Fungicide Sensitivity Research Articles

Genetic diversity in Monilinia laxa populations in stone fruit species in Hungary

The objectives of this study were firstly, to determine the genetic diversity of Monilinia laxa isolates from Hungary, using the PCR-based inter-simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) technique; secondly, to prepare genetic diversity groups based on the dendrograms; and finally, to select some relevant isolates to study their fungicide sensitivity. 55 and 77 random amplified polymorphic ISSR and RAPD markers, of which 23 and 18 were polymorphic and 32 and 59 monomorphic, respectively, were used to assess the genetic diversity and to study the structure of M. laxa populations in Hungary. 27 isolates out of 57 ones were confirmed as M. laxa from several orchards (subpopulations) in three geographical regions, in various inoculum sources and in various hosts, were used. 10 fungicides and 12 isolates selected from genetic diversity groups based on the ISSR dendrograms were used to determine the fungicide sensitivity of the selected isolates. The analysis of population structure revealed that genetic diversity within locations, inoculum sources and host (H(S)) accounted for 99 % of the total genetic diversity (H(T)), while genetic diversity among locations, inoculum sources and host represented only 1 %. The relative magnitude of gene differentiation between subpopulations (G(ST)) and the estimate of the number of migrants per generation (Nm) averaged 0.005-0.009 and 53.9-99.2, respectively, for both ISSR and RAPD data set. The results obtained in dendrograms were in accordance with the gene diversity analysis. Grouping of isolates in the dendrograms was irrespective of whether they came from the same or different geographical locations. There was no relationship between clustering among isolates from inoculum sources and hosts. In the fungicide sensitivity tests, five isolates out of 12 were partly insensitive to boscalid+piraclostrobin, cyprodinil, fenhexamid or prochloraz. Obtained results in genetic diversity of M. laxa populations are discussed together with implications for the management of brown rot.

Fungicide Efficacy Against Calonectria pseudonaviculata, Causal Agent of Boxwood Blight.

Calonectria pseudonaviculata causes leaf spot and stem lesions resulting in defoliation and dieback of boxwood. Fungicides representing 20 different active ingredients from 13 different Fungicide Resistance Action Committee groups were evaluated for their effects on conidial germination and mycelial growth using in vitro assays, and the concentration that suppressed fungal growth to 15% of that on unamended media (EC85) values were determined. A number of fungicides strongly inhibited mycelial growth of C. pseudonaviculata. Four demethylation inhibitor fungicides had EC85 values of 1.2 μg a.i./ml or less. Thiophanate-methyl, fludioxonil, pyraclostrobin, trifloxystrobin, kresoxim-methyl, mancozeb, and chlorothalonil also had activity against mycelial growth. Fludioxonil + cyprodinil had a lower EC85 than the same rate of fludioxonil alone, suggesting that cyprodinil had activity against mycelial growth. Fungicides that inhibited C. pseudonaviculata conidial germination include pyraclostrobin, trifloxystrobin, and kresoxim-methyl as well as fludioxonil, mancozeb, chlorothalonil, and boscalid. Quinoxyfen, etridiazole, fenhexamid, hymexazol, famoxadone, and cymoxanil did not inhibit either C. pseudonaviculata conidial germination or mycelial growth. In comparison with values found in the literature, EC50 values for kresoxim-methyl were up to 10 times higher than reported previously, suggesting that fungicide insensitivity may have developed. Protectant fungicides with activity against conidial germination and systemic fungicides with activity against mycelial growth, such as those identified here, may be complementary to achieve the high levels of pathogen management required for control of this disease. In addition, multiple fungicide active ingredients from different mode-of-action groups used in mixtures or over time may also act to slow selection for fungicide insensitivity.

Fungicide Sensitivity Assessed in Monilinia vaccinii-corymbosi Isolates from Lowbush Blueberry Fields in Maine

The fungicide sensitivity in populations of Monilinia vacciniicorymbosi (Reade) Honey, the fungus that causes mummy berry, may be affected by the use of fungicides for control of this fungus. The sensitivity of M. vaccinii-corymbosi isolates from conventionally and organically managed fields and an unmanaged, wild area was determined for the fungicides propiconazole and fenbuconazole. Propiconazole has been used for more than 14 years to control mummy berry, and fenbuconazole has become more widely used in the last 4 years than previously. The baseline EC50 for isolates from the unmanaged area was significantly higher for propiconazole (0.016 μg/ml) than fenbuconazole (0.006 μg/ml). The propiconazole EC50 for isolates from conventionally managed fields (0.020 μg/ml) was significantly higher than for isolates from the unmanaged area (0.016 μg/ml), but not from the organically managed fields (0.018 μg/ml). No significant differences in the fenbuconazole EC50 were found among management types. The biennial long-term use of the fungicide propiconazole on populations of M. vaccinii-corymbosi may contribute to the development of fungicide insensitivity over time. Accepted 15 August 2014. Published 24 September 2014.

