Fungicide Resistance Phenotypes Research Articles

Comparative fitness of Monilinia fructicola isolates with multiple fungicide-resistant phenotypes.

This study characterized 52 isolates of Monilinia fructicola from peach and nectarine orchards for their multi-resistance patterns to thiophanate-methyl (TF), tebuconazole (TEB), and azoxystrobin (AZO) using in vitro sensitivity assays and molecular analysis. The radial growth of M. fructicola isolates was measured on media amended with a single discriminatory dose of 1 µg/ml for TF and AZO and 0.3 µg/ml for TEB. Cyt b, CYP51, and ß-tubulin were tested for point mutations that confer resistance to quinone outside inhibitors (QoIs), demethylation inhibitors (DMIs), and methyl benzimidazole carbamates (MBCs), respectively. Eight phenotypes were identified including isolates with single, double, and triple in vitro resistance to QoI, MBC, and DMI fungicides. All resistant phenotypes to TF and TEB presented the H6Y mutation in ß-tubulin and the G641S mutation in CYP51. None of the point mutations typically linked to QoI resistance were present in the Monilinia isolates examined. Moreover, fitness of the M. fructicola phenotypes was examined in vitro and detached fruit assays. Phenotypes with single-resistance displayed equal fitness in in vitro and fruit assays compared to the wild-type. In contrast, the dual and triple-resistance phenotypes suffered fitness penalties based on osmotic sensitivity and aggressiveness on peach fruit. In this study, multiple resistance to MBC, DMI, and QoI fungicide groups was confirmed in M. fructicola. Results suggest that Monilinia populations with multiple resistance phenotypes are likely to be less competitive in the field than those with single resistance, thereby impeding their establishment over time and facilitating disease management.

Heat Treatment as a Possible Means to Reduce Botrytis Inoculum on Strawberry Transplants

Nursery transplants have been reported as one of the major sources of Botrytis cinerea primary inoculum causing Botrytis fruit rot of strawberry in Florida. In this study, we evaluated the efficacy of heat treatment to reduce Botrytis spp. on strawberry transplants. In laboratory experiments, conidia and sclerotia of four isolates with different fungicide resistance phenotypes were exposed to 44, 48, 52, and 56°C for 1, 5, 10, 30, 60 (1 h), 120 (2 h), and 240 (4 h) min. Conidial germination was completely inhibited after exposure for 5 min at 48°C or higher temperatures, or after 30 min at 44°C. Sclerotial germination varied for different isolates but in general required higher exposure times and temperature for inhibition. Based on a previous protocol developed to target Xanthomonas fragariae, strawberry transplants were treated at 44°C for 2 or 4 h with or without a preheat step of 37°C for 1 h and a cooling step of 1 h. After exposure to the heat treatments, detached leaf assays showed that the treatments significantly reduced pathogen incidence on leaves. Treatments at 44°C for 4 h, with or without preheat, significantly reduced Botrytis spp. incidence by 95.3 to 100% on noninoculated and 88.9 to 100% on inoculated transplants. In field trials, early fruit production was not affected by the heat treatments. Our results suggest that heat treatment could reduce the inoculum load of Botrytis spp. on strawberry transplants and potentially help improve the efficacy of fungicides to control the disease in production fields.

Natamycin as a postharvest treatment to control gray mold on stored blueberry fruit caused by multi-fungicide resistant Botrytis cinerea

Botrytis cinerea is the cause of gray mold, a major postharvest disease of blueberries grown in California. Control of gray mold is largely dependent on the use of synthetic fungicides. However, multiple resistance to different classes of fungicides is commonly present in B. cinerea populations from blueberry in the region. Alternatives to conventional chemical fungicides are needed to control postharvest gray mold. Natamycin, a naturally occurring substance listed as biofungicide, is registered for postharvest use in certain fresh fruit and it was highly effective to control various postharvest fungal diseases. In this study, natamycin was evaluated for its effectiveness to control gray mold caused by B. cinerea with resistance to various commonly used fungicides as well as its effectiveness to reduce decays in general that resulted from natural infections. Our results showed that as low as a quarter rate of the label rate of natamycin reduced gray mold incidence and lesion size regardless of fungicide resistance phenotypes. Natamycin also reduced overall fruit decay caused by natural infections and it had no adverse effect on fruit quality. Natamycin can be a promising tool to control postharvest gray mold of blueberry fruit while maintaining fruit quality.

