Fungicide Selection Research Articles

Assessing the predictability of fungicide resistance evolution through in vitro selection

Plant pathogens are highly adaptable, and have evolved to overcome control measures including multiple classes of fungicides. More effective management requires a thorough understanding of the evolutionary drivers leading to resistance. Experimental evolution can be used to investigate evolutionary processes over a compressed timescale. For fungicide resistance, applications include predicting resistance ahead of its emergence in the field, testing potential outcomes under multiple different fungicide usage scenarios or comparing resistance management strategies. This review considers different experimental approaches to in vitro selection, and their suitability for addressing different questions relating to fungicide resistance. When aiming to predict the evolution of new variants, mutational supply is especially important. When assessing the relative fitness of different variants under fungicide selection, growth conditions such as temperature may affect the results as well as fungicide choice and dose. Other considerations include population size, transfer interval, competition between genotypes and pathogen reproductive mode. However, resistance evolution in field populations has proven to be less repeatable for some fungicide classes than others. Therefore, even with optimal experimental design, in some cases the most accurate prediction from experimental evolution may be that the exact evolutionary trajectory of resistance will be unpredictable.

Influence of fungicide selection and application timing on take‐all root rot management under field and greenhouse conditions

AbstractTake‐all root rot (TARR) is a detrimental disease of ultradwarf bermudagrass (Cynodon dactylon × Cynodon transvaalensis; UDB) putting greens frequently diagnosed where warm‐season grasses are grown. Since this disease is largely aggregated and variable under field conditions, field research is difficult and often yields inconsistent results. Multiple pathogens have only recently been associated with this disease, so practical management solutions such as fungicide efficacy and fungicide application timing have not been thoroughly investigated. Therefore, the objectives of this research were to determine the influence of fungicide selection and fungicide application timing on take‐all root rot management under field and greenhouse conditions. In general, fungicides from the quinone outside inhibitor and/or demethylation inhibitor chemical classes provided the greatest reduction in take‐all root rot severity. Fungicide applications that were made when soil temperatures were between 77–86°F provided the greatest disease suppression. The in vivo greenhouse method developed in this research proved to be an efficient and consistent method to evaluate management practices such as fungicide efficacy on take‐all root rot. This research improves our understanding of fungicide efficacy and fungicide application timing for take‐all root rot management on ultradwarf bermudagrass.

TaqMan-MGB PCR Method for Rapid Detection of QoI Fungicide Resistance in Chinese Populations of Plasmopara viticola.

Grape downy mildew caused by Plasmopara viticola is one of the most devastating diseases of grapevine worldwide. Quinone outside inhibitor (QoI) fungicides are commonly used for the control of the pathogen in grape fields across China. However, their recurrent use could lead to the emergence of resistance against these compounds. Based on the most common mutation in resistant isolates, a glycine to alanine substitution at amino acid position 143 (G143A) in the cytochrome b protein, a TaqMan-MGB PCR was developed for the rapid detection of resistance to the QoI fungicide azoxystrobin in P. viticola. Specificity and sensitivity of this method showed it could specifically detect the point mutations linked with QoI resistance in P. viticola, and the detection limit was 0.2 pg. It could also quantify the resistance allele even in isolate mixtures containing as little as 5% QoI-resistant P. viticola strains. With this method, a large P. viticola population (n = 2,373) was screened, and QoI-resistant isolates were identified for the first time in China. The average frequencies of the resistant genotype from eight major-grapevine regions were up to 66%. Taken together, the results not only provide a novel tool for the rapid distinction and quantification of the QoI-resistant allele in P. viticola but also provide important references for fungicide selection and application, which will facilitate resistance management of grape downy mildew and improve grape production systems in Chinese vineyards.