Resistance of Kansas Sclerotinia homoeocarpa Isolates to Thiophanate-Methyl and Determination of Associated β-Tubulin Mutation

Eighty-two isolates of Sclerotinia homoeocarpa from 12 sites in Kansas were evaluated for in vitro sensitivity to the methyl benzimidazole carbamate (MBC) fungicide thiophanate-methyl at the discriminatory dose of 10 μg/ml. Seventeen isolates were sensitive to thiophanate-methyl and the remaining isolates were resistant. Of the 65 isolates from golf course putting greens, two isolates were sensitive and the remaining 63 isolates were resistant. Six resistant and five sensitive isolates were also evaluated in greenhouse assays on fungicide-treated plants. The isolates that were sensitive to thiophanate-methyl in vitro did not cause any disease on thiophanate-methyl-treated plants, and those that were resistant in vitro caused blighting on treated plants equivalent to the nontreated controls. The entire β-tubulin gene was sequenced for four resistant and four sensitive isolates. The resistant isolates all harbored a substitution of alanine for glutamic acid at codon 198 (E198A). These results provide a starting point for further surveys and monitoring of fungicide sensitivity. Accepted 7 April 2014. Published 6 June 2014.

Chemical management in fungicide sensitivity of Mycosphaerella fijiensis collected from banana fields in México.

The chemical management of the black leaf streak disease in banana caused by Mycosphaerella fijiensis (Morelet) requires numerous applications of fungicides per year. However this has led to fungicide resistance in the field. The present study evaluated the activities of six fungicides against the mycelial growth by determination of EC50 values of strains collected from fields with different fungicide management programs: Rustic management (RM) without applications and Intensive management (IM) more than 25 fungicide application/year. Results showed a decreased sensitivity to all fungicides in isolates collected from IM. Means of EC50 values in mg L(-1) for RM and IM were: 13.25 ± 18.24 and 51.58 ± 46.14 for azoxystrobin, 81.40 ± 56.50 and 1.8575 ± 2.11 for carbendazim, 1.225 ± 0.945 and 10.01 ± 8.55 for propiconazole, 220 ± 67.66 vs. 368 ± 62.76 for vinclozolin, 9.862 ± 3.24 and 54.5 ± 21.08 for fludioxonil, 49.2125 ± 34.11 and 112.25 ± 51.20 for mancozeb. A molecular analysis for β-tubulin revealed a mutation at codon 198 in these strains having an EC50 greater than 10 mg L(-1) for carbendazim. Our data indicate a consistency between fungicide resistance and intensive chemical management in banana fields, however indicative values for resistance were also found in strains collected from rustic fields, suggesting that proximity among fields may be causing a fungus interchange, where rustic fields are breeding grounds for development of resistant strains. Urgent actions are required in order to avoid fungicide resistance in Mexican populations of M. fijiensis due to fungicide management practices.