Multifungicide resistance profiles and biocontrol in Lasiodiplodia theobromae from mango fields

Stem-end rot (SER) caused by Lasiodiplodia theobromae is a major postharvest disease of mango. To better understand the occurrence of multifungicides resistance in field populations, the sensitivity to five fungicides was investigated in 224 L. theobromae isolates collected from the mango growing region of Hainan Province in 2014–2018. The biocontrol activity of Bacillus amyloliquefaciens HBR-2 against resistant phenotypes of L. theobromae was evaluated using in vitro and in vivo tests. Our studies have established the baseline sensitivity of L. theobromae to difenoconazole, prochloraz and iprodione. Most isolates tested were sensitive to difenoconazole, prochloraz and iprodione. The overall resistance frequencies for difenoconazole, prochloraz and iprodione were 24.11, 15.18 and 1.34%, respectively. In addition, the overall resistance frequencies of L. theobromae for carbendazim and pyraclostrobin were 71.43% and 57.14%, respectively. In Hainan, 16 fungicide-resistant phenotypes were detected: 29.02, 60.27, 7.14, and 0.45% of the isolates were resistant to one, two, three, and four fungicides, respectively. The novel isolate B. amyloliquefaciens HBR-2 was found to have strong antagonistic activity against different resistant phenotypes of L. theobromae. The cell culture broth was the most effective treatment for controlling mango fruit rot in vivo. Multifungicide-resistant isolates of L. theobromae were widespread in Hainan. Biocontrol could be a valuable additional tool to manage the SER of mango fruit in the future. These findings would be useful in designing and implementing fungicide resistance management spray programs for the control of SER in mango.

Efficacy of Natamycin Against Gray Mold of Stored Mandarin Fruit Caused by Isolates of Botrytis cinerea With Multiple Fungicide Resistance.

Gray mold caused by Botrytis cinerea is an emerging postharvest disease of mandarin fruit in California. Management of postharvest diseases of mandarins relies on postharvest fungicides; however, multiple resistance to fungicides of different modes of action is common in B. cinerea populations from mandarin, leading to their failure to control decay. Natamycin is commonly used in the food industry as an additive, and it has been registered as a biofungicide for postharvest use on citrus and some other fruits. Sensitivity to natamycin of 64 isolates of B. cinerea from decayed mandarin fruit with known resistance phenotypes to other citrus postharvest fungicides (azoxystrobin, fludioxonil, pyrimethanil, and thiabendazole) was tested. Effective concentrations of natamycin to cause a 50% reduction relative to the control for conidial germination were from 0.324 to 0.567 µg/ml (mean of 0.444 µg/ml), and those for mycelial growth were 1.021 to 2.007 µg/ml (mean of 1.578 µg/ml). Minimum inhibitory concentrations where no fungal growth was present were 0.7 to 1.0 µg/ml for conidial germination and 5.0 to 10.0 µg/ml for mycelial growth. No cross-resistance between natamycin and other citrus postharvest fungicides was detected. Decay control efficacy tests with natamycin were conducted on mandarin fruit inoculated with B. cinerea isolates exhibiting five different fungicide resistance phenotypes, and natamycin significantly reduced incidence and lesion size of gray mold on fruit, regardless of fungicide resistance phenotypes. Natamycin has the potential to be an effective tool for integration into postharvest fungicide programs to control gray mold and manage B. cinerea isolates resistant to fungicides with other modes of action.

A Genome Resource for Several North American Venturia inaequalis Isolates with Multiple Fungicide Resistance Phenotypes.

The apple scab pathogen, Venturia inaequalis, is among the most economically important fungal pathogens that affects apples. Fungicide applications are an essential part of disease management. Implementation of cultural practices and genetic sources of resistance in the host are vital components of scab management. This is the first presentation of multiple, high quality, well-annotated genomes of four North American V. inaequalis isolates having both sensitive and multiple fungicide resistance phenotypes. We envision that these isolates will enable investigations into fungicide resistance mechanisms, exploring fungal virulence factors and delineating phylogenomic relationships among apple scab isolates from around the world.