In Vitro Fungicide Sensitivity and Effect of Organic Matter Concentration on Fungicide Bioavailability in Take-All Root Rot Pathogens Isolated from North Carolina

Take-all root rot (TARR) of ultradwarf bermudagrass is caused by Gaeumannomyces graminis (Gg), Gaeumannomyces graminicola (Ggram), Candidacolonium cynodontis (Cc), and Magnaporthiopsis cynodontis (Mc). Multiple pathogens have recently been associated with this disease, and biological parameters such as fungicide sensitivity have not been explored in ultradwarf bermudagrass. Although fungicides are commonly used to mitigate disease development, high organic matter present in the turfgrass system could limit the bioavailability of fungicides. Fungicide bioavailability can be influenced by organic matter concentration, and the physicochemical properties of fungicides could provide insight into their binding affinity. However, the influence of organic matter content on fungicide bioavailability has not been investigated. Therefore, the in vitro sensitivity of Gg, Ggram, Cc, and Mc to 14 different fungicides across three chemical classes was determined. An in vitro bioavailability assay was developed using three fungicides and three organic matter concentrations. Generally, demethylation inhibitor and quinone outside inhibitor fungicides provided the greatest reduction in mycelial growth, whereas succinate dehydrogenase inhibitors did not reduce mycelial growth. These data can serve as a foundation for TARR pathogen sensitivity to inform in vitro fungicide sensitivity studies and field efficacy trials. Pyraclostrobin and propiconazole have a high affinity to bind to organic matter, which was evident as more fungicide was required to inhibit Gg growth as organic matter concentration increased. This was not observed when evaluating azoxystrobin, which has a lower binding affinity. Understanding how TARR pathogens respond to fungicide in vitro and how organic matter concentration affects in vitro sensitivity will improve fungicide selection for management of TARR.

Baseline sensitivity of Coniella vitis to fluazinam and resistance risk assessment

AbstractGrape white rot is a destructive fungal disease caused by the pathogenic fungus Coniella vitis. The sensitivity of C. vitis to fluazinam was tested to select an efficient fungicide to control this disease. Furthermore, the baseline sensitivity of C. vitis populations to fluazinam was established based on the EC50 of 62 C. vitis strains collected from different regions in which grapevines are grown in China. The C. vitis populations were extremely sensitive to fluazinam. The efficiency of fluazinam to grape white rot was determined with detached grapevine leaves. Fluazinam was highly effective in controlling this disease. Moreover, the risk of the C. vitis strains developing resistance to fluazinam was assessed with five mutant strains resistant to fluazinam obtained by a fungicide selection method in the laboratory. The fluazinam‐resistant mutant strains were deficient in their hyphal growth, stress and virulence compared with the parental strain. This led to the conclusion that the C. vitis populations are highly sensitive to fluazinam, and C. vitis is unlikely to develop resistance to fluazinam under natural conditions. Thus, fluazinam is a potential fungicide to efficiently control grape white rot.

Benzimidazole-Resistant Isolates with E198A/V/K Mutations in the β-Tubulin Gene Possess Different Fitness and Competitive Ability in Botrytis cinerea.

Previous studies in Botrytis cinerea showed that resistance to methyl benzimidazole carbamates (MBCs) was mainly related to E198A/V/K and F200Y mutations of the β-tubulin gene, and E198V was the dominant mutation in the resistant subpopulation in Hubei Province of China, indicating that resistant mutations might influence fitness. However, little is known about the effect of each E198A/V/K mutation on fitness. In this study, the fitness and competitive ability of isolates with E198A/V/K mutations were investigated. Results showed that E198A/V/K isolates and wild-type isolates shared similar fitness components in terms of virulence, sporulation, conidial germination, oxidative sensitivity, and sclerotial production and viability. However, slower mycelial growth at 4°C, higher sensitivity to 4% NaCl, and increased sclerotial production percentage at 4°C were observed in the isolates with E198V, E198K, and E198A mutations, respectively. Competitive analysis showed that the wild-type subpopulation became dominant after three disease cycles in the absence of fungicide selection pressure, whereas the resistant subpopulation seized the space of the sensitive subpopulation upon MBC application. Unexpectedly, the frequency of E198V isolates decreased dramatically after the first disease cycle with or without fungicide selection pressure. These results suggest that MBC-resistant isolates suffer little fitness penalty but possess competitive disadvantages in the absence of fungicide selection pressure. Under fungicide selection pressure, E198V isolates could not compete with E198A/K isolates. According to the current results, there is a great possibility that the E198V mutation will lose dominance in the future in China.