잔디에 Rhizoctonia 마름병을 유발하는 Rhizoctonia spp.의 침투성 살균제에 대한 반응

Rhizoctonia spp. 에 의한 Rhizoctonia 마름병은 잔디에서 중요한 병이며, 골프장에서 잔디의 좋은 품질을 유지하기 위하여 살균제를 사용하고 있다. 본시험은 포장에서 분리한 R. solani AG-1 IB, R. cerealis 및 R. solani AG2-2에 대한 침투성 살균제인 flutolanil, pyraclostrobine, and hexaconazole에 대하여 실내에서 상대적인 균사생장을 효과적으로 50%억제하는 <TEX>$EC_{50}$</TEX>농도를 결정하는 시험을 수행하였다. 각각 살균제에 대한 감수성은 3종의 Rhizoctonia균주에 대하여 5 수준의 농도를 사용하였다. 3종류의 살균제에 대한 <TEX>$EC_{50}$</TEX> 값은 R. solani AG-1 IB에서 가장 낮았다. 하지만, R. solani AG2-2에 대한 살균제 감수성은 pyraclostrobine 과 flutolanil에서 평균 <TEX>$EC_{50}$</TEX> 값이 0.026 <TEX>${\mu}g\;a.i.\;ml^{-1}$</TEX> 와 0.044 <TEX>${\mu}g\;a.i.\;ml^{-1}$</TEX> 로 가장 높았다. R. cerealis은 hexaconazole에서 <TEX>$EC_{50}$</TEX> 값이 0.022 <TEX>${\mu}g\;a.i.\;ml^{-1}$</TEX>로 가장 감수성이 낮았다. 각각의 살균제의 <TEX>$EC_{50}$</TEX>값을 사용하여 두 농약을 혼용한 3종의 조합에서 3종의 병원균에 대한 균사 생장억제율은 R. solani AG2-2에서 가장 높았다. 본 연구결과는 포장에서 Rhizoctonia 마름병을 방제하기 위하여 실내에서 침투성 살균제에 대한 반응을 확인할 수 있었다. The Rhizoctonia blight causing by Rhizoctonia spp. is an important disease of turfgrass, requiring fungicide application to maintain acceptable conditions for turfgrass good qualities in the golf course. The experiment was conducted to determine the mean 50% effective concentration inhibiting mycelial growth (<TEX>$EC_{50}$</TEX>) value of to flutolanil, pyraclostrobine and hexaconazole to Rhizoctonia solani AG-1 IB, Rhizoctonia cerealis and Rhizoctonia solani AG2-2IV isolated from Gyeongbuk province of Korea in vitro. Five discriminatory concentrations of each fungicide were used to detect in vitro sensitivity. The mean of <TEX>$EC_{50}$</TEX> values to three systemic fungicides was the lowest isolate of R. solani AG-1 IB. However, the sensitivity of fungicides to Rhizoctonia solani AG2-2IV were higher mean <TEX>$EC_{50}$</TEX> value of 0.026 <TEX>${\mu}g\;a.i.\;ml^{-1}$</TEX> of pyraclostrobine and 0.044 <TEX>${\mu}g\;a.i.\;ml^{-1}$</TEX> of flutolanil. R. cerealis was the lowest sensitivity to hexaconazole which was an average <TEX>$EC_{50}$</TEX> value of 0.022 <TEX>${\mu}g\;a.i.\;ml^{-1}$</TEX>. Inhibition of mycelial growth rate (%) by three combine fungicides using the <TEX>$EC_{50}$</TEX> value of each fungicide was the highest R. solani AG2-2IV. Results of this study were may confirmed in vitro response fungicide of three Rhizoctonia species for control of Rhizoctonia blight in the field.

First Report of a Binucleate Rhizoctonia (AG-A) from Potato Stems Infecting Potatoes and Sugarbeet in the Pacific Northwest.

Rhizoctonia solani causes economically important diseases on potatoes and sugarbeet throughout the world (2). R. solani is a species complex of 13 anastomosis groups (AGs) of which R. solani AG3-PT is most commonly associated with potato and AG2-2 and AG4 with sugarbeet. However, several AGs, including AG2-2 and AG4, have been recorded causing potato diseases (2,3). In summer 2012, plants in potato fields in Idaho were sampled for R. solani. Isolations were attempted from symptomatic plants. DNA extracted from the resulting pure Rhizoctonia cultures was screened using a real-time PCR assay for AG3-PT (3). For the isolates that tested negative for AG3-PT, AG was determined by amplifying and sequencing the rDNA internal transcribed spacer (ITS) region using the primers ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The resulting sequences of two isolates (isolates 204 and 206, GenBank Accession No. KC782951) shared 99% identity with other AG-A isolates (AY927358 and AY927356). Koch's postulates were confirmed for isolate 206 by placing five 10-mm plugs, from 10-day-old potato dextrose agar (PDA) cultures, onto the surface of a soil-less potting mix (composed of peat moss, perlite, and sand) of 1-liter pots, where non-inoculated PDA plugs served as a control. Each pot contained a 'Rosara' seed tuber or three ungerminated (BETASEED - BTS 27RR10) sugarbeet seeds (n = 5). Pots were incubated in a glasshouse between 18 and 22°C for 1 month and then assessed for disease. For potatoes, a pigmented necrosis was observed at the soil interface in 88% of the stems and plants were stunted relative to the non-inoculated controls. A significant reduction in root growth was observed in 60% of the germinated sugarbeet plants. Control plants of both potatoes and beets were asymptomatic. For reisolation, 1-cm sections were taken from each potato stem and germinated beet plant, surface sterilized, and placed on alkaline water agar. The reisolated fungi were identified using morphology and a subset was confirmed by sequencing. Isolate 206 was successfully recovered from 84% of the potato stems and from 20% of the sugarbeet seedlings. In a similar experiment, 2-month-old potato and sugarbeet plants were inoculated using 50 g of autoclaved barley grains (inoculated with isolate 206) per 1-liter pot. Between 40 and 60% of inoculated plants appeared stunted in both cases. Pigmented necrosis was observed at the soil interface on 45% of the potato stems and reduced root growth was observed in the 50% of the sugarbeet plants. Control plants were asymptomatic. To our knowledge, this is the first report of the binucleate AG-A causing disease in Idaho on potato stems. BNR species have previously been isolated from potato (4) and sugarbeet plants (1). The binucleate Rhizoctonia AG-A caused disease on potato stems and sugarbeet roots and was readily reisolated. Since sugarbeet is commonly grown in rotation with potato in Idaho, such a rotation could increase the risk of soilborne infection to either crop by AG-A. It is known that AGs can differ in fungicide sensitivity (2), and thus a knowledge of which AGs may be present is important when considering disease management strategies.

