Resist en CIA a Qoi Em Ascochyta

8/3/2019 Resist en CIA a Qoi Em Ascochyta

1/9

528 Plant Disease / Vol. 93 No. 5

Resistance to QoI Fungicides in Ascochyta rabiei from Chickpeain the Northern Great Plains

K. A. Wise, Department of Plant Pathology, North Dakota State University, Fargo 58105; C. A. Bradley, Depart-ment of Crop Sciences, University of Illinois, 1102 S. Goodwin Ave., Urbana 61801; and J. S. Pasche andN. C. Gudmestad, Department of Plant Pathology, North Dakota State University, Fargo 58105

Ascochyta blight, caused by the fungus Ascochyta rabiei (Pass.) Labr. (teleo-morph: Didymella rabiei (Kovacheski) v.Arx.), is an important disease of chickpea(Cicer arietinum L.) throughout the world(22). A. rabiei can infect chickpea at allstages of plant phenology and can causeover 50% yield reduction under conditionsfavorable for disease development (13,17).Within the United States and Canada, As-cochyta blight epidemics are common,making it the most important disease of chickpea in these regions (8,13).

Management of Ascochyta blight re-quires an integrated approach that includescrop rotation and burial of debris from theprevious crop to reduce overwinteringinoculum. Chickpea cultivars with moder-ate levels of resistance are available foruse, but none have complete resistance tothe dominant pathotype of A. rabiei in theUnited States (8,10,32) . Current resistancelevels are often insufficient to prevent dis-ease development and economic loss in the

Northern Great Plains (15). Fungicide seedtreatments are used to control seed-borne

A. rabiei (29), and several applications of foliar fungicides often are required in theNorthern Great Plains of the United Statesand in the Canadian prairies to manageAscochyta blight (7).

Chlorothalonil and maneb are fungicideswith multi-site mode of action and broad-spectrum protectant activity, and are typi-cally applied prior to flowering to delaythe onset of Ascochyta blight. However,once blight symptoms are present, applica-tions of chlorothalonil or maneb alone donot control disease, forcing producers toemploy fungicides with both pre- and post-infection modes of action (11,13). Prior to2007, only two classes of fungicide chem-istry with post-infection activity were reg-istered for control of Ascochyta blight onchickpea in the United States: the quinoneoutside inhibitor (QoI) class (azoxystrobinand pyraclostrobin) and the carboximideclass (boscalid). In 2002, the UnitedStates Environmental Protection Agency(EPA) granted a section 18 emergencyexemption for use of azoxystrobin onchickpea to control Ascochyta blight inNorth Dakota. In 2003, azoxystrobin,boscalid, and pyraclostrobin were grantedfull section 3 registrations on chickpea inthe United States. In 2007, prothiocona-zole, a sterol-demethylation inhibitor(DMI) fungicide, was registered for thecontrol of Ascochyta blight. All of thesefungicides have a single-site mode of

action and are at risk for fungicide resis-tance development.

Currently, of these fungicides, QoI fun-gicides play an important role in manage-ment of Ascochyta blight. These fungi-cides inhibit mitochondrial respiration bybinding to the center of the Qo site of thecytochrome bc 1 complex (complex III) onthe positive side of the inner mitochondrialmembrane (4,5). While this class of fun-gicides is extremely effective at managinga broad range of diseases on many crops,the site-specific mode of action may in-crease the potential for selection of resis-tant mutants of fungal pathogens (3). QoIfungicide resistance was first reported in

Erysiphe graminis on wheat just 2 yearsafter the class was registered for use inEurope (4).

Since 1998, field resistance to QoI com-pounds has been documented for importantpathogens of horticulture and field crops(14,1416,19,22,25,30,31,35). Until re-cently, the mechanism of resistance hasbeen attributed to single-point mutationresulting in amino acid substitution at oneof two positions in the cytochrome b gene.In the majority of pathogens, glycine isreplaced by alanine at codon 143 (G143A),resulting in expression of resistant pheno-types (3,5,12,14,16,18,19,34), while asecond mutation results in a phenylalanineto leucine change on amino acid 129(F129L), and is found in Pyriculariagrisea (19), Pyrenophora tritici-repentis and Pyrenophora teres (27), and Alter-naria solani (23). In 2007, a third cyto-chrome b mutation resulting in a glycine toarginine change at amino acid position 137was reported. This G137R mutation hasrecently been observed in two isolates of Pyrenophora tritici-repentis (27). The typeof mutation present in a fungal populationgreatly influences the level of disease con-trol obtained with QoI fungicide applica-tions (14,23,27). Fungal isolates with theG143A mutation typically have completeresistance, meaning that applications of allQoI fungicides are ineffective at control-ling disease (14). The presence of theF129L or G137R mutation results in re-duced sensitivity and levels of diseasecontrol obtained by QoI fungicide applica-tions (19,23,24,27).

Since the registration of QoI fungicidesfor use on chickpea in 2003, fungicideapplications in North Dakota for As-cochyta blight control have relied almost

ABSTRACTWise, K. A., Bradley, C. A., Pasche, J. S., and Gudmestad, N. C. 2009. Resistance to QoI fungi-cides in Ascochyta rabiei from chickpea in the Northern Great Plains. Plant Dis. 93:528-536.

Ascochyta blight, caused by Ascochyta rabiei (teleomorph: Didymella rabiei ), is an importantfungal disease of chickpea ( Cicer arietinum ). A monitoring program was established in 2005 todetermine the sensitivity of A. rabiei isolates to the QoI (strobilurin) fungicides azoxystrobin andpyraclostrobin. A total of 403 isolates of A. rabiei from the Northern Great Plains and the PacificNorthwest were tested. Ninety-eight isolates collected between 2005 and 2007 were tested usingan in vitro spore germination assay to determine the effective fungicide concentration at which50% of conidial germination was inhibited (EC 50) for each isolatefungicide combination. Adiscriminatory dose of 1 g/ml azoxystrobin was established and used to test 305 isolates from2006 and 2007 for in vitro QoI fungicide sensitivity. Sixty-five percent of isolates collected fromNorth Dakota in 2005, 2006, and 2007 and from Montana in 2007 were found to exhibit a mean100-fold decrease in sensitivity to both azoxystrobin and pyraclostrobin when compared to sen-sitive isolates, and were considered to be resistant to azoxystrobin and pyraclostrobin. Undergreenhouse conditions, QoI-resistant isolates of A. rabiei caused significantly higher amounts of disease than sensitive isolates on azoxystrobin- or pyraclostrobin-amended plants. These resultssuggest that disease control may be inadequate at locations where resistant isolates are present.