Colletotrichum Species Associated with Peaches in China

Colletotrichum is regarded as one of the 10 most important genera of plant pathogens in the world. It causes diseases in a wide range of economically important plants, including peaches. China is the largest producer of peaches in the world but little is known about the Colletotrichum spp. affecting the crop. In 2017 and 2018, a total of 286 Colletotrichum isolates were isolated from symptomatic fruit and leaves in 11 peach production provinces of China. Based on multilocus phylogenetic analyses (ITS, ACT, CAL, CHS-1, GAPDH, TUB2, and HIS3) and morphological characterization, the isolates were identified to be C. nymphaeae, C. fioriniae, and C. godetiae of the C. acutatum species complex, C. fructicola and C. siamense of the C. gloeosporioides species complex, C. karsti of the C. boninense species complex, and one newly identified species, C. folicola sp. nov. This study is the first report of C. karsti and C. godetiae in peaches, and the first report of C. nymphaeae, C. fioriniae, C. fructicola, and C. siamense in peaches in China. C. nymphaeae is the most prevalent species of Colletotrichum in peaches in China, which may be the result of fungicide selection. Pathogenicity tests revealed that all species found in this study were pathogenic on both the leaves and fruit of peaches, except for C. folicola, which only infected the leaves. The present study substantially improves our understanding of the causal agents of anthracnose on peaches in China.

Rust (Puccinia emaculata) Management and Impact on Biomass Yield in Switchgrass.

Rust, putatively caused by Puccinia emaculata, is a widespread and potentially damaging disease of switchgrass, a crop produced as feedstock for livestock and bioenergy. Azoxystrobin, chlorothalonil, and myclobutanil were applied at 1-, 2-, 3-, or 4-week intervals for 12 to 14 weeks to the vegetatively propagated switchgrass cultivar Cloud Nine to assess fungicide selection and application interval for the control of rust as well as the impact of this disease on switchgrass biomass yield. Although rust severity significantly differed among study years, azoxystrobin and myclobutanil were often equally and more effective than chlorothalonil at controlling rust, with superior disease control coming at shorter application intervals compared with extended application intervals. Year, product, application interval, and product × interval significantly impacted dry biomass yield, which was greatest in 2016 and lowest in 2014. Dry biomass yield protection was significantly better with azoxystrobin and myclobutanil applications than with chlorothalonil or no fungicide. Linear regression models with the final disease rating, as well as with the area under disease progress curve in each year, were significant, but coefficients of determination were low to moderate (0.21 < R2 < 0.60), indicating that rust response and subsequent disease impact on dry biomass yield were impacted by other factors. From our models, an estimated 3 to 5% biomass decline was calculated for each 10% increment in rust-related leaf necrosis observed at the final September rating date. With rust-related leaf necrosis ≥80% by 1 September in each of 4 study years, biomass yield may be reduced by 24 to 40% if rust problems are not managed in switchgrass crops.

Competitive ability of multi-fungicide resistant Botrytis cinerea in a blackberry planting over three years

Botrytis cinerea isolates with multi-fungicide resistance have frequently been isolated from small fruit fields such as strawberries and blackberries. Individual B. cinerea isolates have been found resistant to up to seven chemical classes of fungicides. Fitness costs and less competitiveness have been observed in multi-fungicide resistant isolates, but this has not been examined under field conditions. In the spring of 2016, flowers of field-grown blackberries were either not inoculated or inoculated with B. cinerea isolates sensitive (0CCR), resistant to five or six chemical classes excluding phenylpyrroles (5CCR), or resistant to six or seven chemical classes including phenylpyrroles (6CCR/MDR1h). The experimental field was left unsprayed for the duration of this study and isolates of B. cinerea were collected from flowers and/or fruit in each of the three experimental years. Isolates collected in summer of 2016 revealed resistance phenotypes in each plot closely matching those of the respective inoculum, with 95% 0CCR, 55% 5CCR, and 91% 6CCR/MDR1h isolates recovered from 0CCR, 5CCR, and 6CCR/MDR1h inoculation plots, respectively. In the 2017 and 2018 isolate collections, 6CCR/MDR1h resistance phenotypes were found in plots inoculated and non-inoculated with this phenotype, indicating their persistence and movement between plots. Resistance phenotypes different from the inoculum were also recovered each year, indicating that the inoculum was successfully competing with a native Botrytis population. Despite the competition, 6CCR/MDR1h isolates were recovered in high frequency from all inoculated plots in 2018. G3pdh and mrr1 sequences of 6CCR/MDR1h isolates collected in 2018 were identical to the sequences of the inoculum, indicating that these isolates likely descended from the inoculum. This study demonstrates that isolates carrying multi-fungicide resistant phenotypes, specifically 6CCR/MDR1h, are competitive in the absence of fungicide selection pressure.

