf disks infected was performed as described. Pathogen free potato tubers (cv. Snowden), approximately 6.35 cm diameter, were surface sterilized as described (CHAPTER TWO). Colonized portions of rye B agar, 1.0 cm9, were excised from the margin of an actively growing colony of P. infestans and placed into a sterile 1.0 ml syringe with an 18% gauge needle. A wound approximately 1.0 cm from the apex of the tuber and 0.5 cm deep into the tissue was created by stabbing the tuber with a pair of sterile forceps. Three tubers jper :replicate ‘were inoculated by extruding approximately 0.1 ml of macerated colonized agar through the needle into the wound and placed into a covered plastic container. There were three replicates per Inulemxi the experiment was repeated twice. The tubers were incubated in the dark for seven days at 18%: and tuber surface and. cross—sections were evaluated for 83 symptoms and signs of infection by PR infestans. Pathogen presence was verified with a compound microscope when applicable. Tuber inoculations were not performed on ethidium bromide / UV generated strains. An analysis of variance on the number of tubers infected was performed as described. Results Following ethidium tmomide / [A] light treatment, all isolates except Pi88 grew on non—amended media rye B media (data not shown). Vflmxl cultured. on. non—amended. media, treated cultures exhibited disrupted colony morphology and lower growth rates compared to untreated cultures. Of the isolates that grew following ethidium bromide / UV light treatment, only Pi458 and Pi98-2 failed to grow on media amended with 10.0 pg ml’l dimethomorph. However, most isolates required more than 14 days to develop sufficient growth for sub—culturing. Sectors with increased growth rates on dimethomorph amended media were common, but not always present. For all isolates, regardless of species, the rate of growth on media amended with 1.0 pg mld‘ dimethomorph (strain 8L) increased with sub—culture number, e.g. Pi213 (Figure 5). With most isolates, the largest increase 84 Figure 5. Representative colony growth, relative to the untreated control, in diameter (mm) dayd’vs. cycle number for isolate Pi213 when grown on rye B media amended with 0.0 or 1.0 pg mld'dimethomorph. Error bars represent one standard deviation for replicate plates. . 0.0 pg ml’l dimethomorph O 1.0 pg ml'l dimethomorph 1.0-E E I I I I i E E I Colony Diameter Growth (mm dayq) Relative to the Untreated Control 0.0 I I T T l I I I I I 1. 2 3 (4 5 6 '7 8 9 10 Cycle Number 85 occurred between the initial and second sub—cultures, while changes at subsequent sub-cultures were smaller or absent. A fast growing sector appeared from one isolate of P. erythroseptica, Pe00-1, following sub—culture five (x1 dimethomorph amended media, hereafter denoted strain “RS”. PeOO—IM; exhibited different colony morphology than all other strains of Pe00-l, and consistently died when cultured on non—amended media, or stored for more than 21 days at 18°C. The final colony diameter (FCD) of the sub—cultured control strain of each P. infestans isolate *was either similar to or significantly larger (Fischer’s LSD, a = 0.05), than the corresponding wild-type strain on non- amended media, while the ethidium bromide / UV generated strain.luui the smallest FCD (Figure 6). The sub-cultured on dimethomorph amended media strains of P188, Pi95-5, and Pi97-2 had a significantly smaller FCD than both wild-type and control sub—cultured strains on non-amended media. (kl media amended with 0.1 pg mflfl dimethomorph, differences in FCD between wild-type and strains sub—cultured on control or dimethomorph amended media became smaller, or non- significant, while the ethidium bromide / UV generated strains had the lowest FCD values. At 1.0 pg mld' 86 Figure 6 (a—k). Final colony diameter of P. infestans isolates for the wild—type strains (O), repeated culturing control strains ([3), repeated culturing on 1.0 pg ml'l dimethomorph amended media strains (A), and UV / ethidium bromide generated strains (0) at 11 days after inoculation” Isolates were grown on.13maIB media amended with 0.0, 0.1, 1.0, and 10.0 pg ml'l dimethomorph. Error bars represent Fischer’s LSD (a = 0.05). 87 11 Days After Inoculation (mm. Final Colony Diameter 50 40~ 30: 20~ 10: 50 40: 30- 20- 10- 50 40- 30- 20: 10- a. P188 + Strain -4P— Strain -4¥- Strain -<>— Strain WT CT SL UV LSD = 4.00 0.05 d. Pi458 Concentration Dimethomorph (#9 ml”) 88 O 0.1 1 10 9. 9194—4 k. P198-2 50 soflumHSUOGH kuwd when Ha .AEEV 7 3 _ 1 5 2 9 .1 .1 P P f h 0 AU no 0 AU no 0 nu nu 0 nu no 0 nu no 0 4 a3 92 l R. 4 .3 oz 1 :3 4 «3 oz 1 Hmumficao ScoHou Hmcflm 10 10 Concentration Dimethomorph (pg mlq) 89 dimethomorph, the sub—cultured on dimethomorph amended media strain of each isolate typically had a significantly larger FCD than all other strains. If growth occurred on media amended with 10.0 pg ml‘l dimethomorph, it was always a sub-cultured on dimethomorph amended media or ethidium bromide / UV generated strain, with the latter typically having the larger FCD. Of the other Phytophthora species examined, FCD curves were similar to those of P. infestans, and wild—type and sub—cultured on non-amended media strains had similar FCD values at each concentration (Figure 7). The sub- cultured on dimethomorph amended media strains typically exhibited larger FCD values at 1.0 pg ml’l dimethomorph, but not other concentrations. The only strains that grew at 10.0 pg ml"1 dimethomorph were the sub—cultured on dimethomorph amended media strains of Pcap A2 and Pe96-2, Pcap A231, and Pe96—2SL, and the strain that spontaneously sectored from PeOO—lsL, PeOO—lps. The P. infestans isolates Pi458, Pi94—4, and Pi98—1 had significantly larger EC50 values for the sub-cultured on dimethomorph amended media strain than both wild—type and sub—cultured on control media strains (Table 7). The calculated EC50 values for in Vitro growth of sub-cultured 90 Figure 7 (a—e). Final colony diameter at 5 days after inoculation of the wild-type strains (0), repeated culturing control strains (El), repeated culturing on 1.0 pg ml'1 dimethomorph amended media strains (A), and fast growing sector of P. erythroseptica that spontaneously appeared (0) for P. capcisi (Pcap), P. cactorum (Pcac), or P. erythroseptica (Pe). Isolates were grown on rye B media amended with 0.0, 0.1, 1.0, and 10.0 pg ml"1 dimethomorph. Error bars represent Fischer's LSD (OL = 0.05). 