Inaequalis Isolates Research Articles

<i>In Vitro</i> Study of Fitness Parameters in Fungicide-Resistant and -Sensitive <i>Venturia inaequalis</i> Isolates

The developing resistance of Venturia inaequalis to toxicants commonly used in systemic fungicides against apple scab has reduced their effectiveness, causing substantial fruit loss in orchards. To improve the situation and manage the resistance, a thorough analysis of the fitness potential among different pathogen biotypes, particularly those resistant to fungicides, is needed. In this study, the mycelial growth of V. inaequalis isolates with baseline sensitivity and resistance to one or more fungicides was assessed in vitro at four temperatures (6, 18, 27, and 30°C) and three agar concentrations in the nutrient medium (2, 4, and 6% m/V). Except for the mycelial growth at 27°C, the indicators of fitness predicted in vitro did not differ significantly between the V. inaequalis isolates with multiple resistance to fungicides and the biotypes with baseline sensitivity.

Genetic variation and population differentiation in North American, central Asian and European isolates of Venturia inaequalis

AbstractVenturia inaequalis, the causal fungal pathogen of apple scab, has evolved with its Malus hosts during apple domestication. This co‐evolution has resulted in a genetic structure in V. inaequalis populations from Europe and central Asia based on host species and geographic isolation. However, it is not yet clear if geographic isolation has led to population differentiation in North American isolates. We resequenced the genomes of 54 V. inaequalis isolates from North America and analysed them with publicly available genome sequences of 90 European and central Asian isolates for variant discovery and population structure. A total of 204,566 high‐quality single‐nucleotide polymorphisms (SNPs) were identified and used to assess population genetic structure and genomic diversity across 123 Venturia spp. isolates from around the world. Population genetic analysis identified four clusters based on Malus and non‐Malus hosts and Venturia species; this differentiation was supported by genetic diversity parameters including FST, Nei's π and Tajima's D. Genetic structure analysis did not reveal a distinct subpopulation of North American V. inaequalis isolates within the global isolates. Additionally, structure was not observed in V. inaequalis isolates collected on M. × domestica between North America and Europe. These observations indicate that geographical isolation has not contributed to the population differentiation between North America and Europe/central Asia. These results will enhance our understanding of the evolution of V. inaequalis, the emergence of virulent isolates and their implications for managing apple scab effectively.

Sensitivity of Krasnodar Venturia inaequalis Populations to the Sterol Demethylation Inhibitor Difenoconazole.

Difenoconazole sensitivity was assessed in three populations of the apple scab agent Venturia inaequalis of the Krasnodar region. One of the populations was fungicide naïve population; its sample was collected in natural habitats of the eastern crabapple Malus orientalis in foothills of the Severskii raion. The two other populations were from commercial orchards of the apple variety Reinette Simirenko (the Krasnoarmeiskii and Dinskoi raions), where fungicide treatments with various agents, including those with difenoconazole as an active ingredient, were performed on a regular basis. Single-spore V. inaequalis isolates were isolated from fresh leaves with signs of the disease or fallen leaves with fungal fruiting bodies. The median effective concentration (EC50) was defined as the concentration that halves the colony growth and was estimated in a series of in vitro experiments with 120 isolates. Difenoconazole (Score EC, 250 mg/L, Syngenta) was used at six concentrations: 0.005, 0.01, 0.025, 0.05, 0.5, and 1 mg a.i./L. Mean EC50 values were 0.0078mg a.i./L in the natural population and 0.12 and 0.25 mg a.i./L in the orchard populations. Fungicide sensitivity was lower in both of the orchard populations; their resistance factors were estimated at 16 and 32. The proportion of sensitive and low-sensitive isolates differed between the two orchard populations. A discriminatory dose, or single-assessment concentration (SAC), of 0.01 mg a.i./L was proposed to simplify the laboratory monitoring of the difenoconazole sensitivity in V. inaequalis by using a test for relative growth (RG) of the mycelium. Comparable results were obtained with EC50 and RG at the discriminatory dose. The portion of isolates with RGs exceeding the threshold (RG > 70%) was 97% in one of the orchard populations. The results indicate that difenoconazole resistance develops in V. inaequalis populations from commercial orchards of the Krasnodar region.

Genetic Diversity of Venturia inaequalis in Latvia Revealed by Microsatellite Markers.

Apple scab caused by the ascomycete Venturia inaequalis is an economically significant disease worldwide. The annual sexual reproduction of V. inaequalis leads to high variation, changes in the population’s genetic structure and adaptations to the changing environment, including overcoming the host’s resistance. The objective of this study is to characterise and assess the genetic diversity of V. inaequalis populations in two main apple-growing regions in Latvia. In total, 143 V. inaequalis isolates were collected from Latvia, six reference strains with known virulence were obtained from other countries, and all strains were genotyped by 12 SSR markers. The SSR markers were highly variable and informative, identifying 158 alleles that ranged from two to 29 per locus. The Bayesian clustering identified three genetic lineages among the Latvian isolates that did not correlate to the geographic origin, host genotype, organ (leaves or fruits) from which the pathogen was isolated, time of collection, and type of isolation (single conidium or ascospore). The possible relatedness to virulence was detected when reference strains with known virulence were included in the analysis. Our findings correspond with previous studies demonstrating that V. inaequalis in Europe has a high genetic diversity within populations, but low diversity among the populations.