Corresponding author: N. C. GudmestadE-mail: [email protected]

Current address of K. A. Wise: Department of Botany and Plant Pathology, Purdue University,West Lafayette, IN 47907.

Accepted for publication 29 January 2009.

doi:10.1094 / PDIS-93-5-0528 2009 The American Phytopathological Society


2/9

Plant Disease / May 2009 529

exclusively on fungicides within the QoIclass. Applications of fungicides with post-infection activity typically begin whendisease is first observed in a field, andcontinue on a 10- to 14-day schedule untilconditions are no longer favorable fordisease development. In the NorthernGreat Plains, favorable environmentalconditions can often persist throughout thegrowing season, and in some instances upto six sequential applications of QoI fungi-cides have been made to a chickpea field ina single growing season. Ascochyta blightis a polycyclic disease (29), and the con-tinuous use of QoI fungicides in this re-gion increases the frequency of selectionand pathogen population exposed to thisfungicide class during a growing season,potentially contributing to the developmentof fungicide resistance. QoI-resistant iso-lates of A. rabiei have been identifiedthrough in vitro testing in Canada since2004 (7,15), and the risk of fungicide re-sistance development in the NorthernGreat Plains populations is high. In 2006,anecdotal reports from chickpea producersin western North Dakota indicated thatapplications of QoI fungicides were notproviding adequate control of Ascochytablight.

Because of the nearly exclusive use of this chemistry, the limited number of fun-gicide chemistries with different modes of action, the identification of resistance inCanada, and anecdotal reports of reducedfungicide efficacy in North Dakota, QoIresistance development for A. rabiei hasbeen identified as a major concern in theNorthern Great Plains region. Baselinesensitivity of A. rabiei to azoxystrobin andpyraclostrobin was determined in a previ-ous study (33), facilitating the develop-ment of a regional fungicide sensitivitymonitoring program. The overall objec-tives of this study were to (i) determine if ashift in sensitivity to QoI fungicides hasoccurred in the North Dakota A. rabieipopulation, (ii) establish an in vitro singlediscriminatory dose testing method usingazoxystrobin, and ( iii) determine if isolatesexhibiting in vitro QoI fungicide resistancewere controlled less by QoI fungicides invivo using greenhouse experiments.

MATERIALS AND METHODSCollection of A. rabiei isolates. Isolates

of A. rabiei were obtained from chickpeaproduction fields in North Dakota receiv-ing QoI fungicide applications during2005, 2006, or 2007. Chickpea plants withsymptoms of Ascochyta blight were sam-pled on a cross-diagonal transect pattern(X), with samples taken at set intervals of approximately 15 m. Isolates also wereobtained from diseased chickpea in re-search plots located at North Dakota StateUniversity Research Extension Centers inHettinger, Minot, and Williston, ND. Dis-ease samples were bulked by field or re-search plot and returned to the laboratory

for isolation. Additional isolates were re-covered from infected plant material fromSouth Dakota and Nebraska from MartinDraper (South Dakota State University,Brookings) and Robert Harveson (Univer-sity of Nebraska, Scottsbluff) in 2005 andMary Burrows in Montana (Montana StateUniversity, Bozeman) in 2006 and 2007.Isolates were obtained from Idaho andWashington in 2005 from the A. rabiei culture collection of Weidong Chen at theUnited States Department of Agriculture-Agricultural Research Service (USDA-ARS) in Pullman, WA.

Isolates of A. rabiei tested for in vitrofungicide sensitivity screening were ob-tained by cutting symptomatic chickpeastems into 2- to 3-cm sections. Stem sec-tions were placed in a 95% ethanol solu-tion for 1 min, followed by a rinse in ster-ile distilled water (SDW) for 1 minfollowed by 0.5% NaOCl solution for 1min, and rinsed again for 1 min in SDW.Sterilized stem sections were air-dried in alaminar flow hood for 30 s on autoclavedpaper towels and placed on potato dextroseagar (PDA) (Difco Laboratories, Detroit,MI) amended with 10 mg/liter streptomy-cin sulfate (Sigma-Aldrich, St. Louis, MO)in petri plates. Conidial and mycelialgrowth was observed from plated stemsections after 3 to 6 days of incubation at

20C under a diurnal cycle of cool whitefluorescent light (12 h light followed by 12h dark). The presence of A. rabiei wasconfirmed by microscopic observation of conidia at 100 magnification. An individ-ual conidium from each sterilized stemsection was considered a distinct isolate,and was incubated on fresh PDA under theconditions previously described. Single-spore isolates were stored for short-termuse (up to 6 months) by removing three to

four 0.25-cm-diameter plugs of agar cov-ered with sporulating growth from each14-day-old culture and placing plugs in a1.5-ml centrifuge tube with 1 ml of SDW.Tubes were sealed with Parafilm andstored at 4C. Isolates were preserved forlong-term storage as conidia and myceliaon sterile filter paper at 20C (33).