Serine O-acetyltransferase is important, but not essential for cysteine–methionine synthesis in Fusarium graminearum

O-acetyltransferase (SAT) is a key enzyme converting serine into O-acetylserine in the synthesis of sulphur-containing amino acids. To characterize the function of FgSAT in Fusarium graminearum, three deletion mutants of FgSAT (ΔFgSAT-1, -2 and -18) were obtained using a gene replacement strategy. The three mutants did not show recognizable phenotypic changes on potato dextrose agar medium, but exhibited a very weak growth on fructose gelatin agar (FGA) medium containing SO₄²⁻ as sole sulfur source. Supplementation of O-acetylserine, cysteine, or methionine, but not serine, rescued the defect of mycelial growth in FgSAT deletion mutants, indicating that FgSAT is involved in conversion of serine into O-acetylserine. The three mutants had a decrease in conidiation in mung bean liquid, but not in carboxymethyl cellulose. Virulence, deoxynivalenol production and fungicide sensitivity assays found that the three mutants showed no significant difference from wild-type progenitor PH-1. Real-time PCR assays detected an increase in expression levels of FgOAHS, FgCBS and FgCGL genes involved in the alternative pathway in FgSAT deletion mutants, suggesting that the alternative pathway in F. graminearum is present and can operate. Addition of homoserine, the upstream substrate of the alternative pathway, also restored the normal mycelial growth of FgSAT deletion mutants on FGA, indicating that the alternative pathway in F. graminearum might be positively regulated by homoserine.

Fungicide Resistance Profiles for 13 Botrytis cinerea Isolates from Strawberry in Southeastern Louisiana

Fungicidal sprays are widely used for control of Botrytis fruit rot; however, the pathogen often develops resistance to frequently used fungicides. A 96-well plate micro-dilution broth bioassay developed for fungicide discovery was used to provide strawberry growers with a rapid assessment of the fungicide sensitivity of Botrytis isolates against 16 fungicides. Three sensitivity phenotypes were identified: benzimidazole and dicarboximide resistant, benzimidazole resistant and dicarboximide sensitive, and an intermediate response to both fungicides. Codon at position 198 in the β-tubulin gene confirmed benomyl resistance. This bioassay rapidly identifies fungicide resistance and allows growers to quickly adjust their disease management strategy.

QoI Resistance in Fusicladium carpophilum Populations from Almond in California and Evaluation of Molecular Resistance Mechanisms.