Rapid Detection of Fungicide Resistance Phenotypes Among Populations of Nothopassalora personata in South Carolina Peanut Fields

We evaluated the occurrence of fungicide resistance phenotypes in populations of Nothopassalora personata, the causal agent of late leaf spot (LLS), sampled from South Carolina peanut fields in 2018 using a modified detached leaf assay (DLA). Spore suspensions obtained from each of 72 groups of LLS-symptomatic leaves collected from nine counties were used in the DLA to examine phenotypic resistance to four site-specific fungicides (azoxystrobin, benzovindiflupyr, prothioconazole, and thiophanate-methyl) and one multisite contact fungicide (chlorothalonil). Lesion development was measured as a binary event in which presence of a lesion indicated control failure (phenotypic resistance) and absence of a lesion indicated successful control (sensitivity). Phenotypic resistance probabilities of N. personata samples to each site-specific fungicide were compared against the control fungicide (chlorothalonil). Variation in phenotypic resistance probabilities to the four single-site fungicides was observed from most counties (6 of 9), with frequencies of phenotypic resistance having differed among counties. The DLA facilitated rapid evaluation of N. personata phenotypic resistance to azoxystrobin, benzovindiflupyr, prothioconazole, and thiophanate-methyl. This assay has potential to be used as an alternative method for routine monitoring of phenotypic resistance, identification of populations to test for the presence of resistance genes, and improving reliability of current fungicide recommendations.

Synergy between Cu-NPs and fungicides against Botrytis cinerea

Combating drug-resistance is a daunting task, especially due to the shortage of available drug alternatives with multisite modes of action. In this study, the potential of copper nanoparticles (Cu-NPs) to suppress 15 Botrytis cinerea isolates, which are sensitive or resistant to fungicides, alone or in combination with conventional fungicides, was tested in vitro and in vivo. Sensitivity screening in vitro revealed two fungicide resistance phenotypes, resulting from target site mutations. DNA sequencing revealed three B. cinerea isolates highly resistant to benzimidazoles (BEN-R), thiophanare methyl (TM), and carbendazim, bearing the E198A resistance mutation in the β-tubulin gene, and four isolates highly resistant to the QoI pyraclostrobin (PYR-R) with a G143A mutation in the cytb gene. Cu-NPs were equally effective against sensitive and resistant isolates. An additive/synergistic effect was observed between Cu-NPs and TM in the case of BEN-S isolates both in vitro and when applied in apple fruit. A positive correlation was observed between TM and TM + Cu-NPs treatments, suggesting that an increased TM availability in the target site could be related with the observed additive/synergistic action. No correlation between Cu(OH)2 and Cu-NPs sensitivity was found, indicating that different mechanisms govern the fungitoxic activity between nano and bulk counterparts. A synergistic profile was observed between Cu-NPs and fluazinam (FM) - an oxidative phosphorylation inhibitor - in all isolates regardless of resistance phenotype, suggesting that ATP metabolism could be involved in the mode of action of Cu-NPs. Furthermore, the observed cross sensitivity and antagonistic action between Cu-NPs and NaCl also provided evidence for copper ions contribution to the fungitoxic action of Cu-NPs. The results suggested that Cu-NPs in combination with conventional fungicides can provide the means for an environmentally safe, sustainable resistance management strategy by reducing fungicide use and combating resistance against B. cinerea.

Fungicide-resistant phenotypes in Botrytis cinerea populations and their impact on control of gray mold on stored table grapes in California

Gray mold caused by Botrytis cinerea is the major postharvest disease in table grapes grown in the Central Valley of California. Preharvest use of fungicide sprays may provide an alternative to the control of postharvest gray mold. However, fungicide resistance in B. cinerea can result in the failure of disease control. In this study, 212 isolates of B. cinerea were collected from table grape vineyards in three table grape-producing counties in the region and tested for resistance to selected fungicides on fungicide-amended media. In addition, 80 isolates were tested to establish baseline sensitivity to the newer fungicide fluopyram. Seven fungicide-resistant phenotypes were detected; 85.0%, 23.1%, 13.7%, and 94.8% of the isolates were resistant to boscalid, cyprodinil, fenhexamid, and pyraclostrobin, respectively. All isolates were sensitive to fludioxonil. Only 5.2% of the isolates were sensitive to all fungicides tested, whereas 8.9%, 56.1%, 23.6% and 6.1% were resistant to one, two, three, and four modes-of-action fungicides, respectively. Of the 80 isolates tested, all were sensitive to fluopyram with EC50 values ranging from 0.001 to 0.054 μg/mL. Most fungicides failed to control gray mold on detached table grapes inoculated with respective fungicide-resistant phenotypes. Our results suggest that alternation of sprays using different classes of fungicides will be needed to control postharvest gray mold, and that fludioxonil and fluopyram could be effective fungicides integrated into a preharvest fungicide spray program for control of gray mold in table grapes in the Central Valley of California.