In vitro fungicide sensitivity assay.Fungicide sensitivity to azoxystrobin andpyraclostrobin was determined in vitro for98 isolates of A. rabiei collected from2005 to 2007 using previously publishedmethods (33) (Table 1). Stock solutions of technical grade formulations of azox-ystrobin (97.6% active; Syngenta CropProtection, Greensboro, NC) and pyraclos-trobin (99% active; BASF Corporation,Research Triangle Park, NC) were pre-pared at concentrations of 100 mg/ml anddiluted serially in acetone. Fungicide sen-

Table 1. Collection information and results of in vitro Ascochyta rabiei isolate sensitivity assays toazoxystrobin and pyraclostrobin in 2005, 2006, and 2007

EC 50 (g/ml) a

Azoxystrobin PyraclostrobinCollection locationby county Number of isolates Range Range

2005

North DakotaCass 2 0.033-0.034 0.0032-0.0039Foster 5 0.030-0.039 0.0019-0.0101Ward 7 0.026-19.0 0.0035-0.5473

Total 14 0.026-19.0 0.0019-0.5473South Dakota 1 0.032 0.0037Nebraska 1 0.033 0.0043Idaho 1 0.033 0.0044Washington 2 0.031-0.032 0.0077-0.0182Overall total 19 0.026-19.0 0.0019-0.5473

2006North Dakota

Cass 4 3.81-5.82 0.2100-2.730Foster 3 0.030-16.2 0.0027-2.400McClean 5 5.87-29.0 2.380-3.233Mountrail 8 3.22-25.7 0.3470-3.090Renville 10 5.68-16.5 0.3710-3.145Ward 17 0.032-32.4 0.0029-2.970Williams 1 5.94 0.5900

Total 48 0.030-37.7 0.0027-3.233Montana 1 0.032 0.0032Overall total 49 0.030-37.7 0.0027-3.233

2007North Dakota

Adams 1 3.40 2.927Hettinger 1 0.032 0.0030McClean 6 3.30-31.9 2.740-3.549Mountrail 5 0.029-28.4 0.0034-3.613Williams 17 0.032-32.4 0.0030-3.780

Total 30 0.029-32.4 0.0030-3.780a Fungicide sensitivity was determined by calculating the mean effective fungicide concentration in-

hibiting spore germination by 50% of the nontreated control (EC 50 value; g/ml).


3/9


sitivity was determined for 2005 and 2006isolates by evaluating A. rabiei conidialgermination on PDA amended with 0,0.001, 0.01, 0.1, 1, 10, and 100 g/ml of each fungicide. Sensitivity of isolates col-lected in 2007 was determined on azox-ystrobin-amended PDA at concentrationsof 0, 0.01, 0.1, 1, 10, and 100 g/ml andpyraclostrobin-amended PDA at 0, 0.001,0.1, 1, and 10 g/ml. Salicylhydroxamicacid (SHAM; Sigma-Aldrich) was dis-solved in methanol and added to all fungi-cide-amended media at a concentration100 g/ml to minimize the effects of thealternative oxidative pathways that somefungi use to overcome QoI fungicide toxic-ity in fungicide sensitivity assays in vitro(4,9,33). A. rabiei is able to use this alter-native pathway in the presence of QoIfungicides, and SHAM has been deter-mined to have no effect on conidial germi-nation (33). In all experiments, the 0 g/mltreatment served as a control and wasamended with 100 g/ml SHAM, 1 ml of acetone, and 1 ml of methanol per liter.

A. rabiei isolates in all experiments wereprepared using previously reported meth-ods (33). Briefly, a conidial suspensionwas obtained by adding sterile 0.05%Tween 20 (Sigma-Aldrich) solution inwater and dislodging conidia of a 7-day-old culture of A. rabiei with a sterile glassrod. The concentration of the conidialsuspension for each isolate was determinedwith the aid of a hemacytometer, adjustedto 2 10 5 conidia/ml, and 100 l of thesuspension was pipetted onto each of tworeplicate petri plates (60 15 mm). Plateswere incubated at 20C for 18 h in thedark, and subsequently examined at 100magnification under a compound micro-scope. Percent germination was recordedfor at least 100 conidia per isolate. A co-nidium was considered germinated if thegerm tube was at least as long as the co-nidium (33). Percent conidial germinationwas converted into percent inhibition cal-culated as 100 [(% germination of fungi-cide-amended media/mean % germinationof nonamended control) 100]. From this,EC 50 values (the fungicide concentrationthat inhibits conidial germination by 50%of the nonamended control) for each iso-late were calculated using a linear interpo-lation method (23,24,33). The resistancefactor of individual isolates relative tosensitive isolates was calculated by divid-ing the EC 50 value of individual isolates bythe mean EC 50 values of the baseline popu-lation to azoxystrobin (0.0272 g/ml) andpyraclostrobin (0.0023 g/ml) (33).

A. rabiei isolates were tested in groupswith 5 to 12 isolates per group. In eachgroup, at least one internal control isolatewas included to ensure assay reproducibil-ity (23,24,33,34,36). In the in vitro fungi-cide sensitivity trials conducted on 2005 A.rabiei isolates, a known QoI-sensitiveisolate (AR666) was selected from thepreviously established baseline (33) to

serve as an internal control; for those col-lected in 2006 and 2007, a QoI-resistantinternal control isolate (06BWEF2-46) wasalso included. The internal controls weretested in five separate trials as describedabove, and the mean, standard error, and95% confidence intervals were calculatedbased on the resulting EC 50 values(33,34,36). If the internal control isolateEC 50 values were within the previouslydetermined 95% confidence intervals,trials were combined for statistical analy-sis. Only trials that satisfied the assay re-producibility requirements were includedin analysis. Isolates were combined into asingle experiment by year of isolate collec-tion. Isolate EC 50 values were analyzedusing the general linear models (PROCGLM) in SAS (version 9.1, SAS institute,Inc., Cary, NC), following a completelyrandomized design. The experiment wasrepeated, and F tests were conducted todetermine if variances were homogeneous(P 0.05) between experiments. Correla-tion analysis was performed on EC 50 valuesfor azoxystrobin and pyraclostrobin usingPearsons correlation coefficient (PROCCORR). Mean EC 50 values were comparedusing Fishers protected least significantdifference (LSD) test ( = 0.05).