Disease management failures have been reported in California for almond scab caused by Fusicladium carpophilum following quinone outside inhibitor (QoI) applications. Resistance in the pathogen populations was found to be common and at high incidence in the major almond-growing regions beginning in 2003, 4 years after registration of azoxystrobin on this crop. Two levels of azoxystrobin resistance, moderate and high, were identified with 50% effective concentration (EC50) values between 0.15 and 10 μg/ml or >40 μg/ml, respectively. Reference isolates collected before resistance was detected had EC50 values <0.05 μg/ml. High-resistance was associated with a G143A mutation in the mitochondrial cytochrome b gene. For the less commonly found moderately resistant isolates, no mutations in the gene were detected between codons 122 and 212. Using primers targeting the G143A mutation or the cytochrome b gene of all F. carpophilum isolates in quantitative polymerase chain reaction (qPCR) analyses, the frequency of highly resistant isolates was accurately determined in mixtures of conidia with selected ratios of sensitive and resistant isolates. The frequency of high resistance in bulked samples of scab lesions, however, was generally underestimated compared with in vitro testing of fungicide sensitivity of fungal isolates from the same lesions. Competition experiments using conidial suspensions demonstrated stability of the highly resistant genotype in the presence of different amounts of sensitive and moderately resistant genotypes. Analysis of covariance of linear regressions of cycle threshold values on DNA concentrations derived from qPCR amplifications using two primer pairs for cytochrome b alleles with and without the G143 mutation showed that several isolates differed in their slopes and midpoints. Thus, heteroplasmy of mitochondrial-inherited QoI resistance is suggested as a likely cause for incongruence in estimating resistance frequencies using the two methods.

Sensitivity of Corynespora cassiicola from soybean to carbendazim and prothioconazole

The incidence of target spot disease on soybean has increased in recent years in Brazil even with intensive use of fungicides, and fungal resistance has been reported in recent studies. The objective of this study was to determine the fungicide sensitivity to carbendazim and prothioconazole in a sample of 24 isolates of Corynespora cassiicola from soybean collected from 1996 to 2011 in the states of Parana, Mato Grosso and Sao Paulo ( Brazil) and Corpus Christi (Paraguay). The 50% effective concentration (EC50) values were estimated by the relative mycelium growth reduction on fungicide-amended medium with the doses of 0, 0.5, 1, 10, 100 and 1000 µg of active ingredient/ mL. For carbendazim, four highly resistant isolates (EC50>50 µg/mL) were observed from samples collected from Mato Grosso in 2008 and from Parana and Mato Grosso in 2011. The EC50 values for prothioconazole ranged from 0.47 µg/mL to 26.44 µg/mL (mean: 5.02 µg/ ml). The results reinforce the occurrence of C. cassiicola resistance to benzimidazole in Parana and Mato Grosso states.

Constraints on the evolution of azole resistance in plant pathogenic fungi

The durability of azole fungicides in controlling agriculturally important pathogenic fungi is unique amongst modern single site fungicides. Today, azoles are still relied on to control pathogens of many crops including cereals, fruits and vegetables, canola and soybeans. Significantly, this widespread use continues despite many reports of azole‐resistant fungal strains. In this review, recent reports of azole resistance and the mechanisms associated with resistant phenotypes are discussed. The example of the complex evolution of the azole target sterol 14α‐demethylase (CYP51) enzyme in modern European populations of the wheat pathogen Mycosphaerella graminicola is used to describe the quantitative and epistatic effects on fungicide sensitivity and enzyme function of target site mutations, and to explore the hypothesis that constraints on CYP51 evolution have ensured the longevity of azoles. In addition, the threats posed by alternative resistance mechanisms causing cross‐resistance to all azoles or even unrelated fungicides are discussed, and postulations are made on how using new genomic technologies to gain a greater understanding of azole resistance evolution should enhance the ability to control azole‐resistant strains of plant pathogenic fungi in the future.

Rhizoctonia solani Anastomosis Group 3 is Predominant in Potato Tubers in Poland.

Rhizoctonia solani is a species complex of 13 related but genetically distinct anastomosis groups (AGs). In potato, R. solani can infect the stems, stolons, and roots, resulting in quantitative losses. It can also cause qualitative losses through blemishes occurring on progeny tubers, such as black scurf and elephant hide (corky cracking). Knowledge of the AG in local populations is important because they differ in host range, fungicide sensitivity, and disease severity (2). To determine the AGs present in Poland, 54 tuber samples displaying typical R. solani symptoms were taken from six different fields in 2011. The fields were representative of five different administrative regions of Poland and from at least 10 different varieties. Rhizoctonia was isolated from tubers by placing symptomatic material on to tap water agar amended with streptomycin and penicillin and after 2 to 3 days Rhizoctonia colonies were identified and hyphal tips of these transferred to potato dextrose agar. Rhizoctonia was successfully isolated from 48 tubers displaying black scurf and two tubers displaying elephant hide symptoms. DNA was extracted from Rhizoctonia cultures using a Wizard Food kit (Promega) and the AG was determined using specific real-time PCR assays (1). All Rhizoctonia isolates were determined to be AG3 and this was confirmed for 10 selected isolates by observing hyphal fusion with a known AG3 tester isolate (Rs08) as described previously (3). Pairings were also conducted amongst the 10 Polish isolates, C2 reactions were typically observed indicating numerous vegetative compatible groups are present. This study shows that AG3 is likely to be the predominant AG in potato tubers in Poland. This is similar to other studies in Europe, which have all determined that AG3 accounts for at least 92% of isolates from potato (2,3). AG2-1, 4, and 5 have also been found in tubers worldwide and climate and certain crop rotations can influence the presence of these other AGs in potato tubers (2). However, climate and crop rotations in Poland are similar to other parts of Europe so the predominance of AG3 is expected. AG3 was also isolated from elephant hide symptoms; however, it was more frequently isolated from sclerotia. The ability of AG3 to prolifically produce sclerotia and thereby survive on seed tubers may explain its predominance in potato crops (4). Therefore, studies focusing on the management of Rhizoctonia potato disease in Poland should consider AG3 in the first instance.