Evaluation of a Sclerotinia homoeocarpa Population with Multiple Fungicide Resistance Phenotypes Under Differing Selection Pressures.

Dollar spot, caused by Sclerotinia homoeocarpa, is one of the most significant diseases of cool-season turfgrass on golf courses. Resistance to the benzimidazole, dicarboximide, and succinate dehydrogenase inhibitor (SDHI) classes and reduced sensitivity to the sterol-demethylation inhibitor (DMI) in S. homoeocarpa populations have been widely reported in the United States. Moreover, the occurrence of S. homoeocarpa populations with multiple fungicide resistance (MFR) is a growing problem on golf courses. The present study was undertaken to evaluate the efficacy of DMI, dicarboximide, and SDHI against a S. homoeocarpa population with MFR on a Connecticut golf course fairway from 2014 to 2016. Also, because the S. homoeocarpa population consisted of four different phenotypes with differing resistance profiles to benzimidazole, dicarboximide, and DMI, in vitro sensitivity assays were used to understand the dynamics of the MFR population in the presence and absence of fungicide selection pressures. Results indicated that boscalid fungicide (SDHI) was able to provide an acceptable control of the MFR dollar spot population. Propiconazole or iprodione application selected isolates with both DMI and dicarboximide resistance (DMI-R/Dicar-R). In the absence of fungicide selection pressures, the percent frequency of DMI-R/Dicar-R or DMI and benzimidazole resistance (DMI-R/Ben-R) isolates declined in the population. Out of the four phenotypes, the percent frequency of isolates with DMI, dicarboximide, and benzimidazole resistance (DMI-R/Dicar-R/Ben-R) was the lowest in the population regardless of fungicide selection pressures. Our first report of MFR population dynamics will help develop effective strategies for managing MFR and potentially delay the emergence of future resistant populations in S. homoeocarpa.

Fitness Penalties in the Evolution of Fungicide Resistance.

The evolution of resistance poses an ongoing threat to crop protection. Fungicide resistance provides a selective advantage under fungicide selection, but resistance-conferring mutations may also result in fitness penalties, resulting in an evolutionary trade-off. These penalties may result from the functional constraints of an evolving target site or from the resource allocation costs of overexpression or active transport. The extent to which such fitness penalties are present has important implications for resistance management strategies, determining whether resistance persists or declines between treatments, and for resistance risk assessments for new modes of action. Experimental results have proven variable, depending on factors such as temperature, nutrient status, osmotic or oxidative stress, and pathogen life-cycle stage. Functional genetics tools allow pathogen genetic background to be controlled, but this in turn raises the question of epistatic interactions. Combining fitness penalties under various conditions into a field-realistic scenario poses an important future challenge.

Exploring Genotype × Environment × Management synergies to manage fusarium head blight in wheat

Fusarium head blight (FHB) is a devastating disease of wheat because of direct detrimental effects on grain yield, quality, and marketability and production of mycotoxins (e.g. deoxynivalenol or DON). Conditions most favourable for the development of FHB are high humidity, frequent rainfall and relatively warm night temperatures at heading, especially in regions where host crop residues are present. These risk factors have resulted in a significant westward expansion of FHB in the Canadian prairies, while favouring continued development in Ontario, Quebec and the Maritimes. Since cultivar selection is a key integrated pest management (IPM) strategy, a systems approach that couples genetic resistance with management tactics is required. Thus, successful FHB mitigation is an ideal case study in Genotype (G) × Environment (E) × Management (M) interactions where more resistant cultivars (G) are grown in at-risk regions (E), and unique approaches to management (M) for sustainable wheat production are required. Since no cultivar is completely resistant to FHB, greater attention to management strategies is needed. The over-arching principle for FHB management is the manipulation of agronomic factors that facilitate completion of critical crop developmental phases, such as flowering, while doing so rapidly and uniformly, as a consequence of early sowing and increased seeding rates. These strategies and the adoption of practices involving proper fungicide selection, and optimal application timings and methods will lead to improved yield stability and quality in high-risk environments. This paper explores the potential synergies that exist for FHB mitigation when appropriate genetics are combined with an array of key agronomic strategies.