Establishment of a discriminatorydose system. Preliminary experiments todetermine a discriminatory dose forscreening A. rabiei fungicide sensitivityusing azoxystrobin-amended media pre-pared at concentrations of 0, 0.1, and 1g/ml with 100 g/ml SHAM were estab-lished as described above. A. rabiei iso-lates were prepared for testing and evalu-ated for percent germination as describedabove. Results of these experiments dem-onstrated that spore germination of sensi-tive isolates is completely inhibited atfungicide concentrations of 1 g/ml, butresistant isolates had greater than 50%germination at the same fungicide concen-tration ( data not shown ). Based on theseresults, a discriminatory dose of 1 g/ml of azoxystrobin was selected for testing anadditional 22 isolates from 2006, and 283isolates from 2007 for in vitro QoI fungi-cide sensitivity. Isolates were tested asdescribed above in nine groups with 35 to50 isolates per group. The internal controlisolates previously tested were included ineach group. Isolates were considered resis-tant to azoxystrobin if germination wasgreater than 50% at the discriminatorydose. Thirty arbitrarily selected isolatesfrom 2007 were tested for azoxystrobinsensitivity using both the discriminatorydose method, and by calculating EC 50 values using the procedures describedabove. This was done to validate discrimi-natory dose results by determining if iso-lates with high EC 50 values had high ger-mination rates on the discriminatory dose.The experiment was repeated, and percentgermination values for each isolate wereexamined for statistical measures of dis-

persion and normality using PROC UNI-VARIATE of SAS. Due to skewed, non-normal distributions of values, data werearcsine transformed and re-examined fornormality. Because of the nature of thefungicide sensitivity response, transforma-tion of percent values did not reduce skew,and distributions of discriminatory dosedata were compared using the Kolmo-gorov-Smirnov two-sample test in SAS.

Effect of A. rabiei fungicide sensitivityon disease control on chickpea. Green-house trials were performed to determinethe level of in vivo disease control attain-able with QoI fungicides against isolateswith differing QoI-sensitivities based on invitro tests. Two QoI-sensitive A. rabiei isolates (JB9-5 and SHRF12) and threeQoI-resistant isolates (BMXQ65, DF8, andH201-6) were included in the trial. QoIsensitivity of these five isolates was deter-mined using the discriminatory dose of 1g/ml azoxystrobin described above. Co-nidia of each of the three QoI-resistantisolates had over 95% germination on thediscriminatory dose of 1 g/ml, while co-nidia germination of the two sensitiveisolates was completely inhibited at thesame dose ( data not shown ).

Methods established by Pasche et al.(23,24) were used as a basis for perform-ing greenhouse experiments. Briefly,chickpea seeds (cv. Burpee) were sown in473-ml plastic cups filled with SunshineMix 1 (Sun Gro Horticulture Inc., Belle-vue, WA) and grown under 400 watt high-pressure sodium lamps set for an 18-hphotoperiod, at 22 2C. Ten to 14 daysafter planting, chickpea plants were treatedwith commercial formulations of azox-ystrobin (Quadris 2.08 SC; Syngenta CropProtection) or pyraclostrobin (Headline,2.09 EC; BASF Corporation) at concentra-tions of 0, 0.1, 1.0, 10, and 100 g a.i./mlof water. Fungicides were applied to runoff using a CO 2-powered hand-held sprayer.Approximately 24 h after fungicides wereapplied, plants were inoculated with A.rabiei conidial suspensions prepared from14-day-old cultures of selected sensitiveand resistant isolates. Suspensions wereadjusted to a concentration of 3 10 5 co-nidia/ml and applied to chickpea plantswithin an hour after preparation. Inoculumfrom each isolate was applied to plantsusing a hand-held airbrush paint sprayerconnected to a vacuum pump (Welch Dry-Fast Vacuum Pump, Gardner Denver Inc.,Niles, IL). Chickpea plants were placed inseparate mist chambers by isolate and heldat >95% relative humidity for 36 h at a 16-h photoperiod under artificial lightingbefore being placed in enclosed chamberson greenhouse benches. Chambers wereconstructed with 1-m-high polyethyleneplastic barriers between plants inoculatedwith different isolates to reduce the poten-tial for cross-contamination. After 10 days,disease severity for plants was visuallyassessed as the percent area infected of


4/9


whole plant. The experiment was designedas a randomized complete block (RCB)with a split-plot arrangement. Isolate wasconsidered as the whole plot factor and afactorial arrangement of fungicides andfungicide concentrations as the subplot. All

main effects were considered fixed for thepurpose of testing significance. Three rep-licates were included in each experiment,and the average disease severity was calcu-lated for two plants from each experimen-tal unit. Percent disease control was calcu-

lated by: [1 (% diseased tissue/% diseaseon 0 g/ml control)] 100. The experi-ment was repeated, and F tests were con-ducted to determine if variances were ho-mogeneous between the two greenhouseexperiments. Data were converted to per-cent disease control to facilitate directcomparisons between sensitive and resis-tant isolates, and analyzed using PROCGLM in SAS. Mean percent disease sever-ity and control were compared usingFishers protected LSD test ( = 0.05).

RESULTSIn vitro fungicide sensitivity assay. In-

dependent analyses of variance of in vitrofungicide sensitivity experiments for pyra-clostrobin and azoxystrobin EC 50 valuesdetermined that error variances were ho-mogenous ( P = 0.05); thus, experimentswere combined for further analysis. Fre-quency distributions of 19 A. rabiei iso-lates collected in 2005 demonstrated that89 and 63% of isolates had EC 50 values of less than 1 g/ml and 0.005 g/ml forazoxystrobin and pyraclostrobin, respec-tively (Figs. 1 and 2). These isolates wereconsidered to be sensitive to the fungicidestested, and EC 50 values of these isolateswere comparable to previously establishedbaseline values of 0.0272 g/ml for azox-ystrobin and 0.0023 g/ml for pyraclos-trobin (33). EC 50 values for two 2005 iso-lates were well outside the rangeestablished by the baseline; they exhibiteda 539-fold decrease in sensitivity to azox-ystrobin and a 704-fold decrease in sensi-tivity to pyraclostrobin when compared tothe mean sensitivity of baseline isolates.Conversely, in 2006 and 2007, 93.7 and53.1% of A. rabiei isolates were deter-mined to have EC 50 values greater than 1g/ml and 0.005 g/ml for azoxystrobinand pyraclostrobin, respectively (Figs. 1and 2). Correlation analysis revealed apositive association between azoxystrobinand pyraclostrobin EC 50 values ( r = 0.66, P < 0.001, n = 98) (Fig. 3).

Fig. 2. Frequency distributions of effective fungicide concentrations that inhibited spore germinationby 50% (EC 50 value; g/ml) for Ascochyta rabiei isolates to pyraclostrobin in 2005 ( n = 19), 2006 ( n =49), and 2007 ( n = 30). Individual isolates are grouped in class intervals of 0.9 g/ml; values on the x-axis indicate the midpoint of the interval.