Within-Season Shift in Fungicide Resistance Profiles of Botrytis cinerea in California Strawberry Fields.

Sensitivity of Botrytis cinerea to seven fungicide chemical classes was determined for 888 isolates collected in 2016 from 47 California strawberry fields. Isolates were collected early season (minimum fungicide exposure) and late season (maximum fungicide exposure) from the same planting block in each field. Resistance was determined using a mycelial growth assay, and variable frequencies of resistance were observed to each fungicide at both sampling times (early season %, late season %): boscalid (12, 35), cyprodinil (12, 46), fenhexamid (53, 91), fludioxonil (1, 4), fluopyram (2, 7), iprodione (25, 8), isofetamid (0, 1), penthiopyrad (8, 25), pyraclostrobin (77, 98), and thiophanate-methyl (81, 96). Analysis of number of chemical class resistances (CCRs) revealed an increasing shift in CCR from the early to late season. Phenotypes of 40 isolates that were resistant or sensitive to different chemical classes were associated with presence or absence of mutations in target genes. Fungicide-resistance phenotypes determined in the mycelial growth assay closely matched (93.8%) the genotype observed. Previously described resistance-conferring mutations were found for each gene. A survey of fungicide use from 32 of the sampled fields revealed an average of 15 applications of gray mold-labeled fungicides per season at an average interval of 12 days. The most frequently applied fungicides (average number of applications during the 2016 season) were captan (7.3), pyraclostrobin (2.5), cyprodinil (2.3), fludioxonil (2.3), boscalid (1.8), and fenhexamid (1.4). Multifungicide resistance is widespread in California. Resistance management tactics that reduce selection pressure by limiting fungicide use, rotating among Fungicide Resistance Action Committee codes, and mixing/rotating site-specific fungicides with multisite fungicides need to be improved and implemented.

Fungicide Resistance in Botrytis cinerea Populations in California and its Influence on Control of Gray Mold on Stored Mandarin Fruit.

Gray mold caused by Botrytis cinerea is an emerging postharvest disease affecting stored mandarin fruit in California. To develop effective control programs, fungicide sensitivities to four citrus postharvest fungicides were determined. One hundred B. cinerea isolates each in 2015 and 2016 were obtained from decayed fruit collected within packinghouses and tested for resistance to the fungicides. Sensitivity to azoxystrobin was examined based on the point mutation in the cyt b gene using PCR, while resistance to fludioxonil, pyrimethanil, and thiabendazole was examined on fungicide-amended media. For azoxystrobin, 83 and 98% of the isolates were resistant in 2015 and 2016, respectively. For pyrimethanil, 71 and 93% were resistant in 2015 and 2016, respectively. For thiabendazole, 63 and 68% were resistant in 2015 and 2016, respectively. No fludioxonil resistance was detected in both years. Five fungicide-resistant phenotypes were detected, and the most common phenotype was triple resistance to azoxystrobin, pyrimethanil, and thiabendazole, accounting for 59 and 65% in 2015 and 2016, respectively. Of the 200 B. cinerea isolates, 5, 23.5, and 62% were resistant to one, two, or three classes of fungicides, respectively. Inoculation tests were conducted to evaluate if the fungicides at label rates controlled various resistant phenotypes on fruit. Most fungicides failed to control gray mold on mandarin fruit inoculated with the respective fungicide resistant phenotypes. Our results suggest that alternative control methods need to be integrated into existing decay control programs to target this emerging disease on mandarin fruit.