91 11 Days After Inoculation (mm). Final Colony Diameter 50 40: 30- 20- 10: 50 40 30- 2O 10: 50 40~ 30: 20~ 10: a—O—-Strain WT -4r— Strain CT —4¥- Strain SL -<>— Strain RS LSDOfiS = 3.27 Concentration Dimethomorph (pg mlq) 92 10 Table 7. Calculated EC50 for in Vitro hyphal growth (diameter) and resistance factors for all strains of P. infestans isolates examined. Statistical comparisons shown within each isolate using Fischer’s LDS (a,= 0.05). 7 - Isolate Strain1 EC” ,1 Resistargce (pg 1111) Factor P188 WT 0.347 a4 - CT 0.373 a 1.075 SL 0.770 a 2.219 UV *5 * Pi95—5 WT 0.107 b - CT 0.186 b 1.138 SL 0.800 b 7.447 UV 2.479 a 23.168 P1671 WT 0.703 b - CT 1.049 b 1.492 SL 1.083 b 1.541 UV 2.290 a 3.257 Pi458 WT 0.843 b - CT 0.555 b 0.658 SL 1.818 a 2.157 UV * * P1670 WT 0.617 ab — CT 0.544 b 0.882 SL 1.494 a 2.422 UV 1.213 ab 1.966 P1213 WT 0.680 b — CT 0.869 b 1.278 8L 0.979 b 1.439 UV 4.368 a 6.424 93 Table 7 (cont’d). . 1 EC“? Resistance Isolate Strain (ugnfld) Factora P194—4 WT 0.442 c:4 — CT 0.865 c 1.958 SL 2.851 1) 6.452 UV 9.921 a. 22.446 Pi95—7 WT 0.613 13 - CT 0.617 1) 1.007 SL 0.742 k) 1.210 UV 4.430 a 7.227 Pi97—2 WT 0.237 k) - CT 0.370 ab 1.561 SL 0.850 ab 3.586 UV 1.237 a. 5.219 P198—1 WT 0.558 1) — CT 0.535 1) 0.958 SL 1.653 a 2.960 UV 1.984 a 3.556 Pi98-2 WT 0.587 a — CT 1.259 a. 2.145 SL 1.253 a 2.135 UV *5 * 1. WT = wild-type, or CT and SL = sub-cultured 10 times on media amended with 0.0 or 1.0 pg nflfl dimethomorph, respectively, or UV = mutated with ethidium bromide and UV light. 2. Effective Concentration to reduce colony diameter to 50% of fungicide un—amended media. 3. Resistance factor = ECW of manipulated strain / ECm of WT strain. 4. Means followed by the same letter are not significantly different within each isolate using Fischer's LSD (a = 0.05). Comparisons between strains of different isolates are not shown. 5. Attempted nmtagenesis was lethal with this isolate or In) growth occurred on 10.0 pg ml‘1 dimethomorph. 94 on dimethomorph amended media strains were numerically larger than those of the wild-type and sub—cultured on control media strains for nine of 11 isolates of P. infestans, indicating 51 decrease 1J1 sensitivity’ to dimethomorph. through. sub-culturing. The isolates Pi94-4 and Pi98—2 showed large, but not significant, increases in EIho of the sub-cultured on control media strain in comparison to the wild—type strain. The ethidium bromide / UV generated strains from five of the eight isolates had significantly" higher EC“) values than the other strains within each isolate. For all isolates of P. infestans, except P198—2, the: calculated. resistance factor' (RF) ‘was larger for the sub—lethal generated strain than the control strain. The RF values for the ethidium. bromide / UV generated strains were larger than all other strains for seven of the eight isolates with such strains. The isolates Pi95-5 and Pi94-4 had the largest RF factors for sub—lethal and ethidium bromide / UV generated strains with 7.447 and 23.168, and 6.452 and 22.446, respectively. The sub—cultured on dimethomorph amended media strains of the other Phytophthora species examined had significantly larger ECw values compared to both the wild- type and the sub—cultured on control media strains within isolates (Table 8). Within. the isolate Pe00-1, the RS 95 Table 8. Calculated EC50 for in Vitro hyphal growth (diameter) and resistance factor, for all strains of P. capcisi (Pcap) , P. cactorum (Pcac) , and P. erythroseptica (Pe) isolates examined. Statistical comparisons shown within each isolate using Fischer's LDS (a,= 0.05). . 1 ECso2 Resistance Isolate Strain (pg ml") Factor? Pcap A2 WT 0.234 b4 - CT 0.269 b 1.150 SL 1.004 a 4.291 Pcac X WT 0.247 b - CT 0.235 b 0.951 SL 0.662 a 2.680 Pcac Y WT 0.259 b — CT 0.249 b 0.961 SL 0.700 a 2.703 Pe96—2 WT 0.234 b - CT 0.264 b 1.128 SL 0.648 a 2.769 Pe00-1 WT 0.176 c - CT 0.201 c 1.142 SL 0.662 b 3.761 RS 1.253 a 7.119 1. WT = wild—type, or CT and SL sub-cultured 10 times on media amended with 0.0 or 1.0 pg ml‘1 dimethomorph, respectively or R = fast growing sector of P. erythroseptica that spontaneously appeared. 2. Effective Concentration to reduce colony diameter to 50% of fungicide un-amended media. 3. Resistance factor = ECw of manipulated strain / EC“ of WT strain. 4. Means followed by the same letter are not significantly different within each isolate using Fischer’s LSD (a = 0.05). Comparisons between strains of different isolates are not shown. 96 strain had a significantly larger EC“) than the other strains. Pe00-1M;also had the largest resistance factor. The ability to infect and cause symptoms on potato leaf disks (cv. Snowden) was limited for most isolates, regardless of strain, as only four of 11 wild-type strains resulted in 75% of more leaf disks infected (Table 9). The wild—type strains of isolates, Pi95—5, P1671, P1670, Pi94- 4, Pi97—2, and Pi98-2 successfully infected 50% or less of leaf disks, indicating low virulence of the wild—type strain, while only the wild-type strain of Pi458 infected all leaf disks. In comparison to the wild—type strains, the sub—cultured on dimethomorph amended media strains had significantly reduced percent leaf disks infected for five of 11 isolates of P. infestans. However, the sub-cultured on control media strains of isolates Pi458 and Pi95—7 infected significantly fewer leaf disks than the wild—type strain. Infection and symptom development of tubers following inoculation of mycelium was more efficient than leaf disk inoculation with the wild—type strains of eight of 11 isolates causing symptoms in all tubers inoculated. The sub—cultured on dimethomorph amended media strains of seven isolates infected significantly less tubers compared to the wild-type strain of each isolate. Some of the sub-cultured 97 Table 9. Percent of infection by P. infestans isolates and. strains, expressed an; percent incidence, when inoculated onto both excised leaf disks and into whole tubers (>4.0 cm, <6.5 cm). Statistical comparisons shown within each isolate using Fischer’s LSD (a = 0.05). 