Fig. 3. Relationship between in vitro mean effec-tive fungicide concentration that inhibited sporegermination by 50% (EC 50 value; g/ml) forazoxystrobin and pyraclostrobin sensitivity of 98

Ascochyta rabiei isolates from 2005 to 2007.

Fig. 1. Frequency distributions of effective fungicide concentrations that inhibited spore germinationby 50% (EC 50 value; g/ml) for Ascochyta rabiei isolates to azoxystrobin in 2005 ( n = 19), 2006 ( n =49), and 2007 ( n = 30). Individual isolates are grouped in class intervals of 4.9 g/ml; values on the x-axis indicate the midpoint of the interval.


5/9


Establishment of a discriminatorydose. Comparison of distributions of per-cent germination on the discriminatorydose of 1 g/ml by the Kolmogorov-Smirnov two-sample test showed no sig-nificant differences between experiments(KSa = 1.143, P = 0.1466). The selected

discriminatory dose of 1.0 g/ml azox-ystrobin was effective in determining thein vitro fungicide sensitivity of 30 isolatesof A. rabiei from 2007 when compared toEC 50 values generated for the same iso-lates, and a clear differential response inconidial germination was observed be-

tween QoI-resistant and -sensitive isolates(Fig. 4). Sixteen isolates with resistancefactors of approximately 100-fold had amean germination of 94.0% in the pres-ence of 1 g/ml azoxystrobin (Fig. 4). QoI-sensitive control isolates had less than 3%conidial germination on the discriminatorydose, with a mean of 0.3% conidial germi-nation (Fig. 4). Discriminatory dose datafrom the 305 isolates collected in 2006 and2007 determined that the frequency of azoxystrobin resistance in A. rabiei wasover 60% in each year. These results aresimilar to the frequencies determined byEC 50 values generated for isolates col-lected in those same years (Fig. 5).

QoI-resistant isolates (determined byEC 50 values or discriminatory dose meas-urements) were present in only one of three counties sampled in North Dakota in2005, in all seven counties sampled in2006, and in seven of eight counties sam-pled in 2007. QoI-resistant isolates weredetected in four of five counties sampled inMontana in 2007 (Tables 1 and 2).

Effect of A. rabiei fungicide sensitivityon disease control on chickpea. Inde-pendent analysis of disease control ex-periments determined that variances werehomogenous, and experiments were com-bined for further analysis. Significant in-teractions were observed between thewhole plot (isolate) and subplot factors(fungicide and fungicide concentration) ( P < 0.001), as well as between the subplotfactors of fungicide and fungicide concen-tration ( P < 0.001) for percent diseaseseverity and percent disease control of fungicides on Ascochyta blightinfectedchickpea. Significant effects ( P < 0.001)were also observed for isolate, fungicide,and level of fungicide concentration forboth percent disease severity and percentdisease control.

Disease severity was significantlygreater on plants inoculated with QoI-resistant isolates at all concentrations of azoxystrobin and pyraclostrobin, includingthe nontreated control (0 g/ml). QoI-sensitive isolates were completely con-trolled at concentrations of 10 and 100g/ml azoxystrobin and pyraclostrobin(Figs. 6 and 7). Disease control of QoI-resistant isolates was significantly reducedfor azoxystrobin and pyraclostrobin whencompared to QoI-sensitive isolates at allfungicide concentrations (Fig. 7). Pyra-clostrobin provided significantly greaterdisease control of QoI-resistant isolates atconcentrations of 100 g/ml when com-pared to azoxystrobin. However, pyraclos-trobin provided less than 65% diseasecontrol of QoI-resistant isolates, while100% disease control of sensitive isolateswas achieved at the same concentration(Fig. 7).

DISCUSSIONResistance to QoI fungicides was ob-

served in isolates of A. rabiei in North

Fig. 4. Mean in vitro sensitivity of 16 QoI-resistant () and 14 QoI-sensitive (- - -) Ascochyta rabieiisolates from 2007 measured as mean percent germination on azoxystrobin-amended media at differentfungicide concentrations (g/ml) for determination of a discriminatory dose. Values include standarderrors of percent germination.

Fig. 5. Frequency of QoI-sensitive and resistant Ascochyta rabiei isolates in the Northern Great Plainsas determined by a discriminatory dose of 1 g/ml of azoxystrobin-amended media for isolates col-lected in 2006 ( n = 22) and 2007 ( n = 283).


6/9


Dakota in all years of collection and inMontana in 2007. In this study, only two

A. rabiei isolates from one county wereconsidered to be QoI-resistant in 2005,while in 2006 and 2007, QoI-resistantisolates were present at a higher frequencythan sensitive isolates, and resistance waswidespread across the sampling locationsin North Dakota. With the continued appli-cation of QoI fungicides, it would not beexpected for the frequency of resistantisolates to decrease slightly from 2006 to2007, but differences among years canmost likely be explained by the increase inthe numbers of samples and sampling loca-tions from 2006 to 2007.

When the monitoring program was es-tablished in 2005, isolates were availablefrom a limited number of locations. Fungi-cide sensitivity monitoring was expandedin 2006 and 2007 to include a greaternumber of isolates from grower locations,which provided a more complete sensitiv-ity distribution of the A. rabiei populationin these areas. If the frequency of resistantisolates in a population is low at a giventime and location, it is likely that a largenumber of isolates will need to be tested todetect fungicide resistance, especially if aloss in disease control has not been ob-served with a fungicide (26). Subse-quently, it is difficult to determine if apathogen population is truly sensitive tofungicides based on the EC 50 values of oneor a few isolates from a location, and wecannot accurately state that fungicide resis-tance did not exist in some locations sam-pled in the Northern Great Plains and thePacific Northwest, since only a few sam-ples were available for testing. This rein-forces the need for adequate sample num-bers in fungicide sensitivity monitoringprograms, so that determination of isolatesensitivity, and consequently disease man-agement recommendations, are based onadequate and representative data.