First Report of QoI Insensitive Cercospora beticola on Sugar Beet in Ontario, Canada.

Cercospora beticola Sacc. causes Cercospora leaf spot (CLS) of sugar beet (Beta vulgaris L.) and is the most destructive foliar disease of sugar beet worldwide (1). The QoI fungicide pyraclostrobin has been an important management tool for CLS in Canada since 2003. Beginning in 2010, some growers reported poor disease control after applying pyraclostrobin. Leaf disk samples with CLS lesions were collected in September 2012 from 16 commercial fields located in Kent and Lambton Counties, Ontario, Canada. These counties (ca. 300,000 ha) encompass the major commercial sugar beet production area in Ontario (ca. 3,925 ha). CLS severity ranged from low to severe among the sampling sites. Leaf discs with a single leaf spot were cut from leaves using a hole punch. Spots were up to 5 mm in diameter with tan, light brown, or sometimes gray centers. DNA was extracted from leaf discs using a Qiagen DNeasy Plant Mini Kit (Germantown, MD) according to the manufacturer's instructions. PCR was used to amplify a fragment of the C. beticola cytochrome b (CYTB) gene (4). Pure cultures were obtained by placing plant tissue in a moist chamber and transferring single spores to V8 juice agar. PCR products were sequenced for 32 samples at the Genomics Technology Support Facility (Michigan State University, East Lansing, MI) and 25 were confirmed to have 100% identity with the sequence of QoI-resistant C. beticola from Michigan (2) and to QoI-resistant isolates from GenBank (Accession Nos. JQ619933 and JQ360628). The remaining seven had 100% identity with a sensitive isolate (EF176921.1). Each resistant isolate contained a change in codon 143 that is predicted to lead to a substitution of G143A in the cytochrome b gene. This G143A mutation has been associated with QoI resistance in a number of fungi (3). To confirm the result, a conidium germination bioassay was carried out using nine isolates with the G143A mutation on sugar beet leaf agar covered with water agar amended with pyraclostrobin at concentrations ranging from 0 to 54.3 μg/ml and distributed on a spiral gradient using an Eddyjet II spiral plater. The medium was supplemented with salicylhydroxamic acid (SHAM) to block the alternate oxidation pathway. Following incubation at 25°C for 2 days, the distance between the center of the plate at which conidial germination was 50% of the maximum observed growth (EC) and the point at which conidial germination terminated were measured (TEC). The EC50 values were determined from the SGE software for each isolate by entering the EC and TEC values, respectively. The estimated EC50 for a representative wild type (sensitive) isolate was 0.03 μg/ml, while the value for the resistant isolate could not be calculated because it was greater than the highest concentration tested (54.3 μg/ml). Additionally, in the controls with no SHAM or fungicide, the resistant isolate showed a consistent reduced germination rate compared to the sensitive isolate (30.0% and 93.5% germination, respectively). Confirmation of fungicide insensitivity will require a re-evaluation of current management practices in Ontario to minimize economic losses due to CLS.