98 Table 9. Percent Incidence of Infection2 - 1 Isolate Strain Leaf Disks ers P188 WT 58 a3 100 a CT 50 a 33 b SL 58 a 22 b Pi95—5 WT 25 a 33 a CT 8 a 22 ab SL 25 a 0 b P1671 WT 50 a 100 a CT 50 a 100 a SL 17 b 100 a Pi458 WT 100 a 100 a CT 8 b 89 a SL 25 b 44 b P1670 WT 50 a 100 a CT 50 a 67 b SL 33 a 22 c P1213 WT 92 a 100 a CT 75 a 100 a SL 25 b 100 a Pi94-4 WT 42 a 56 a CT 42 a 56 a SL 8 a 44 a Pi95—7 WT 92 a 100 a CT 58 b 78 a SL 42 b 44 b Pi97-2 WT 17 a 67 a CT 17 a 67 a SL 25 a 22 b Pi98—1 WT 92 a 100 a CT 75 a 56 b SL 8 b 33 b Pi98-2 WT 42 a 100 a CT 33 a 100 a SL 33 a 89 a 1. WT = wild-type, or CT and SL = sub-cultured 10 times on media amended with 0.0 or 1.0 pg ml“1 dimethomorph, respectively. 2. Percent of leaf disks (out of four) or tubers (out of three) with symptoms and signs of infection by PL infestans 96 or 168 hours after inoculation with a sporangia / zoospore suspension or mycelial homogenate, respectively. 3. Means followed by the same letter are not significantly different within each isolate using Fischer’s LSD (a = 0.05). Comparisons between strains of different isolates are not shown. 99 on control media strains infected significantly lower numbers of tubers when compared to the corresponding wild- type strains. Discussion The development of insensitivity to dimethomorph was demonstrated in P. infestans following ethidium bromide / UV light exposure of mycelium. Mutagenesis created strains of two P. infestans isolates with resistance factor (RF) values >20. These values are similar to previously reported RF values generated for P. parasitica using UV irradiation (21), and P. capsici using chemical mutagenesis (141). In comparison, the resulting RF cfif.P. capsici for the phenylamide fungicide metalaxyl was >100 in the latter study, and a RF value of 20 for dimethomorph was considered moderate (141). All isolates of P. infestans that survived the mutagenesis treatment had reduced growth rates on non- amended media and disrupted colony morphology compared to the wild-type. On non-amended media, both factors could be attributable to 1) mutations unrelated to dimethomorph insensitivity negatively affecting growth or 2) that the dimethomorph insensitivity mechanism(s) themselves disrupted growth. Particularly in the former situation, one might have expected additional fitness reductions, lOO including virulence (99), as mutations generally reduce the fitness of an organism. The loss of the ability to sporulate supports this idea. Excluding the composition of the cell wall, little is known about the biochemistry involved with cell wall formation in Phytophthora (7) and therefore the genetic basis for insensitivity to dimethomorph may be difficult to discern. The development of insensitivity to dimethomorph was demonstrated in P. infestans and other Phytophthora species following repeated sub—culturing on media amended with a sub—lethal concentration (ME dimethomorph. A. significant decrease in sensitivity (in Vitro growth) occurred in three of 11 isolates of P. infestans and all isolates of the other Phytophthora species examined. Almost all isolates had numerically decreased sensitivity compared to the wild— type. However, the resistance factor for most isolates was less than 3.0. The only strain generated from a distinct sectoring of the colony was formed from the P. erythroseptica isolate Pe00—1. The rapid increase in final colony diameter by the third sub-culture cycle on amended media indicates 1) a physiological adaptation to in Vitro growth on dimethomorph or 2) selection of the genetic factors responsible for insensitivity following the de novo development of nuclear lOl or cytoplasmic insensitivity. Culturing the isolates on non-amended media prior to the in Vitro growth sensitivity assessments was used to reduce the effects of physiological adaptation, but without detailed investigation of cell wall composition and formation it is difficult to discern the true cause and should be further investigated. The ECSO metric of in Vitro colony growth, while commonly used in fungicide sensitivity studies (49,81,141), is sensitive to changes in the growth rate of the fungus because of the percentage inhibition transformation relative to the control colonies. A mutant strain with a reduced growth rate may have the same colony diameter as the wild—type at all concentrations of fungicide and yet the smaller colony diameter on non-amended media results in a lower percent inhibition. Therefore, when calculating the ECso value using percent inhibition versus concentration, a larger ECSO is calculated. Similarly, the RF value, being calculated using the ECSO, is dependent on both reduced growth rates on non-amended media and to extremely sensitive wild—type isolates. For example, the isolate Pi95—5 had reduced growth on non—amended media for both repeated culturing on dimethomorph amended media and ethidium bromide / UV generated strains and the lowest wild-type ECSO of any isolate. The former strain of this 102 isolate had an EC50 that was not significantly different from either the wild—type or repeated culturing on control media strains, and yet had the largest RF value of that strain type for all P. infestans isolates. As an alternative to ECSO, the growth of an isolate at a pre- determined (discriminating) concentration or inoculation of fungicide treated plant tissue would likely be better indicators of sensitivity, as previously described (120). The low virulence of several wild—type strains of P. infestans on leaf disks was not surprising as variable virulence (85) and pathogenic specialization (94) have been documented, even on potato cultivars with no known R—genes, such as Snowden. Possible reductions in virulence of the strains generated by repeated sub-culturing, regardless of fungicide amendments, was expected as P. infestans is known to lose virulence following long-term in Vitro culturing (46). However, since most isolates sub—cultured on non— amended media retained near wild-type virulence, any reduction in virulence of the strains sub-cultured on dimethomorph amended media was probably caused by the mechanism(s) responsible for dimethomorph insensitivity. Phytophthora infestans requires the formation of viable sporangia for pathogenicity on foliar tissue (107), and any putative changes in cell wall formation resulting from the 103 development of dimethomorph insensitivity might be disruptive to sporangia formation. Additionally, cell wall components are known to induce resistance in potato (50) and