QoI sensitivity evaluations via the gen-eration of EC 50 values from percent co-nidial germination is considered to be areliable and established method for de-termining fungicide sensitivity (24,35) andwas utilized to develop the previouslydescribed baseline for A. rabiei to QoIfungicides (33). However, these methodsare very time-consuming, especially con-sidering the large number of samples thatmust typically be examined to detect thetrue level of resistance in a pathogen popu-lation (26). Fungicide sensitivity assaysusing a single discriminatory dose oftenare utilized where fungicide resistance hasbeen identified in a pathogen population(21,25,34). An effective discriminatorydose is typically a fungicide concentrationat which growth of sensitive isolates ismostly or completely inhibited and resis-tant isolates have greater than 50% growth.This screening method allows a largenumber of isolates to be rapidly and accu-rately assessed for fungicide resistance,

Table 2. Collection information and location of 2006 and 2007 Ascochyta rabiei isolates tested for invitro QoI fungicide sensitivity using a discriminatory dose of 1 g/ml azoxystrobin

Collection locationby county

Number of locations sampled

Total numberof isolates

Isolates withQoI resistance a

2006North Dakota

McClean 1 4 0Renville 2 11 11Ward 1 5 3Williams 1 2 0

Total 5 22 14

2007North Dakota

Adams 1 12 12Burke 2 22 22Divide 3 17 17Hettinger 1 4 0McClean 3 27 27Mountrail 2 25 8Ward 1 16 16Williams 11 144 66

Total 24 267 168Montana

Gallatin 1 2 2Richland 1 2 2Sheridan 2 9 8Valley 1 2 0Yellowstone 1 1 1

Total 6 16 13a An isolate was considered resistant if mean conidia germination was >50% on the discriminatory dose.

Fig. 6. Mean in vivo percent disease severity for two QoI-sensitive (- - -) and three QoI-resistant () Ascochyta rabiei isolates to A, azoxystrobin and B, pyraclostrobin at each fungicide concentration(g/ml). Values include standard errors of disease control measurements obtained from two plantsacross three replications.


7/9


and has been used in several pathogensystems (21,25,34). The discriminatorydose of 1 g/ml was very effective in iden-tifying A. rabiei isolates resistant to azox-ystrobin. The development and use of adiscriminatory dose fungicide sensitivityassay for azoxystrobin and pyraclostrobinresistance monitoring facilitated thescreening of a much larger sample size of the A. rabiei population.

Differences in disease control were ob-served when azoxystrobin and pyraclos-trobin were applied to chickpea plantsinfected with QoI-resistant and QoI-sensitive isolates. Applications of azox-ystrobin at a concentration of 100 g/mlprovided less than 50% control of diseaseon plants infected with QoI-resistant iso-lates. This level of control is commer-cially unacceptable, and indicates that invitro fungicide assays are capable of pre-dicting A. rabiei isolate sensitivity invivo. Clear differences in disease severitywere observed between both QoI-sensitive isolates causing significantly

less disease on non-fungicide-treatedplants as compared to the three QoI-resistant isolates used in the study. Thissuggests that QoI-resistant A. rabiei iso-lates may have increased aggressivenesscompared to QoI-sensitive isolates, pos-sibly providing a competitive advantagein nature. These conclusions are based ona limited number of isolates, however,and additional pathogenicity studiesshould be conducted on a larger numberof QoI-sensitive and -resistant isolates todetermine if true differences in aggres-siveness exist.

Since no A. rabiei isolates were col-lected from North Dakota prior to 2005, itcannot be determined if detectable QoIfungicide resistance was present beforethis time. Despite this, QoI fungicide resis-tance was detected in under 3 years of registration and use for azoxystrobin andwithin 2 years for pyraclostrobin. Thisrapid shift in sensitivity has been observedin several other plant pathogens, including

Erysiphe graminis (4), Podosphaera xan-

thii (16), Pyricularia grisea (31), Colleto-trichum cereale (34), and Didymella bry-oniae (28). In each case, resistance to QoIfungicides occurred in two or less years.However, the speed at which resistance toQoI fungicides was expressed in A. rabiei is not necessarily surprising, since curativeapplications of a single chemical classwere applied repeatedly to a pathogen withthe potential for high inoculum productionand genetic diversity.

QoI resistance in A. rabiei was first re-ported in Canada in 2004, and in vitrobaseline sensitivity of Canadian popula-tions of A. rabiei to pyraclostrobin wasreported as 0.25 ppm (7). This value issubstantially higher than the sensitivity of

A. rabiei baseline populations in theNorthern Great Plains (0.0023 g/ml)(33), and due to methodological differ-ences and different baseline populations itis difficult to compare the results of theCanadian work with those of the currentresearch (7,15). Although it is difficult toascertain the effect of methodologicaldifferences on the detection of QoI-resistant phenotypes of A. rabiei , it isclear that standardized testing methodsusing baseline populations, spore germi-nation assays (4), and SHAM (33) arenecessary to provide accurate assessmentsof QoI sensitivity in different chickpeaproduction areas.

Large shifts in magnitude of fungicidesensitivity (>100) and a complete loss of disease control with QoI fungicides aretypical of isolates that have developed theG143A mutation conferring QoI fungicideresistance, and is documented in manypathogens (3,5,6,12,14,16,18,19,34). Whilethe specific mutation conferring QoI resis-tance in A. rabiei has not been deter-mined, greater than 100 sensitivity shiftswere observed in vitro and in our green-house fungicide efficacy study. Bothazoxystrobin and pyraclostrobin appliedat the highest rate (100 g a.i./ml) did notprovide adequate disease control of sus-pected QoI-resistant isolates. This leadsto speculation that the G143A mutation ispresent in A. rabiei , and indicates thatapplications of either QoI fungicide to aresistant A. rabiei population may notprovide the disease control necessary fora profitable crop. The lack of diseasecontrol and magnitude of resistance fac-tors observed with both azoxystrobin andpyraclostrobin with QoI-resistant isolatesindicates that cross-resistance to QoIfungicides is observed in A. rabiei onchickpea, and confirms a previous reportof in vitro cross-sensitivity (33).