Fungicide Selection and Nozzle Arrangement Impact Target Spot Control and Yield of Cotton

Target spot is an emerging disease in cotton. Studies in southwest and central Alabama in 2013 and 2014 assessed Headline 2.09SC and Quadris 2.08SC efficacy against target spot for improving yield when applied in an over-the-top or drop-nozzle configuration to the susceptible Phytogen 499 WRF and partially resistant Deltapine 1252 B2RF varieties. At three location-years, onset occurred in late July and exceeded 50% defoliation by mid-September; dry weather suppressed disease at the fourth location. Final defoliation was lower for the Headline drop than for the over-the-top nozzle arrangement and lower than Quadris regardless of nozzle arrangement. Despite higher defoliation of Phytogen 499 WRF, both varieties had similar yields at two locations in 2013 and inconsistent yields in 2014. Relative area under the disease curve (relAUC) for defoliation differed by fungicide treatment across location-years. Fungicides increased yields at one location in 2013, with greater yields for the Headline and Quadris drop-nozzle treatments than the control. Across three location-years with &gt;50% defoliation, Headline drop-nozzle treatment improved Phytogen 499 WRF yield compared with no treatment. Despite better control, higher costs and maintenance issues will limit drop-nozzle use. Results show that a partially resistant variety is an effective target spot-management tool.

Fitness and Competitive Ability of Botrytis cinerea Isolates with Resistance to Multiple Chemical Classes of Fungicides

Resistance to multiple chemical classes of fungicides in Botrytis cinerea isolates from eastern United States strawberry fields is common and strategies to control them are needed. In this study, we compared fitness and competitive ability of eight sensitive isolates (S), eight isolates resistant to five or six chemical classes of fungicides but not to phenylpyrroles (5CCR), and eight isolates resistant to six or seven chemical classes including phenylpyrroles (6CCR/MDR1h). The latter included the MDR1h phenotype due to overexpression of atrB based on Δ497V/L in mrr1. The 6CCR/MDR1h isolates grew more slowly at 4°C on potato dextrose agar, and both 5CCR and 6CCR/MDR1h isolates were hypersensitive to osmotic stress compared with S isolates. In contrast, no differences were found in oxidative sensitivity, aggressiveness, and spore production in vivo, and sclerotia production and viability in vitro. In competition experiments, the 5CCR and 6CCR/MDR1h isolates were both outcompeted by S isolates and 6CCR/MDR1h isolates were outcompeted by 5CCR isolates in the absence of fungicide pressure. Under selective pressure of a fludioxonil/pyraclostrobin rotation, the 6CCR/MDR1h isolates dominated after coinoculation with 5CCR and S isolates. The competitive disadvantage of 5CCR and especially 6CCR/MDR1h isolates suggest that, in the absence of fungicide selection pressure, S isolates may reduce inoculum potential of multifungicide-resistant isolates under field conditions.

Characterisation of heteroplasmic status at codon 143 of the Botrytis cinerea cytochrome b gene in a semi-quantitative AS-PCR assay.

An in-depth understanding of quinone outside inhibitor (QoI)-fungicide-resistant Botrytis cinerea isolates in a vineyard is expected to contribute to the development of an optimum disease management programme for the control of grape grey mould. The resistance and structure of the cytochrome b gene in B. cinerea collected from a Japanese vineyard were characterised. The semi-quantitative allele-specific primer-polymerase chain reaction (AS-PCR) assay developed in the present study was able to distinguish heteroplasmic status from homoplasmic status at codon 143 of the cytochrome b gene in QoI-fungicide-resistant B. cinerea from vineyards in Japan. With this assay it was demonstrated that the repeated introduction of QoI fungicide selection pressure increased the ratio of G143A-mutated cytochrome b genes in B. cinerea isolates. It is proposed that the semi-quantitative AS-PCR assay is a reliable tool for the detection of QoI fungicide resistance and the evaluation of homoplasmic/heteroplasmic status at codon 143 of the cytochrome b gene in B. cinerea isolates.