it is possible that any changes which occurred as a result of dimethomorph insensitivity allowed for the release of such compounds. The tuber inoculation test, while not biologically accurate (91), was performed because differences in foliar and tuber susceptibility have been noted (41,43) and may be important. The lack of correlation between the foliar and tuber inoculations is probably related to the differences in the assays in that the tuber inoculation did not require the development of viable sporangia and/or zoospores for infection. Instead, it only required the pathogen to overcome any tuber defenses, and since the periderm was eliminated, the major tuber defense mechanism was removed. In situ foliar and tuber infection studies would be required to more fully evaluate this relationship. The generation of insensitivity to dimethomorph in Phytophthora is possible through both repeated selection on sub-lethal amended media and chemical or UV induced mutation. The low amount of insensitivity that developed indicates that resistance may be quantitative and possibly multigenic, as with the dimethylation inhibitor fungicides 104 commonly used to control true fungi (111). If this hypothesis is true, one would expect resistance in the field to develop through directional selection (112) and occur“ in small increments. .Also, resistance :management techniques such as block treatments and co-application of dimethomorph with protectant fungicides would likely be effective. CMrrently, the development of insensitivity to dimethomorph in P. infestans is unlikely for most potato growing regions of the United States because growers rely primarily on protectant fungicide applications and the use of systemic fungicides, including dimethomorph, typically is limited. However, as use of protectant fungicides is restricted because of their larger environmental impact and higher rates required for control in comparison to many systemic fungicides, dimethomorph. usagee:may' increase and active resistance management strategies may require implementation. 105 Ik CHAPTER FOUR FIELD OPTIMIZATION OF DIMETHOMORPH FOR THE CONTROL OF PHYTOPHTHORA INFESTANS ON POTATO: APPLICATION RATE, INTERVAL, AND MIXTURE PARTNERS Abstract Aspects concerning the field use of dimethomorph for the control of foliar and tuber potato late blight were examined. wmen application rate and interval manipulation for a season-long dimethomorph / mancozeb mixture was performed, the rate increasing through the season and 80% of full rate programs had equal final foliar blight control as the full rate jprogram, regardless of interval. ‘Phe minimum application rate for control equivalent to the full 1 week'l. When dimethomorph was rate program was 1.34 kg ha’ tank-mixed with one of three protectant fungicides and integrated into a chlorothalonil—based late blight control program, all programs were as effective as the season-long chlorothalonil program. NOne of the mixture partners were more effective than the others. When tank-mixed with pyraclostrobin and alternated with chlorothalonil applications, rate reduction to 50% of full rate gave foliar' blight control equivalent to 61 full rate, for“ a dimethomorph. / pyraclostrobin. mixture and. pyraclostrobin 106 alone. Introduction Chemical control strategies for potato late blight changed with the migration of phenylamide insensitive strains of Phytophthora infestans into the United States (61,79). In the early 1990’s, several new chemistries were released, including dimethomorph, a cinnamic acid derivative. Dimethomorph was highly selective towards certain members of the Peronosporales (2), had a moderate level of apoplastic systemicity (29), and inhibited many stages of the life cycle of P. infestans via the disruption of cell wall formation (90). Dimethomorph was considered to disrupt one or a few biological processes within the target organisms because it fit the characteristics of other systemic fungicides in that it was biologically active at low concentrations (2) and moderate insensitivity could be generated in Vitro relatively easy (21). To reduce the risk of resistance development, dimethomorph was initially released in the United States for emergency use on potatoes to control P. infestans as a pre—mixed wettable powder consisting of 60% mancozeb and 9% dimethomorph by weight. The optimal usage protocols of the dimethomorph / mancozeb mixture for controlling late blight of potatoes on a chipping variety under Michigan growing conditions had 107 not been completely elucidated. In 2001, dimethomorph was released as a single active ingredient (a.i.) product with a requirement that it must be applied with a fungicide having a different biological mode of action, excluding the phenylamides. The most conunon potato late blight fungicide program used by Michigan growers is a chlorothalonil-based program with applications occurring on a five to ten day interval, depending on weather conditions and epidemic development (88). The incorporation of systemic fungicides into the fungicide program is common. Systemic fungicide applications are often timed to correspond to weather-based disease thresholds (6) and specific points within the growing season, such as at tuber initiation and immediately prior to foliar desiccation. The dominance of chlorothalonil over other protectant fungicides in Michigan and the higher cost of the pre—mixed dimethomorph / mancozeb product encouraged the investigation of dimethomorph applications with other mixture partners and at multiple rates within a chlorothalonil program. Protectant fungicides such as chlorothalonil and mancozeb, two potential mixtures partners for dimethomorph, are considered probable carcinogens by the Environmental Protection Agency (EPA) and their use may eventually be 108 restricted (62). Such restrictions have expedited the development of alternative chemistries with lower environmental impact. The introduction of fungicides that inhibit the Q0 site of the cytochrome bcl complex, including the strobilurins (54), offered growers one such alternative. Strobilurins have both high field efficacy when used in a protectant fashion (24) and a high resistance development risk (119). Strict anti—resistance programs have been mandated to eliminate or delay the onset of resistance including: co—application