Occurrence of the F129L mutation in Alternaria solani populations in Germany in response to QoI application, and its effect on sensitivity

Early blight caused by Alternaria solani is a highly destructive disease of potatoes. Control of early blight mainly relies on the use of preventive fungicide treatments. Because of their high efficacy, azoxystrobin and other quinone outside inhibitors (QoIs) are commonly used to manage early blight. However, loss of sensitivity to QoIs has previously been reported for A. solani in the United States. Two hundred and three A. solani field isolates collected from 81 locations in Germany between 2005 and 2011 were screened for the presence of the F129L mutation in the cytochrome b gene; of these, 74 contained the F129L mutation. Sequence analysis revealed the occurrence of two structurally different cytb genes, which differed in the presence (genotype I) or absence (genotype II) of an intron, with genotype I being the most prevalent (63% of isolates). The F129L mutation was detected only in genotype II isolates, where it occurred in 97%. Sensitivity to azoxystrobin and pyraclostrobin was determined in conidial germination assays. All isolates possessing the F129L mutation had reduced sensitivity to azoxystrobin and, to a lesser extent, to pyraclostrobin. Early blight disease severity on plants treated with azoxystrobin was significantly higher for A. solani isolates with reduced fungicide sensitivity in the conidial germination assay compared with sensitive isolates. Data suggest an accumulation of F129L isolates in the German A. solani population over the years 2009–2011. It is assumed that the application of QoIs has selected for the occurrence of F129L mutations, which may contribute to loss of fungicide efficacy.

Evaluation of Eggplant Rootstocks and Pepper Varieties for Potential Resistance to Isolates of Phytophthora capsici from Michigan and New York.

Phytophthora capsici is a soilborne pathogen of major economic importance in pepper, and of less importance in tomato and eggplant production. As soil fumigation becomes more expensive and limited, and fungicide insensitivity of P. capsici isolates becomes more prevalent, grafting is quickly becoming an industry-favored method to control soilborne diseases. Greenhouse experiments were performed to evaluate an eggplant cultivar (Classic), two eggplant lines (EG195, EG203), a pepper line (CM334), and three pepper cultivars (Paladin, Camelot, and Red Knight) for root rot resistance to 14 P. capsici isolates. The isolates showed various degrees of virulence between pepper and eggplant in both experiments. Both eggplant and one pepper lines showed moderate resistance to the most virulent isolates tested in experiment one. The partially resistant pepper cultivar, Paladin, was significantly more susceptible than CM334 and the eggplant lines, but was still resistant to most isolates. In the second experiment, the eggplant cultivar Classic and the susceptible pepper cultivar Red Knight were both susceptible to most isolates tested, while EG203 and EG195 were resistant to most isolates. The two eggplant breeding lines, EG195 and EG203, showed moderate resistance to all isolates tested in both experiments. This is the first reported evaluation of eggplant resistance to P. capsici. Further research is warranted to test eggplant lines EG195 and EG203 for resistance to a wide range of soilborne pests and to evaluate their usefulness as P. capsici-resistant rootstocks for peppers, tomatoes, and eggplants.

Field Strains of Monilinia fructicola Resistant to Both MBC and DMI Fungicides Isolated from Stone Fruit Orchards in the Eastern United States.

In 2012, significant brown rot disease was observed on stone fruit in Pennsylvania, Maryland, and South Carolina despite preharvest application of methyl benzimidazole carbamate (MBC) and demethylase inhibitor (DMI) fungicides. In total, 140 Monilinia fructicola isolates were collected from diseased orchards and examined for fungicide sensitivity. In addition to isolates resistant to either the DMI propiconazole or the MBC thiophanate-methyl, 22 isolates were discovered that were resistant to both fungicides, including 4 isolates from peach in South Carolina, 12 isolates from peach and sweet cherry in Maryland, and 6 isolates from sweet cherry in Pennsylvania. Analysis of MBC resistance revealed that dual-resistant isolates from South Carolina carried the β-tubulin E198A mutation, whereas isolates from Maryland and Pennsylvania carried E198 mutations not previously described in the Monilinia genus, E198Q or F200Y. The genetic element Mona, associated with DMI fungicide resistance in M. fructicola, was detected in the dual-resistant isolates from South Carolina but not in the isolates from the two more northern states. An investigation into the molecular mechanism of DMI resistance in the latter isolates revealed that resistance was not based on increased expression or mutation of MfCYP51, which encodes the target of DMI fungicides. Label rates of formulated propiconazole or thiophanate-methyl were unable to control dual-resistant isolates on detached peach fruit, confirming field relevance of dual resistance. The same isolates were not affected by fitness penalties based on mycelial growth rate, ability to sporulate, and virulence on detached peach fruit. The emergence of M. fructicola strains resistant to both DMI and MBC fungicides in multiple states and multiple stone fruit crops is a significant development and needs to be considered when designing resistance management strategies in stone fruit orchards.