Once the mutation conferring resistanceis determined for A. rabiei , allele-specificprimers can be designed to distinguishQoI-sensitive isolates from QoI-resistantisolates, and a real-time or quantitativePCR (Q-PCR) assay can be implementedfor fungicide resistance monitoring. Thismethod is preferable to screening isolates

Fig. 7. Mean in vivo percent disease control for two QoI-sensitive (- - -) and three QoI-resistant () Ascochyta rabiei isolates to A, azoxystrobin and B, pyraclostrobin at each fungicide concentration(g/ml). Values include standard errors of disease control measurements obtained from two plantsacross three replications.


8/9


using in vitro spore germination tech-niques, because it is rapid and accurate,and fungicide sensitivity can be deter-mined for a large number of isolates in ashort amount of time. This method wouldalso aid in determining if other resistancegenotypes such as the F129L or G137Rexist in populations of A. rabiei, sincethese mutations may not be easily ob-served with a discriminatory dose assay.Q-PCR has been used in fungicide resis-tance studies in several pathogens(12,18,23,27) and would be a desirablealternative for screening for QoI fungicidesensitivity in A. rabiei isolates in theNorthern Great Plains.

In response to the results presented here,North Dakota State University recom-mended that no applications of QoI fungi-cides be applied to chickpea in North Da-kota in 2007. Instead, it was recommendedthat preventative applications of chlorotha-lonil or maneb be applied prior to flower-ing, followed by a rotation of the fungi-cides boscalid and prothioconazole atflowering, or if conditions were favorablefor disease development. Although theDMI and carboximide fungicides are con-sidered to be at a medium risk for develop-ing resistance, fungicide resistance hasdeveloped within each of these classes inother pathogens (1,6). Thus, extreme careshould be taken to use these fungicides in amanner that prevents further developmentof A. rabiei fungicide resistance in otherfungicide classes. Cross-resistance withinfungicide classes limits the potential of new fungicides from the same chemicalclass for use in chickpea if resistance toone member of that class is already pre-sent. Furthermore, recent work in anothersystem has resulted in further complicationof resistance development: DMI-resistantisolates of Monilinia fructicola were re-ported to develop resistance to the QoIfungicide azoxystrobin more quickly thanDMI-sensitive isolates (20). This informa-tion reinforces the need for fungicide sen-sitivity monitoring in pathogens such as A.rabiei that are predisposed to fungicideresistance due to their biological nature,and are intensively managed with fungi-cide applications.

Until chickpea cultivars with durablelevels of Ascochyta blight resistance areavailable, fungicide applications for dis-ease management will be essential in theNorthern Great Plains. Additional researchis needed on the efficacy of new fungicidalcompounds and/or different chemicalclasses on Ascochyta blight to increase themanagement options available for growersand minimize the selection pressure on thepathogen due to repeated applications of one fungicide class.

ACKNOWLEDGMENTSThis project was funded by a grant from the

United States Department of Agriculture Coop-erative State Research, Education, and ExtensionService (USDA-CSREES) Cool Season Food

Legume Research Program. We thank C. Doetkottfor statistical consultation; N. Anderson, R. Benz,D. Liane, I. Mallik, D. Peterson, R. Sherman, andB. Tarang for technical assistance; and BASF Cor-poration and Syngenta Crop Protection for provid-ing the technical grade formulations of the fungi-cides.

LITERATURE CITED1. Avenot, H. F., and Michailides, T. J. 2007.

Resistance to boscalid fungicide in Alternariaalternata isolates from pistachio in California.Plant Dis. 91:1345-1350.

2. Avenot, H., Morgan, D. P., and Michailides, T.J. 2008. Resistance to pyraclostrobin, boscalid,and multiple resistance to Pristine (pyraclos-trobin + boscalid) fungicide in Alternaria al-ternata causing Alternaria late blight of pista-chios in California. Plant Pathol. 57:135-140.

3. Avila-Adame, C., Olaya, G., and Koller, W.2003. Characterization of Colletotrichumgraminicola isolates resistant to strobilurin-related QoI fungicides. Plant Dis. 87:1426-1432.

4. Bartlett, D. W., Clough, J. M., Godwin, J. R.,Hall, A. A., Hamer, M., and Parr-Dobrzanski,B. 2002. The strobilurin fungicides. Pest Man-age. Sci. 58:649-662.

5. Brasseur, G., Sami Saribas, A., and Daldal, F.1996. A compilation of mutations located inthe cytochrome b subunit of the bacterial and

mitochondrial bc1 complex. Biochim. Biophys.Acta 1275:61-69.6. Brent, K. J., and Holloman, D. W. 2007. Fun-

gicide resistance: The assessment of risk. 2nded. Fungicide Resistance Action Committee.Crop Life, Brussels, Belgium.

7. Chang, K. F., Ahmed, H. U., Hwang, S. F.,Gossen, B. D., Strelkov, S. E., Blade, S. F., andTurnbull, G. D. 2007. Sensitivity of field popu-lations of Ascochyta rabiei to chlorothalonil,mancozeb, and pyraclostrobin fungicides, andeffects of strobilurin fungicides on the progressof Ascochyta blight of chickpea. Can. J. PlantSci. 87:937-944.

8. Chen, W., Coyne, C., Peever, T., and Muehl-bauer, F. 2004. Characterization of chickpeadifferentials for pathogenicity assay of As-cochyta blight and identification of chickpea

accessions resistant to Didymella rabiei . PlantPathol. 53:759-769.9. Chin, K. M., Wirz, M., and Laird, D. 2001.

Sensitivity of Mycosphaerella fijiensis frombanana to trifloxystrobin. Plant Dis. 85:1264-1270.

10. Chongo, G., Gossen, B. D., Buchwaldt, L.,Adhikari, T., and Rimmer, S. R. 2004. Geneticdiversity of Ascochyta rabiei in Canada. PlantDis. 88:4-10.

11. Davidson, J. A., and Kimber, R. B. E. 2007.Integrated disease management of Ascochytablight in pulse crops. Eur. J. Plant Pathol.119:99-110.

12. Fraaije, B. A., Butters, J. A., Coelho, J. M.,Jones, D. R., and Holloman, D. W. 2002. Fol-lowing the dynamics of strobilurin resistancein Blumeria graminis f.sp. tritici using quanti-

tative allele-specific real-time PCR measure-ments with the fluorescent dye SYBR green I.Plant Pathol. 51:45-54.