and alternating applications with fungicides having different modes of action, and limiting the rate and number of applications within a season (13). Dimethomorph has a different mode of action than Qo inhibitors (97). Therefore, there is potential to use dimethomorph as a mixture partner for the recently released strobilurin fungicide pyraclostrobin (F500). Rate reduction of a dimethomorph / pyraclostrobin mixture within a protectant—based late blight control program offers an economically feasible putative resistance Inanagement strategy by reducing the Q0 resistance selective jpressure placed. on the population. However, efficacious rates are undetermined. The objectives of this study were to 1) examine zapplication rate and interval manipulation of a 109 dimethomorph / mancozeb mixture in order to optimize efficacy , 2) compare three non—systemic fungicide mixture partners for dimethomorph when applied at three points within a chlorothalonil based late blight control program, and 3) examine rate reductions of a dimethomorph / pyraclostrobin mixture when alternated with chlorothalonil in order to determine the minimal required for effective late blight control. Materials and Methods Experimental Design and Agronomic Management Cut potato seed (cv. Snowden) was planted at the Michigan State University Muck Soils Experimental Station, Bath, MI, between 3 - 10 June, 1999, 2000, and 2001. Treatments were replicated four times in a randomized complete block design. Inoculation, assessment of foliar late blight, maintenance pesticide application, and harvest dates were similar for all years. Treatments were applied to two—row by 6.10 m plots (90 cm row and 23 cm plant spacing). The two—row plots were separated by a 1.5 m unplanted row. Fungicides were applied with an ATV rear- mounted spray boom (R&D Sprayers, Opelousas, LA, U.S.A.) which traveled at 1 m 5‘1 and delivered 230 1 ha'1 (3.5 kg cm“2 pressure) with three XR11003VS nozzles (Spray Systems, 110 Pomona, CA, U.S.A.) per row positioned 45 cm above the canopy: Fungicide applications commenced approximately 30 days after planting (DAP) and were applied.cn1&a seven-day application interval unless otherwise noted. Plots were irrigated as necessary to maintain canopy and soil moisture conditions conducive to the development of foliar (136) and tuber (91) late hflight using turbine rotary garden sprinklers (Gilmour Group, Somerset, PA, U.S.A.) at 1055 1 (H20) ha“l hr'l. Plots were managed under standard potato agronomic practices for the region (88,124). At 85 and 92 DAP diquat bromide was applied at 0.56 a.i. kg ha‘1 to desiccate foliage and ease harvesting. Plots were harvested approximately 30 days after the initial desiccant application. Inoculation Inoculum for a mixed isolate zoospore suspension of phenylamide—insensitive P. infestans US8/A2 genotype (59) was produced by growing previously isolated and characterized isolates on modified rye B agar as described (CHAPTER TWO). Plots were inoculated. by injecting the zoospore suspension into the irrigation system. 1 x 106 zoospores ha&, 45 - 57 DAP. lll Data Collection and Metric Generation Emergence was evaluated after the majority of tubers had appeared and typically 80% of the seed pieces planted had successfully emerged 21 DAP. It was assumed that missing plants were due to skips during planting, rotted seed tubers, or those lacking eyes. In plots where less than 17 plants had emerged, pre—sprouted tubers were hand planted into the sections lacking sprouts so that all rows had a minimum of 17 plants per row. Subjective visual evaluations of percentage foliar late blight were made four to five days after inoculation and repeated on a five to seven day interval until almost complete defoliation in untreated plots. The Relative Area Under the Disease Progress Curve (RAUDPC) metric was calculated for all plots using the following formula: wikTi-Ti-IXR' —1)+ (Ti—Ti —I)(Pi—Pi - I) RAUDPC= "7’ 2 (Tflnul — To)(100) where T0 was the day of inoculation (zero point for calculation), T1- was the 1th day after inoculation when an estimation of percent foliar late blight was made, Tfinal was the number of days after inoculation at which the final foliar assessment was taken, and Pi was the estimated percent foliar late blight at Ti. 112 About 115 DAP, one row of each plot was mechanically harvested and sorted into three size classes: culled tubers were <4.0 cm in any plane and dropped back onto the ground, B grade tubers were 4.0 - 6.5, and A grade tubers were >6.5 cm. Tubers from A and B size classes were placed into separate mesh bags and incubated for 14 - 21 days at 15%: / 80% relative humidity to encourage the development of symptoms. Tubers within each size class were weighed, counted, and visually assessed for infection In! P. infestans by examining the tuber surface and internal tissue for symptoms and/or signs. The number of tubers per plant for each size class was calculated and used for all calculations involving tuber number or yield in order to reduce variability' due to differences in rthe number of plants per plot. Data Analysis Analysis of variance (ANOVA) was calculated (Proc GLM - SAS/Stat, SAS Institute, Cary, NC, ULS.A.) at (x = 0.05 'via pair—wise comparisons using Fisher’s LSD and treatments were then arranged into statistical groupings. The F Max test (68) was used to determine if data from the repeated trials could be combined. 113 Fungicide Trials Four separate trials were conducted: 1) dimethomorph / mancozeb rate manipulation (DMR), 2) dimethomorph / mancozeb rate and interval manipulation (DMRI) , 3) dimethomorph mixture partner and rate trial (DMP), and 4) dimethomorph / pyraclostrobin rate trial (DSR). Because of the complexity' of fungicide jprograms, abbreviations ‘were created for treatments to ease discussion. The DMR trial was conducted in 1999 and 2001 and examined season-long rate manipulation for a dimethomorph / mancozeb pre-mixed commercial product. The DMRI trial was conducted in 2000 and 2001 and was a nwdified version of the DMR trial that incorporates both five and seven day application intervals in addition to different rate manipulations. For both the INC? and DMRI trials, actives were applied using a commercial product consisting of 60% mancozeb and 9% dimethomorph by weight, pre—mixed wettable powder fungicide. The maximum label rate (U.S.A.) for a single application is 1.74 kg ha'1 of total actives (2.52 kg ha"1 formulated product). The DMP trial was conducted in 2000 and 2001 and examined dimethomorph at two rates when tank— mixed with three different protectant fungicide partners; mancozeb, chlorothalonil , and metiram, within a chlorothalonil season-long late blight control program. 