Fitness and Competitive Ability of Botrytis cinerea Field Isolates with Dual Resistance to SDHI and QoI Fungicides, Associated with Several sdhB and the cytb G143A Mutations

Respiration inhibitors such as the succinate dehydrogenase inhibitors (SDHIs) and the quinone outside inhibitors (QoIs) are fungicide classes with increasing relevance in gray mold control. However, recent studies have shown that dual resistance to both fungicide classes is a common trait in Botrytis cinerea populations from several hosts throughout the world. Resistance of B. cinerea to SDHIs is associated with several mutations in the sdhB, sdhC, and sdhD genes, while resistance to QoIs, in most cases, is associated with the G143A mutation in the cytb gene. The objective of the current study was to investigate the fitness and the competitive ability of B. cinerea field strains possessing one of the H272Y/R/L, N230I, or P225F sdhB substitutions and the G143A mutation of cytb. Fitness parameters measured were (i) mycelial growth and conidia germination in vitro, (ii) aggressiveness and sporulation capacity in vivo, (iii) sclerotia production in vitro and sclerotia viability under different storage conditions, and (iv) sensitivity to oxidative stress imposed by diquat treatments. The competitive ability of the resistant isolates was measured in the absence and presence of the SDHI fungicides boscalid and fluopyram selection pressure. The measurements of individual fitness components showed that the H272R/G143A isolates had the lower differences compared with the sensitive isolates. In contrast, the groups of H272Y/L/G143A, N230I/G143A, and P225F/G143A isolates showed reduced fitness values compared with the sensitive isolates. Isolates possessing only the cytb G143A substitution did not show any fitness cost. The competition experiments showed that, in the absence of fungicide selection pressure, after four disease cycles on apple fruit, the sensitive isolates dominated in the population in all the mixtures tested. In contrast, when the competition experiment was conducted under the selection pressure of boscalid, a gradual decrease in the frequency of sensitive isolates was observed, whereas the frequency of H272L and P225F isolates was increased. When the competition experiment was conducted in the presence of fluopyram, the sensitive isolates were eliminated even after the first disease cycle and the P225F mutants dominated in the population. Such results suggest that the sdhB mutations may have adverse effects on the mutants. The observed dominance of sensitive isolates in the competition experiments conducted in the absence of fungicides suggest that the application of SDHIs in alternation schemes may delay the selection or reduce the frequency of SDHI-resistant mutants.

Fungicide Selection and Application Timing for Management of Peanut Pod Rot

Aims: To evaluate the effect of fungicide regimes and application timing on pod rot incidence, peanut yield, grades, and diseased kernels. Study Design: Randomized complete block design with nine treatments and four replicates. Place and Duration of Study: Studies were conducted during the 2010 and 2011 growing seasons on producers farms located west of Seminole, Texas in Gaines County. Methodology: Rows were spaced 101.6 cm apart on raised beds. Plot size was four rows wide by 304.8 m in length. Fungicide regimes comprised of azoxystrobin and/or mefenoxam, or flutolanil were evaluated. Initial applications were made 68 or 82 days after planting (DAP) with subsequent applications occurring 30 days later. All fungicides were applied in a 50.8 cm band. Results: In 2010, the percentage of pods affected and percent damaged kernels were reduced by early applications of mefenoxam and azoxystrobin. The flutolanil treatment in 2010 showed the greatest improvement in yield compared to the control. The 2011 trial was greatly affected by drought, and no appreciable differences were seen for any of the

On-Farm Evaluations of Reduced Input Fungicide Programs in Peanut Fields with Low, Moderate, or High Levels of Disease Risk

ABSTRACT In 2003, 2004, and 2005 standard and reduced input fungicide programs were evaluated throughout the peanut production region of Georgia for control of early leaf spot (Cercospora arachidicola), late leaf spot (Cercosporidium personatum), and southern stem rot (Sclerotium rolfsii). Disease risk was determined for each field based on the cultural practices implemented and ranged from low to high. Six to eight fungicide applications were made in the standard programs, versus three to six applications in reduced programs. Leaf spot ratings were higher for the reduced programs in five of the fourteen trials with substantial defoliation occurring in one trial. Overall, southern stem rot control for the reduced programs was equal to or better than that for the standard program, which could be attributed to the differences in fungicide selection or timing. Pod yields for the reduced programs were equal to or greater than the standard programs in all but one trial. Net returns were higher for the reduced programs in half of the trials; however, the reduced program resulted in lower net returns in one trial in 2004. Our results indicate that reduced input fungicide programs can be used to adequately manage fungal diseases of peanut without compromising yield or profitability, and that the use of cultivars with moderate levels of disease resistance may enhance disease control.