13. Gan, Y. T., Siddique, K. H. M., MacLeod, W.J., and Jayakumar, P. 2006. Management op-tions for minimizing the damage by As-cochyta blight ( Ascochyta rabiei ) in chickpea(Cicer arietinum L.). Field Crops Res.97:121-134.

14. Gisi, U., Sierotzke, H., Cook, A., and McCaf-fery, A. 2002. Mechanisms influencing theevolution of resistance to Qo inhibitor fungi-cides. Pest Manage. Sci. 58:859-867.

15. Gossen, B. D., and Anderson, K. L. 2004. Firstreport of resistance to strobilurin fungicides in

Didymella rabiei . (Abstr.) Can. J. Plant Pathol.26:404.

16. Ishii, H., Fraaije, B. A., Sugiyama, T., Nogu-chi, K., Nishimura, K., Takeda, T., Amano, T.,and Hollomon, D. W. 2001. Occurrence andmolecular characterization of strobilurin resis-tance in cucumber powdery mildew and downymildew. Phytopathology 91:1166-1171.

17. Kaiser, W. J. 1997. Inter- and intranationalspread of Ascochyta pathogens of chickpea,faba bean, and lentil. Can. J. Plant Pathol.19:215-224.

18. Kianianmomeni, A., Schwarz, G., Felsenstein,F. G., and Wenzel, G. 2007. Validation of areal-time PCR for the quantitative estimation

of a G143A mutation in the cytochrome bc1 gene of Pyrenophora teres . Pest Manage. Sci.63:219-224.

19. Kim, Y. S., Dixon, E. W., Vincelli, P., andFarman, M. L. 2003. Field resistance to stro-bilurin (QoI) fungicides in Pyricularia grisea caused by mutations in the mitochondrial cy-tochrome b gene. Phytopathology 93:891-900.

20. Luo, C., and Schnabel, G. 2008. Adaptation tofungicides in Monilinia fructicola isolates withdifferent fungicide resistance phenotypes. Phy-topathology 98:230-238.

21. Mondal, S. N., Bhatia, A., Shilts, T., andTimmer, L. W. 2005. Baseline sensitivities of fungal pathogens of fruit and foliage of citrusto azoxystrobin, pyraclostrobin, and fenbu-conazole. Plant Dis. 89:1186-1194.

22. Nene, Y. L., and Reddy, M. V. 1987. Chickpea

diseases and their control. Pages 233-270 in:The Chickpea. M. C. Saxena and K. B. Singh,eds. CAB International, Oxon, UK.

23. Pasche, J. S., Piche, L. M., and Gudmestad, N.C. 2005. Effect of the F129L mutation in Al-ternaria solani on fungicides affecting mito-chondrial respiration. Plant Dis. 89:269-278.

24. Pasche, J. S., Wharam, C. M., and Gudmestad,N. C. 2004. Shift in sensitivity of Alternariasolani in response to QoI fungicides. Plant Dis.88:181-187.

25. Rebollar-Alviter, A., Madden, L. V., Jeffers, S.N., and Ellis, M. A. 2007. Baseline and differ-ential sensitivity to two QoI fungicides amongisolates of Phytophthora cactorum that causeleather rot and crown rot on strawberry. PlantDis. 91:1625-1637.

26. Russell, P. E. 2004. Sensitivity baselines in

fungicide resistance research and management.2nd ed. Fungicide Resistance Action Commit-tee. Crop Life, Brussels, Belgium.

27. Sierotzki, H., Frey, R., Wullschleger, J., Pal-ermo, S., Karlin, S., Godwin, J., and Gisi, U.2007. Cytochrome b gene sequence and struc-ture of Pyrenophora teres and P. tritici-repentis and implications for QoI resistance.Pest. Manage. Sci. 63:225-233.

28. Stevenson, K. L., Langston, D. B., Jr., andSeebold, K. W. 2004. Resistance to azox-ystrobin in the gummy stem blight pathogendocumented in Georgia. Online. Plant HealthProgress doi:10.1094/PHP-2004-1207-01-RS.

29. Tivoli, B., and Banniza, S. 2007. Comparisonof the epidemiology of Ascochyta blights ongrain legumes. Eur. J. Plant Pathol. 119:59-

76.30. Udupa, S. M., Weigand, F., Saxena, M. C., andKahl, G. 1998. Genotyping with RAPD andmicrosatellite markers resolves pathotype di-versity in the Ascochyta blight pathogen of chickpea. Theor. Appl. Genet. 97:299-307.

31. Vincelli, P., and Dixon, E. 2002. Resistance toQoI (strobilurin-like) fungicides in isolates of Pyricularia grisea from perennial ryegrass.Plant Dis. 86:235-240.

32. Wise, K., Bradley C., Henson B., McKay, K.,Chen, W., and Dugan, F. 2006. Pathotypes andfungicide sensitivity levels of Ascochyta rabiei isolates in the United States. (Abstr.) Proc. Int.Ascochyta Workshop Grain Legumes, 1st. LeTronchet, France. p. D-3.

33. Wise, K. A., Bradley, C. A., Pasche, J. S.,


9/9


Gudmestad, N. C., Dugan, F. M., and Chen, W.2008. Baseline sensitivity of Ascochyta rabiei to azoxystrobin, pyraclostrobin, and boscalid.Plant Dis. 92:295-300.

34. Wong, F. P., Midland, S. L., and de la Cerda,K. A. 2007. Occurrence and distribution of

QoI-resistant isolates of Colletotrichum ce-reale from annual bluegrass in California.Plant Dis. 91:1536-1546.

35. Wong, F. P., and Wilcox, W. F. 2000. Distribu-tion of baseline sensitivities to azoxystrobinamong isolates of Plasmopara viticola . Plant

Dis. 84:275-281.36. Wong, F. P., and Wilcox, W. F. 2002. Sensitiv-

ity to azoxystrobin among isolates of Uncinulanecator : Baseline distribution and relationshipto myclobutanil sensitivity. Plant Dis. 86:394-404.

Documents

Resist en CIA a Qoi Em Ascochyta