114 Applications of the dimethomorph / protectant fungicide mixtures occurred corresponding to the primary inoculation event and immediately prior to desiccation. The DSR trial was conducted only in 2001, and examines dimethomorph / pyraclostrobin mixtures at varying rates when alternated with chlorothalonil . Details of all combinations , application frequencies and rates are included in Tables 1- 4 . Results Dimethomorph/Mancozeb Rate Manipulation (DMR) All experimental factors passed the F Max test and data from different years were combined (data not shown). All fungicide programs, regardless of application rate or schedule had significantly lower (Fischer's LSD, on = 0.05) FFLB and RAUDPC values than the untreated control (Table 10a). The FR program had the numerically lowest final percentage of the foliage with late blight lesions (FFLB), but was only significantly lower than the HF and RI programs. All programs had significantly lower RAUDPC than the RI program, but were not different from each other. All programs had significantly higher class A and total yield (metric tons ha’l) than the untreated control, but were not different from each other. Only the FR program 115 Table 10 (a-b). The effect of increasing, decreasing and maintaining a constant rate of active ingredient of mancozeb 7’ dimethomorph fungicide pmograms (n1 (a) final foliar late blight, disease progress, yield, (b) infected marketable tubers plantd, marketable tubers plantd, anmi mean mass (g tuberd) for 1999 and 2001 combined. Statistical comparisons performed using Fischer’s LSD (a = 0.05). 116 .mCMummwcw .m 0n cofluowucfi mo mEOuQEhm mcfl3onm muwnsu momma magnumxumz 0 .A00.0 u 50 qu m.umnumflm mcfims ucmuwmwflp haucmoflwflcmflm Doc mum umuuwa TEMW may >3 UmSOHHow mcmwz 0 .>Hw>fluowamwn .soflmcwEflp >Cm CA Eu 0.0 I 0.0 man so 0.0A mums muwnsu mpmum m can < 0 .m>uso mmmumoum wmmwmflp Tau umpc: mwnm w>flumem .m .mcofimma unmflan wumH nuflz 0000000 may we mmmucmonmm Hmcfim v .820 Hmnm .ms< vane .ms< 0am .Hsn Hmnm .Hsn «mum .Hsn 0Hno .Hsn oaum .Hsn mua "mmumc ceaumuflaaam .m .A3\30 00 nauofionuweflp \ 000 nmNoocmE .muCTCOQEoo mo .fl.m pwcflnEoo 7mg 0x 00.H mmz mums cofiumoflammm 117 Hasm .:\5 >3 UTDMUAGCA wpfloflmcsw HmfloumEEoo pmxflEIqu .nmu0805uwfiflp u EEO .nmuoocmfi N am: .0 .Honucou nmm I 0: .mumu coflumoflammm Hazm I mm .wumu coflumoflaaam Hana mo mam: I mm .commmm IUHE um xmwm wumn I mm .commwm :msounu mcflmmmuocfl mush I Hm .commmm cmsounu mcflmmwuomp 0.53 I am .0 A 0.000 nm 0.00 Q «.0 m 0.0 D 00.0 ........... AHI<0 00.0 "Houucoo 030 UD 0 0.000 D0 0.00 m 0.0 o 0.0 m 00.0 ............. AHIdv 00.0 "EEO \ cm: .mm m 0.000 nm 0.00 m H.w nm 0.0 D0 00.0 ............. AHIdv 00.0 NEED \ cm: mm m 0.000 m 0.00 m 0.0 on 0.H cm 00.0 ........... Am.ov v0.0 .Am.uv .0H.H .A0.m0 00.0 .Am.<0 00.0 "EEO \ cm: mm m 0.000 D 0.00 m 0.0 nm 0.0 gm 00.0 ........... Am.ov 00.H .Am.m0 .0H.H .AQ.OV 00.0 .Am.fluo¢..mGOaUd«>ounn< Eduuoum .nOH magma n 0.3 n 0.3 0 000.0 m 0.00 ..................... 3.5 00.0 HHouucou 0N0 on m 0.00 m 0.00 0 000.0 0 0.00 ....................... AHIcv v0.H "ESQ \ cmz .mm m 0.00 m H.m0 0 000.0 0 0.00 ....................... AHIounn¢ auuoum .moH magma had a significantly“ higher number of infected. A tubers plant"1 than the untreated control, while remaining programs were not different (Table 10b). A shift in the number of tubers plant"l within. each size class and. mean. mass (g tuber—1) occurred with fungicide application, as the untreated control had the numerically highest number of B tubers plantq, and a significantly lower number of A tubers plantd‘and mean mass A (g tuberJ), while fungicide programs had less B tubers plant“1 and more A tubers plant'l. No significant change occurred in the mean mass of B tubers. Dimethomorph/Mancozeb Rate and Interval Manipulation (DMRI) All experimental factors passed the F Max test and data from different years were combined (data not shown). All fungicide programs, regardless of application rate or interval had significantly lower FFLB and RAUDPC values than the untreated control (Table 11a). Programs with a five—day application interval generally had numerically lower FFLB and RAUDPC values than the corresponding seven- day interval programs, but only the L5 program had significantly less FFLB than the corresponding seven-day program (L7). The untreated control had the numerically lowest class A and total yield, and was significantly lower than all of 118 Table 11 (a—b). The effect of increasing, decreasing and maintaining a constant rate of active ingredient of mancozeb / dimethomorph fungicide programs with a five or seven day application interval on (a) final foliar late blight, disease progress, yield, (b) infected marketable tubers plantd, marketable tubers plantd, and mean mass (g tuber”) for 2000 enmi 2001 combined. Statistical comparisons performed using Fischer’s LSD (a = 0.05). 119 .A00.0 n 60 0mg m4um£om0m 0:0m3 ucwumwm0p >0ucmo0m0cm0m Doc mum umuum0 06mm 0:» >3 pm300000 mcmwz .00m>0uommmmn .050 00nd .030 00n0 .OS¢ bmum .Hss ¢mum .13\30 Hash ..\. >0 nmumoflocfi cw>wm Mo w>0w m ucwmwuawu ..0s .m:< 00um .050 0000 mm nanofionumfi0p \ 000 Dmnoucmfi mp0o0mC:w paw .C00mcw50p ham G0 EU 0.0 I 0.v paw EU 0.0A wum3 mumnsu wpmum m can < .050 00um .w>u:o mmwnmoua mmmwm0p wnu prc: mwnm m>0u00mm .mC00mm0 u£m00n wDMO £u03 0000000 03» mo wmmDCwoqu 0MC0m .05< .msm 00uz .054 0nd .054 mum .050 0muw .020 .Hso mane .Hsn OHIU .Hsn mum .Hsn mna "mmumc coflumuflflam< .m .wucwCOQEou 00 .0.0 pwc0nfiou m: ox v0.0 mm3 wumn :00um000mmm 0m0oumEEoo UmX0EImuQ EEG .anoocmE u an: .0 40m>umuC0 C00umo00mmm 0mm .Commwm nmsounu Q'anb .msd 00uO .mdd V0nz fil .nmuofionumfiwp n .00w>0uummmwu 0C0mmwuowp 0000 I Q .cowmwm LGDOHSD mC0mmwuos0 mumn I D .wumn 0030 I m .wumuup0E I 2 .mumu um0300 I A .0 p 0.00 p 0.00 m 000.0 m 0.00 .................... 