Thiophanate-methyl resistance in Sclerotinia homoeocarpa from golf courses in China

Sclerotinia homoeocarpa causes dollar spot disease on many turfgrass species and is a significant problem worldwide. Thiophanate-methyl (TM), a methyl benzimidazole carbamate (MBC) fungicide, has been used for over forty years to manage dollar spot. Here we describe genetic mutations linked to three distinct TM fungicide resistance phenotypes: sensitive (S), moderately resistant (MR) and highly resistant (HR). These were established using multiple doses of TM, compared to previous studies using single discriminatory doses. In total, 19 S, 3 MR and 22 HR isolates were detected. Analysis of the β-tubulin gene revealed the MR isolates had a point mutation from T to A at codon 200 changing phenylalanine (TTC) to tyrosine (TAC). Twenty HR isolates had a mutation at codon 198 changing glutamic acid (GAG) to alanine (GCG) and two HR isolates had a mutation at codon 198 changing glutamic acid (GAG) to lysine (AAG). Allele-specific PCR assays were developed for rapid detection of these mutations in isolates of S. homoeocarpa. In addition, our results suggest a two-dose system for in vitro screening provides useful information for monitoring the development of resistance.

Fungicide resistance profile and genetic structure of Botrytis cinerea from greenhouse crops in Cyprus

Botrytis cinerea is a complex species prone to fungicide resistance and characterized by enormous genetic diversity. During 2013, 220 B. cinerea isolates causing gray mold were collected from greenhouse-grown crops in the regions of Ammochostos, Larnaca, and Limassol (Cyprus). Sensitivities of the sampled populations to seven botryticides with different modes of action were screened in vitro. The results of this in vitro screening highlighted the widespread phenomenon of fungicide resistance in greenhouses, since only 8.6 % of the isolates were sensitive to all botryticides. Resistance to thiophanate-methyl was the most prevalent, with frequencies ranging from 53.8 % to 80 %. Similarly, high resistance frequencies were observed for pyraclostrobin (27.1 to 78.9 %) and boscalid (28.2 to 66.2 %). Multiple fungicide resistant phenotypes were predominant, covering 67.3 % of the population, with frequencies of 80.0, 37.5, 53.8, 83.1, and 60.2 % in cucumber, eggplant, green bean, strawberry, and tomato, respectively. No fludioxonil-resistant isolates were observed. Botrytis cinerea and Botrytis group S genotypes comprised the gray mold population. B. cinerea was predominant within cucumber, eggplant and strawberry, whereas both genotypes were in equilibrium in green bean and tomato. However, Botrytis group S was found in all hosts. B. cinerea was the most prevalent in the majority of fungicide resistance phenotypes from strawberry, while genotype distributions within tomato were generally more balanced. B. pseudocinerea was not detected in the sampled population. Overall, frequency of the mating type allele MAT1–1 was higher to MAT1–2, underlying their unequal distribution in the population. However, cases of 1:1 distribution were apparent within particular subpopulations, suggesting that mating in the field cannot be excluded.

Fungicide Resistance Profiling inBotrytis cinereaPopulations from Blueberry in California and Washington and Their Impact on Control of Gray Mold

Gray mold caused by Botrytis cinerea is a major postharvest disease of blueberry grown in the Central Valley of California and western Washington State. Sensitivities to boscalid, cyprodinil, fenhexamid, fludioxonil, and pyraclostrobin, representing five different fungicide classes, were examined for 249 (California) and 106 (Washington) B. cinerea isolates recovered from decayed blueberry fruit or flowers. In California and Washington, 7 and 17 fungicide-resistant phenotypes, respectively, were detected: 66 and 49% of the isolates were resistant to boscalid, 20 and 29% were moderately resistant to cyprodinil, 29 and 29% were resistant to fenhexamid, and 66 and 55% were resistant to pyraclostrobin. All isolates from California were sensitive to fludioxonil, whereas 70% of the isolates from Washington showed reduced sensitivity to fludioxonil. In California, 26 and 30% of the isolates were resistant to two and three classes of fungicides, respectively. In Washington, 31, 14, 16, and 9% of the isolates were resistant to two, three, four, and five classes of fungicides, respectively. Inherent risk of the development of resistance to quinone outside inhibitor (QoI) fungicides was assessed by detecting the presence of the Bcbi-143/144 intron in gene cytb. The intron was detected in 11.8 and 40% of the isolates in California and Washington, respectively, suggesting that the risk of QoI resistance is higher in California than in Washington. On detached blueberry fruit inoculated with 11 isolates exhibiting different fungicide-resistant phenotypes, most fungicides failed to control gray mold on fruit inoculated with the respective resistant phenotypes but the mixture of cyprodinil and fludioxonil was effective against all fungicide-resistant phenotypes tested. Our findings would be useful in designing and implementing fungicide resistance management spray programs for control of gray mold in blueberry.