A two‐component histidine kinase Shk1 controls stress response, sclerotial formation and fungicide resistance in Sclerotinia sclerotiorum

Fungal histidine kinases (HKs) are involved in osmotic and oxidative stress responses, hyphal development, fungicide sensitivity and virulence. Members of HK class III are known to signal through the high-osmolarity glycerol mitogen-activated protein kinase (HOG MAPK). In this study, we characterized the Shk1 gene (SS1G_12694.3), which encodes a putative class III HK, from the plant pathogen Sclerotinia sclerotiorum. Disruption of Shk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides and increased sensitivity to hyperosmotic stress and H2 O2 -induced oxidative stress. The Shk1 mutant showed a significant reduction in vegetative hyphal growth and was unable to produce sclerotia. Quantitative real-time polymerase chain reaction (qRT-PCR and glycerol determination assays showed that the expression of SsHOG1 (the last kinase of the Hog pathway) and glycerol accumulation were regulated by the Shk1 gene, but PAK (p21-activated kinase) was not. In addition, the Shk1 mutant showed no change in virulence. All the defects were restored by genetic complementation of the Shk1 deletion mutant with the wild-type Shk1 gene. These findings indicate that Shk1 is involved in vegetative differentiation, sclerotial formation, glycerol accumulation and adaption to hyperosmotic and oxidative stresses, and to fungicides, in S. sclerotiorum. Taken together, our results demonstrate, for the first time, the role of two-component HKs in Sclerotinia.

Screening and characterization of resistance to succinate dehydrogenase inhibitors in Alternaria solani

Field isolates of Alternaria solani, which causes early blight of potato in Idaho, USA were evaluated in vitro for their sensitivity towards the succinate dehydrogenase inhibitor (SDHI) fungicides boscalid, fluopyram and penthiopyrad. A total of 20 isolates were collected from foliar‐infected tissue in 2009, 26 in 2010 and 49 in 2011. Fungicide sensitivity was tested using the spiral‐gradient end point dilution method. The frequency of boscalid‐resistant isolates (&gt;50% relative growth when using a spiral dilution gradient starting at 507 mg L−1) drastically increased over the duration of this study (15% in 2009, 62% in 2010 and 80% in 2011). Increasing resistance to fluopyram and penthiopyrad was observed. However, cross‐resistance was only observed between boscalid and penthiopyrad. The target site of this fungicide class is the succinate dehydrogenase (SDH) enzyme complex, which is vital for fungal respiration. Sequence analysis of the SDH complex revealed mutations in the subunits B and D that were correlated with the emergence of boscalid resistance in potato fields in Idaho. In particular, H277R and H133R were identified in SDH subunits B and D, respectively. The presence of restriction sites in the gene sequences allowed the development of a rapid PCR‐RFLP method to assess boscalid sensitivity in A. solani populations.

Assessing fungicide resistance in populations of Alternaria in Idaho potato fields

Early blight, caused by the fungus Alternaria solani and brown leaf spot, caused by Alternaria alternata, are important diseases of potato crops in Idaho. In recent years growers have reported a reduction in efficacy of fungicides traditionally used in the past decade to control early blight. In 2009, a collection of A. solani 39 isolates were screened for resistance to azoxystrobin, pyraclostrobin, boscalid and famoxadone. Fungicide sensitivity testing was done using spiral plate dilution gradients. Results showed that of 39 isolates screened, all were resistant to azoxystrobin and three were resistant to boscalid. None were resistant to pyraclostrobin or famoxadone. In summer 2010, more isolates were collected (9 A. alternata and 26 A. solani) and the survey was expanded to include more fungicides with four different modes of action that targeted succinate dehydrogenase (SDH), methionine biosynthesis, mitochondrial respiration and multi-site contact activity. New isolates of A. solani and A. alternata were also collected from two additional sites. The results showed that 57% of the isolates were resistant to boscalid as well as an average of 63% of the isolates being resistant to the strobilurin fungicides. Seven and 15% of isolates were resistant to penthiopyrad (an SDH inhibitor), and pyrimethanil (a methionine biosynthesis inhibitor), respectively. However, none of the isolates were resistant to fluopyram (an SDH inhibitor) or a mixture of fluopyram and pyrimethanil. Although there appears to be cross resistance developing in Alternaria spp. to some of the new SDH inhibitors like penthiopyrad, others such as fluopyram are still showing limited to no resistance development in Alternaria spp. in Idaho.