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Am.o.o.z.q.x.0.U.m.o.m.0uo<_.mfiOfluu«>ouan4.fiduu0Hm .800 00089 120 mmum mmuH 00 0900800000500 \ 000 Q0N0000E 00000009000 QDOQ 00 0.0 000080.000 .000.0 n 00 Q00 0.0000000 0:005 000000000 0000000000000 000 000 000000 0800 000 0Q 00300000 00002 .0 .000>0uu0am0u .000000800 >00 :0 Eu 0.0 I 0.0 000 EU 0.0A 0003 mH0Q5u 00000 m 000 d .0 .054 0mnm .050 00mm .054 mmuo .050 mmuw .mmmummmcw .m 0Q 000000000 00 08000600 0C03onm mn0Q5u 00000 00Q000x00z .0 .050 .054 0mu0 .054 00uo .054 00nz .054 m0": .054 0H0 .054 mum .050 0mu0 .050 .050 m0n0 .050 00um .050 m0u0 .050 O0n0 .050 mum .050 mn4 "00000 00000000004 .0 .03\30 .T0Q mx 00.0 003 0000 00000000QQ0 0050 ..\= 0Q 000000000 000000050 0000008500 00X0EI0HQ .000050300600 n EEO .Q0NOUC0E n 002 .0 .>00>0000&m00 .00>00000 00000000QQ0 >00 C0>0m Ho 0>0w 0 000000000 ..0: 000 00: 0 AQ 00300000 0000000>0HQQ4 .0000000 030 I U .Com00m £0500Qu 0000000000 0000 I 0 .000000 0050000 0000000000 0000 I D .0000 0050 I m .0000I00E I z .0000 000300 I 0 .0 0 0.000 Q 0.00 0 0.0 0 0.0 0 00.0 ............. 00.0.0.2.0.M.0.o.m.o.m.Q 00300000 00002 .0 .000>00000000 .000000800 >00 00 80 0.0 I 0.0 000 80 0.0A 0003 000Q50 00000 0 000 0 .m .0>050 00000000 0000000 000 0000s 0000 0>00000m .0 .0000000 000000 0000 0003 0000000 000 00 0000000000 00000 .0 .050 00n0 .050 00mm .054 v0uo .05< 0H0 .050 0mnm .050 00mm .050 00nu .050 00um .050 mu< "00000 00000000000 .0 .00x08 #000 000 2+: 0 0003 000000000 00>000< .200>00000000 .0000 0050 00 300 00 000080000800 I 00 0o 00 0003 000 00 .Q0000002 <2 .8000002 I 02 00050000 000 8000000 00000000000000 0000:000000 0 0000 000000000000 000 08000000 000000800 .0000000 000 I 0.0.0 .0000I000000 00000000000000 I .000 .0 0 0.00 0 0.00 0 000.0 0 0.00 .............. AHIdv oo.o H0000000 030 090 00 0.00 0Q 0.00 Q 000.0 Q 0.00 ........ 00.0.00 00.0 H880 + 00.0 ”002 .Am.0.0.uv 00.0 ”000 .0m.¢v m0.o "000 0002 0 0.00 0 0.00 0Q 000.0 Q 0.00 ........ 00.0.00 00.0 “880 + 00.0 H002 .Am.0.0.uv 00.0 H000 .Am.0v m0.o H00o 0042 0Q 0.00 Q 0.00 0Q 000.0 Q 0.00 ........ 00.0.00 00.0 H880 + m0.0 H000 .Am.o.m.uv 00.0 ”000 .0m.0000..m000000bounnd.8000000 .000 00000 124 .Amo.o n 0V 000 0.0000000 00005 000000000 >000000000000 000 000 000000 0800 000 >Q 00300000 00002 .m mNHH .m:< 0muz .00X08 0000 000 =+= 0 0003 000000000 00>0000 .050 v0nO .>00>00000000 .020 hum . WCMUWmNQfl .050 00mm .000000800 >00 00 80 m.m I 0.0 000 80 m.mA 0003 000Q50 00000 0 000 0 23‘ .0 >Q 000000000 00 080008>0 0003000 000Q50 00000 00Q000x002 .m .050 ”00000 00000000000 .N .050 vaD .050 b0uU .050 O0nm .>00>00000000 .000 mu0 .0000 0050 00 300 00 000080000800 I 00 00 00 0003 000 00 .Q0000002 I 02 .8000002 I 02 “0050000 000 8000000 00000000000000 0000I000000 0 0000 000000000000 000 08000000 000000800 .0000000 000 I 0.0.0 .0000I000000 00000000000000 I 000 .0 Q m.m00 Q 0.00 Q v.m 00 0.0 Q0 00.0 .............. 00I0V 00.0 H0000000 0:0 090 Q m.m00 Q0 o.mv Q0 m.m Q 0.0 Q 00.0 ........ 00.0.00 mm.o H880 + 00.0 H002 .Am.0.0.uv m0.0 H000 .00.0v mm.o "000 0002 Q0 0.000 Q0 p.00 0 0.0 Q0 h.m Q0 00.0 ........ 00.0.00 m0.o H880 + 00.0 H002 .Am.w.0.uv m0.0 H00,0 .00.0v mo.o ”000 0002 0 0.000 Q0 0.00 Q0 m.m Q0 0.0 Q0 v0.0 ........ 00.0.00 mm.o H880 + m0.0 "000 .Am.0.0.0v m0.0 n000 .Am.o0 0 m.mv Q0 w.m Q0 0.0 Q0 m0.o ........ 00.0.00 mm.o "880 + 00.0 H002 .Am.o.0.uv m0.0 H000 .Am.0v mw.o ”000 0002 0 m.mo0 0 m.mv Q0 m.m Q0 m.m Q0 00.0 ........ 00.0.00 m0.o H880 + 00.0 H002 .Am.o.0.uv m0.0 H00.0 .00.0v mw.o H000 0002 Q 0.000 0 v.mv 0 0.0 0 o.m m0 0N.o ..... AHIUV m0.0 H00o .00.0v no.0 H00o 000 0 0 0 .0 .700000 000059 «00000000 0R0 6700 000 0000 0700059 00 0002 0002 vuq¢Hm muonaa 40 ”03033 00000000000 "00>0000..0000000>00000.8000000 .000 00000 125 long chlorothalonil program (CLDl and CLD2) had the numerically lowest RAUDPC values , regardless of dimethomorph rate. The untreated control had the numerically lowest class A and total yield (metric tons ha’l) and was significantly lower than all programs except the MEDl, CLDl, and MAD2. The CHL program had the numerically highest number of infected A tubers plant-1, but was not significantly higher than any treatment except the CLDl and MAD2 programs (Table 12b). No distinct trends were apparent in the number of A and B tubers plant'1 or mean mass A and E (g tuber'l). None of the replacement programs were more effective at controlling foliar late blight or resulted in higher yield than the season—long chlorothalonil program (CHL). Of the dimethomorph mixture partners, chlorothalonil was only numerically superior to mancozeb and metiram at controlling foliar late blight but no yield differences were apparent. No distinct trends were present in terms of foliar late blight or yield between the two rates of dimethomorph. Dimethomorph]Pyraclostrobin Rate Manipulation (DSR) All fungicide programs had significantly lower FFLB and RAUDPC than the untreated control (Table 13a). Numerical rate responsetrends in FFLB were measured for 126 Table 13 (a—b). The effect of dimethomorph and pyraclostrobin at varying rates when incorporated into a season—long chlorothalonil program on (a) final foliar late blight, disease progress, yield, (b) infected marketable tubers plant'l, marketable tubers plant'l, and mean mass “3 tuber‘U for 2001. Statistical comparisons performed using Fischer’s LSD (a = 0.05). 127 L. .Amo.o u 6v 0mg w.uw£omflm mcfims ucwanMHp maucmofiwficmflm uoc mum uwuuwH wEMm wnu >8 pwsoHH0m mcmwz . m .>Hm>fluomamwu .coflmcmfiwp xcm CM EU m.m I o.v paw Eu m.mA wuwz muonsu mpmnm m can 4 .m .m>usu mmwumoum mmmwmfio mnu umpco mwud m>flumHmm .v .mcoflmma unmflam mama £ua3 mmmflaom mnu mo monusmonmm Hmcfim .m .ms< .m mNuH .m5¢ Hmum .m54 anU .de nnm rHSh Hmum .Hsh «mum .H:h bHuU .Hdh oaum .Hdh mn< “mwumw CofluMUflammd .waflE xCMu mum e+s m nufls Umumnmmwm mw>fiuu¢ .Qfinouumhx0N4 I ONd pcm .wumn Hmnma HHDM may mo wooa paw .wmb .wom .wmm um Canouuonumuhm I ooam paw \mrm .omm ‘mmm .mwumu Hanna Hana wnu mo wooa paw .wmh .wom .wmm um SQHOEonquflQ + CanouumoHumu>m I ooaom Ucm .mbmm .omam ‘mmam uqmu nufl3 Umumcnmuam mum mEmumouQ mcflcfimfimu .Houucoo 03* I 490 .mcoHICommmm HflcofimnuouoHno I qmu .H n m.am n o.mH m omm.o m m.mm ..... . ........ 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Amrmro.mo mm.o "a + sfi.o um coaom AH.o.m.ov MH.H ”Hno .Aav mo.o "ago an m.om gm m.mm n omo.o mun m.ov .......... lm.m‘armv sa.o no + NH.o ”a mean AHro.mrov MH.H ”ago .r4c mm.o ”ago an m.om m m.mm n mmo.o on 0.0m .......... im.m.ormv Hfi.o Ha + mo.o "a Oman AHrwrm.ov MH.H ”ago .AHnnoowm«cmfim non who Hmuuwa mEom mnu >n pm3oHH0m mcooz .m .