Reduced Virulence of Azoxystrobin-Resistant Zymoseptoria tritici Populations in Greenhouse Assays.

The development of resistance to multiple fungicide classes is currently limiting disease management options for many pathogens, while the discovery of new fungicide classes may become less frequent. In light of this, more research is needed to quantify virulence trade-offs of fungicide resistance in order to more fully understand the implications of fungicide resistance on pathogen fitness. The purpose of this study was to measure the virulence of azoxystrobin-resistant and -sensitive Zymoseptoria tritici populations collected from North and South Willamette Valley, Oregon, in 2012 and 2015. Inoculum mixtures of known fungicide-resistant phenotypes were used to simulate natural field conditions, where multiple genotypes exist and interact in close proximity. Six greenhouse inoculations were conducted over 2 years, and virulence of the isolate mixtures was evaluated in planta. We considered virulence to be "the degree of pathology caused by the organism" and visually estimated the percent area of leaf necrosis as a measure of virulence. In greenhouse conditions, a consistent association of reduced virulence with azoxystrobin-resistant Z. tritici isolate mixtures was observed. North Willamette Valley and South Willamette Valley populations did not differ in virulence.

RIM15 antagonistic pleiotropy is responsible for differences in fermentation and stress response kinetics in budding yeast.

Different natural yeast populations have faced dissimilar selective pressures due to the heterogeneous fermentation substrates available around the world; this increases the genetic and phenotypic diversity in Saccharomyces cerevisiae In this context, we expect prominent differences between isolates when exposed to a particular condition, such as wine or sake musts. To better comprehend the mechanisms underlying niche adaptation between two S. cerevisiae isolates obtained from wine and sake fermentation processes, we evaluated fermentative and fungicide resistance phenotypes and identify the molecular origin of such adaptive variation. Multiple regions were associated with fermentation rate under different nitrogen conditions and fungicide resistance, with a single QTL co-localizing in all traits. Analysis around this region identified RIM15 as the causative locus driving fungicide sensitivity, together with efficient nitrogen utilization and glycerol production in the wine strain. A null RIM15 variant confers a greater fermentation rate through the utilization of available glucose instead of its storage. However, this variant has a detrimental effect on fungicide resistance since complex sugars are not synthesized and transported into the membrane. Together, our results reveal the antagonist pleiotropic nature of a RIM15 null variant, positively affecting a series of fermentation related phenotypes, but apparently detrimental in the wild.

Multiple-fungicide resistance to carbendazim, diethofencarb, procymidone, and pyrimethanil in field isolates of Botrytis cinerea from tomato in Henan Province, China

Botrytis cinerea, the fungal pathogen responsible for causing gray mold in tomatoes, frequently exhibits multiple fungicide resistance. In this study, the sensitivity to carbendazim (Car), diethofencarb (Die), procymidone (Prc), and pyrimethanil (Pyr) was determined for 263 isolates of B. cinerea from diseased fruit and leaves of tomato plants from 87 commercial greenhouses in Henan Province of China during 2013 and 2014. Of the isolates tested, 89%, 93%, 85%, and 86% were resistant to Car, Die, Prc, and Pyr, respectively. Twelve phenotypes were found having different sensitivity patterns, and multi-fungicide resistance was found to one, two, three or four fungicides. The percentage of multiple fungicide resistant isolates (CarRDieRPrcRPyrR) was 68%. Mycelial growth, spore production, and mycelial dry weight were not significantly different between resistant and sensitive phenotypes of the B. cinerea isolates. However, the virulence of CarSDieRPrcSPyrS isolates was higher than that of the other fungicide-resistance phenotypes. All of the carbendazim and diethofencarb resistant phenotypes carried a point mutation at codon 198 (E198A or E198K) or 200 (F200Y) in the β-tubulin gene, and all of the procymidone resistant phenotypes carried point mutations at codons 369 (Q369P) and 373 (N373S) in the Bos1 gene. These results provided important reference data for assessment of resistance risk of B. cinerea isolates from commercial tomato transplants in Henan Province, indicating the existence of a high frequency of resistance to carbendazim, diethofencarb, procymidone, and pyrimethanil revealing the necessity of a more integrated control of gray mold.