Evaluation of the incidence of the G143A mutation and cytb intron presence in the cytochrome bc‐1 gene conferring QoI resistance in Botrytis cinerea populations from several hosts

Previous studies have shown that resistance of Botrytis cinerea to QoI fungicides has been attributed to the G143A mutation in the cytochrome b (cytb) gene, while, in a part of the fungal population, an intron has been detected at codon 143 of the gene, preventing QoI resistance. During 2005-2009, 304 grey mould isolates were collected from strawberry, tomato, grape, kiwifruit, cucumber and apple in Greece and screened for resistance to pyraclostrobin and for the presence of the cytb intron, using a novel real-time TaqMan PCR assay developed in the present study. QoI-resistant phenotypes existed only within the population collected from strawberries. All resistant isolates possessed the G143A mutation. Differences were observed in the genotypic structure of cytb. Individuals possessing the intron were found at high incidence in apple fruit and greenhouse-grown tomato and cucumber populations, whereas in the strawberry population the intron frequency was lower. Cultivation of QoI-resistant and QoI-sensitive isolates for ten culture cycles on artificial nutrient medium in the presence or absence of fungicide selection showed that QoI resistance was stable. The results of the study suggest that a high risk for selection of QoI-resistant strains exists in crops heavily treated with QoIs, in spite of the widespread occurrence of the cytb intron in B. cinerea populations. The developed real-time TaqMan PCR constitutes a powerful tool to streamline detection of the mutation by reducing pre- and post-amplification manipulations, and can be used for rapid screening and quantification of QoI resistance.

Competitive Parasitic Fitness of Mefenoxam-Sensitive and -Resistant Isolates of Phytophthora erythroseptica under Fungicide Selection Pressure.

A 2-year field and laboratory experiment was initiated to study the competitive parasitic fitness of mefenoxam-resistant (50% effective concentration [EC50] > 100 μg ml-1) and mefenoxam-sensitive (EC50 = 0.07 μg ml-1) isolates of Phytophthora erythroseptica with equal aggressiveness. The competitive ability of the mefenoxam-resistant and -sensitive isolates was tested under no selection pressure (nonfungicide treated) as well as under the influence of mefenoxam and non-mefenoxam (phosphorous acid) fungicides. P. erythroseptica isolates were combined in four ratios of mefenoxam-resistant (R) to mefenoxam-susceptible (S) (0R:0S, 1R:1S, 3R:1S, and 1R:3S) and subsequently infested into the soil at the time of planting. In-furrow mefenoxam applications were applied to the soil immediately following infestation with P. erythroseptica. Phosphorous acid was applied at tuber initiation and 14 days after tuber initiation. Noninfested, nonfungicide-treated plots served as controls. P. erythroseptica isolates recovered from field-infected pink rot tubers at harvest and 3 to 4 weeks after harvest were tested for mefenoxam sensitivity in vitro. In vivo studies were performed by challenge inoculating a zoospore suspension in the four ratios described above onto potato tubers harvested from nontreated, phosphorous acid-treated, or mefenoxam-treated field plots. These field plots were not infested with P. erythroseptica at planting. Results from both field and in vivo studies demonstrate that mefenoxam-resistant isolates of P. erythroseptica are as fit as sensitive isolates in the absence of selection pressure or in the presence of a phosphorous acid fungicide treatment. Under mefenoxam selection pressure, mefenoxam-resistant P. erythroseptica isolates were more parasitically fit than -sensitive isolates. These studies suggest the lack of an apparent fitness penalty in mefenoxam-resistant P. erythroseptica populations under field conditions and that these isolates could be stable in most agroecological systems. Based on these results, mefenoxam-based fungicides are no longer recommended for the management of pink rot once mefenoxam-resistant P. erythroseptica populations are detected in a specific field.