>Hm>fluommmmu .coflmcmeflp xno an Eu m.w I o.v pco Eu m.mA mumz muons» mponm m pco d .v .mcoummwcw .m >n cofluowwcfl mo mEouQE>m mcflzonm muonsu wponm wanoumxuoz .m .msd wmuH .m24 Hmum .mso «duo rms< sum .Hso Hmum raga qmno .Hso nHuo .Hso oaum ‘Hso muo ummuoo acnuoUnHaao .m .waflE xcou who :+s o nnflz Umuouommm mm>fluod .cflnonum>on4 I 0N4 pco .wuoH Honoa Hazw wnu mo wooa poo .wmh ~wow .wmm no nanouumoHoonxm I ooam pco .mhm romm .mmm .mwuou Honoa HHSw mnu mo mooa pco .wmb .wom .wmm no nQHoEonumEflo + canouumoHooH>m I ooaom Uco .mnom .omom .mmom unmu nufiz pwuocumuao who mEonoum mcflnflofimn .Houucoo can I new ~mcoHIcomomm aficoHonuouoHnU I nmu .H n v.mOH no H.mv n H.m o w.m n 00.0 .............. AHIdv oo.o "Houucoo 03* new no a.mHH no m.mv no m.m n m.H no Ho.o .................. Az.m.a.mv mm.o ”Ond 0N4 AH.w.mrUv MH.H UHnU .A4v mm.o HHnU no m.oHH o m.v¢ no m.m no m.H n 00.0 .................... Az‘mrn.mv bH.o “m ooam AH.wrm~Uv mH.H Mano .Adv no.0 "HnU no m.mHH n H.mm no v.0 n m.H n 00.0 .................... Am.mrormv NH.o ”m mom AH.U.m.UV MH.H ”HnU .Adv no.0 .Hnu no m.mHH no m.mv no m.m no m.H n 00.0 .................... Am.m.o.mv mo.o ”m omm AH.o.mruv mH.H uHnU .Adv mm.o "HnU no H.mmfi no m.mv no o.m n m.H no no.0 .................... Am.m.o.mv «0.0 .m mmm AHiw‘m.Uv mH.H HHnU .ndv no.0 .Hnu no b.mmfi no m.mo o o.> n v.H no mo.o .......... Am.mrormv mm.o "a + ha.o .m ooaom AH.O.m~UV MH.H HHnU .ndv mm.o "anu no m.mHH o H.mv no H.m no o.m no Ho.o .......... A$.m.o.mv na.o “Q + NH.o ”a mean AH.w~m~UV MH.H HHnU .Adv mo.o "HnU no «.mma o m.w¢ no m.m n m.H n 00.0 .......... Am.mrnrmv HH.o "Q + mo.o “m omom AHro.mrUV MH.H nHnU .n n m.H o qo.o .......... Am.m.orm. wo.o "Q + v0.0 “m med AH.o.m.UV mH.H UHnU .Adv mm.o "ano o w.mma no m.av no m.m no o.m mno no.0 ....... AHImv mH.H HHnU rndv mm.o ”HnU nmu 4 d .m Tufludm muonfla «oanwonom can .?dn uxv ovum AThondB av and: com: Tufldnm muonfla 4 «wauuouGH Goauou«HQQ< "mo>«u04..mflOdud«>0unn-—.—.—-—.—~—.—l III. Attempt to generate insensitivity to the fungicide, determine the ease of generation, quantify and characterize the insensitivity, and examine the fitness and virulence of resistant strains. IV. Using the results of in Vitro and in vivo activity, examine field usage of the fungicide, including: specific positions within the disease cycle for application, application rates and intervals, resistance management parameters, such as potential_mixture partners, etc. Revise the usage recommendations of the fungicide for the control of the plant disease in question. 135 rate and alternative mixture partners and rate reduction information was not available. This research project initially examined the baseline sensitivity of isolates from various genetic backgrounds to dimethomorph at all stages of the P. infestans asexual life cycle and the protectant, curative, and antisporulation activity when applied to potato leaf disks. The Exgo'values calculated for each stage of the asexual life cycle were similar to those previously reported and the variability of the P. infestans isolates examined was similar to those reported for other Oomycete fungicides. Under in Vitro conditions, the stages responsible for infection by Phytophthora species were all highly sensitive to dimethomorph. However, when applied to leaf disks, much higher concentrations of dimethomorph were required for inhibition of symptom development, and little curative activity was found. The difference between the required concentrations is probably’ a result of the affinity of dimethomorph to the leaf cuticle and the low water solubility and systemicity of dimethomorph in the leaf. Antisporulation activity was present, but lower than that previously reported. In general, concentrations higher than 1000.0 ug ml‘1 should be used under field conditions to control potato late blight. 136 I J Next, the generation of insensitivity of dimethomorph in P. infestans isolates from various genetic backgrounds was attempted. Ethidium bromide / UV mutagenesis was more successful in generating strains with moderate levels of insensitivity than repeated culturing on sub-lethal media. In both cases, the saprophytic fitness of the insensitive strains was generally less than those of the wild-type. Additionally, the virulence of the most insensitive strains generated from repeated culturing was reduced from that of the wild-type on both leaf disks and in whole tubers. The generation of dimethomorph insensitive strains was possible, but the level of insensitivity was low and the resulting strains typically were less virulent. With the currently limited use of dimethomorph for the control of potato late blight in most potato growing regions of the United States and the negative aspects associated with insensitivity, it is unlikely that wide—spread field insensitivity will occur. Finally, the pre—mixed dimethomorph / mancozeb commercial product and alternative mixture partners for dimethomorph were examined. Application rate and interval manipulation was possible with the pre—mixed dimethomorph / mancozeb jproduct” The examination. of such. a fungicide program in conjunction with weather-based late blight 137 forecasting systems could allow for a reduction in overall fungicide use. In general, dimethomorph at the full label rate (U.S.A.) of 0.22 kg ha”1 did not measurably increase the efficacy of the mixture partners examined, when applied in the absence of dimethomorph. The high activity of dimethomorph under controlled conditions supports the use for potato late blight control. However, until the field efficacy of dimethomorph can be increased, use will remain limited. Increasing the application rate and possibly the efficiency of crop coverage, will likely increase field efficacy. Dimethomorph has potential for use in resistance management schemes with other single mode of action fungicides, such as the strobilurins. Future restrictions on the use of multi—site fungicides with high environmental impact may encourage the changes in the label rate of dimethomorph and allow for the fungicide to be more effective in the control of potato late blight. 138 L ITERATURE C ITED Albert, G., J. Curtze, and C.A. Drandarevski. 1988. 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