Fungal endophytes of invasive Phagramites australis populations vary in species composition and fungicide susceptibility

Phragmites australis subsp. australis (Poaceae) is an aggressively invasive reed that threatens both freshwater and saltwater ecosystems in North America. We sampled P.a. australis plants for fungal endophytes at seven sites across a short geographic range near a freshwater lake in Michigan. Compared to previous studies, our data reveal novel variation in the diversity and abundance offungal endophytes withinP.a. australis. Within each sampling site we observed 4-10 mor- phologically unique, culturable fungi. Since fungal endophytes can confer significant benefits to their plant host, we hypothe- sized that fungal endophytes are important for mediating plant invasions. To test this hypothesis we first had to establish a protocol to experimentally control fungal endophytes within P.a. australis, which does not easily grow from sterile seed. We therefore investigated the effect of fungicides as a potential method for eliminating fungi from living plant tissue such as rhizomes. We selected the ten most abundant fungi isolated from P.a. australis and tested their susceptibility to three com- monly available fungicides. Response to the fungicides varied across fungal isolates, demonstrating physiological variation and fungicide-resistant phenotypes.

Detection and characterization of fungicide resistant phenotypes of Botrytis cinerea in lettuce crops in Greece

The development of resistance to chemical control agents needs continuous monitoring in Botrytis cinerea. 790 isolates from lettuce and other vegetable crops were collected from six widely separated sites in Greece and tested for their sensitivity to 11 fungicides from nine unrelated chemical groups. 44 of the isolates exhibited multiple resistance to fenhexamid (hydroxyanilides), azoxystrobin and pyraclostrobin (QoI’s), boscalid (SDHI’s), cyprodinil and pyrimethanil (anilinopyrimidines), fludioxonil (phenylpyrroles), carbendazim (benzimidazoles) and iprodione (dicarboximides). Thirty per cent of such phenotypes were detected in an experimental glasshouse with lettuce crops, the third year after commencing fungicide applications. The average resistance factor (Rf) for mycelial growth to fenhexamid, pyraclostrobin, boscalid, cyprodinil and fludioxonil, was over 40, 1,000, 100, 700 and 50, respectively. Some strains with high resistance to anilinopyrimidines (14 %) or moderate to fludioxonil (7 %) were detected even in isolates collected from vegetable crops prior to commercial use of these fungicides in Greece. Isolates with fludioxonil moderate resistance and fenhexamid high resistance, were detected for the first time in Greece. The results suggested the high risk in chemical control of grey mould due to development of resistance to most fungicides with site-specific modes of action. Isolates with resistance to fluazinam (phenylpyridinamines) and to chlorothalonil (phthalonitriles) were not found. The inclusion of appropriate multi-site inhibitors like chlorothalonil in fungicide anti-resistance strategies was indispensable.

Two Promoter Rearrangements in a Drug Efflux Transporter Gene Are Responsible for the Appearance and Spread of Multidrug Resistance Phenotype MDR2 inBotrytis cinereaIsolates in French and German Vineyards

In French and German vineyards, Botrytis cinerea isolates with multiple fungicide resistance phenotypes have been observed with increasing frequencies. Multidrug resistance (MDR) results from mutations that lead to constitutive overexpression of genes encoding drug efflux transporters. In MDR2 and MDR3 strains, overexpression of the major facilitator superfamily transporter gene mfsM2 has been found to result from a rearrangement in the mfsM2 promoter (type A), caused by insertion of a retroelement (RE)-derived sequence. Here, we report the discovery of another, similar RE-induced rearrangement of the mfsM2 promoter (type B) in a subpopulation of French MDR2 isolates. MDR2 isolates harboring either type A or type B mutations in mfsM2 show the same resistance phenotypes and similar levels of mfsM2 overexpression. RE sequences similar to those in mfsM2 were found in low copy numbers in other but not all B. cinerea strains analyzed, including non-MDR2 strains. Population genetic analyses support the hypothesis that the two rearrangement mutations have only occurred once, and are responsible for the appearance and subsequent spread of all known MDR2 and MDR3 strains in French and German wine-growing regions.