Genetic diversity and population structure of Venturia inaequalis isolates in apple orchards from Turkey

Apple scab caused by Venturia inaequalis is one of the major diseases of apple orchards worldwide. Genetic variability in pathogen populations present new strategies for management the disease and breeding studies. In this study aimed to evaluate genetic diversity and population structure of 81 V. inaequalis isolates obtained from six provinces of Turkey. Twenty-six iPBS and 20 ISSR primers were investigated for their ability to produce polymorphic bands among the pathogen isolates. The mean percentage of polymorphic bands per primer was 85.48 and 84.87 for iPBS and ISSR markers, respectively. The dendrogram derived from UPGMA cluster analysis based on the combined dataset of iPBS and ISSR markers classified the isolates into two main groups with a genetic similarity of 69%, which no relationship between distribution and geographical origin of the isolates. The dendrogram of Nei's genetic distances Isparta and Sivas populations clustered separately other populations. STRUCTURE analysis showed four populations (K = 4) with the highest mean value of Ln likelihood (−3067.0). The average genetic differentiation (GST) among all populations was 0.105. The overall gene flow (NM) was high level of 4.260. The analysis of molecular variance (AMOVA) showed that 96% of the variation was between the isolates within populations, compared to only 4% was among populations. iPBS marker based on the LTR retrotransposons has been tried for the first time to evaluate intraspecific variation among the isolates of V. inaequalis. Both iPBS and ISSR markers were useful, informative and reliable tools for investigating the genetic diversity of V. inaequalis. The results obtained from the present study provide detailed information about population genetic structure of V. inaequalis in Turkey. These study also will provide new approaches for plant breeders and disease management strategies.

Polyploidy affects the development of Venturia inaequalis in scab-resistant and -susceptible apple cultivars

Apple (Malus × domestica Borkh.) production is significantly affected by apple scab disease caused by the fungus Venturia inaequalis. Currently, management of the disease is largely based on fungicide applications and less on the inherent resistance in apple cultivars carrying resistance (Rvi) genes. Moreover, the durability of both approaches is questionable, since new virulent strains with resistance to fungicides and/or Rvi genes can develop. Hence, a combination of complementary management strategies is needed to tackle apple scab. To broaden the management options, a viable approach might be the breeding for polyploid cultivars and their implementation in commercial orchards. Apple germplasm is substantially characterized by polyploidy, which throughout the evolution of angiosperms often led to improved adaptation to adverse climatic conditions and abiotic stresses. However, the role of polyploidy in conferring resistance to biotic challenges is less clear. Here, we show that autopolyploidy in two apple cultivars, i.e. the susceptible ‘Gala’, and the resistant ‘Makali’ which carries the Rvi6 scab resistance gene, can reduce severity of apple scab symptoms upon inoculation with two different V. inaequalis isolates. Leaves of tetraploid plants show reduced sporulation symptoms in comparison with their diploid counterparts upon infection with both pathogenic strains, attributed to a reduced presence of V. inaequalis. These results suggest that polyploidy enhances resistance of apple cultivars against different V. inaequalis isolates. However, the degree of the enhanced resistance depends on a plant-pathogen-specific interaction. The knowledge provided here offers a relevant framework for improving our understanding of polyploidy-enhanced resistance to biotic stress in plants and may set the stage for implementing polyploidy breeding for further genetic improvement of apple.

Genetic diversity of Venturia inaequalis isolates (Apple scab) in China and U.K. determined by SSR markers.

Apple scab caused by Venturia inaequalis is a serious disease of cultivated apple worldwide. In this study, we collected 132 V. inaequalis isolates from Shaanxi, Gansu, Xinjiang, and the U.K. and analyzed their genetic diversity by using 13 microsatellite markers. Cluster analysis based on population structure and genetic distances suggested high similarity among the four regions. Population differentiation values ranged from 0.044 to 0.155, indicating there is a high level of kinship among the four regions. All isolates could be divided into 5 lineages with a 0.76 similarity coefficient. Among the four regions, Shaanxi had only one lineage, Group II; Gansu had four lineages, Group I, Group II, Group IV, and Group V; Xinjiang had all five lineages, Group I, Group II, Group III, Group IV, and Group V; and the U.K. had three lineages, Group I, Group II and Group IV. High molecular variance was detected for populations in the four regions, with 91% of the variance occurring within the populations and 9% among the populations. Structure analysis there are three common ancestors of these four regions. The results of the present study shed light on the genetic diversity of V. inaequalis in Shaanxi, Gansu and Xinjiang, which will lead to the development of more effective management strategies and new resistant apple cultivars through molecular marker-assisted selection.

Population structure of Venturia inaequalis, a hemibiotrophic fungus, under different host resistance specificities in the Kashmir valley.

Venturia inaequalis is a notorious fungal pathogen and show classical gene for gene interaction with its apple host. Neutral markers provide clues about history, evolutionary potential, genetic diversity and population structure of V. inaequalis. The genetic diversity and population structure of fungus indicates that the pathogen is highly diverse with the capacity to breach the scab resistance genes. In the present study, we collected 108 V. inaequalis isolates from three apple cultivars differing in Rvi1 resistance gene. Based on the AMOVA, the variation was mostly distributed among the isolates, providing evidence of non-existence of subpopulation in orchards thus founder population is difficult to arise in Kashmir apple orchards. Pair wise genetic differentiation is less due to regular occurrence of gene flow between the populations residing on different orchard as infected material is transported without stringent quarantine measures. Based on principal coordinate analysis and clustering algorithm as implemented in STRUCTURE, we observed admixture between the two subpopulations, which is quite low, suggesting the existence of pre-zygotic and post-zygotic barriers to gene flow and we cannot rule out the existence of other structures shared by accessions belonging to different varieties. Due to the continuous increase in introduction and monoculture of apple varieties, mixed orchard with different host resistance specificities are more suitable for managing the apple scab in Kashmir valley.

Point mutation in CYP51A1 of Venturia inaequalis is associated with low sensitivity to sterol demethylation inhibitors

In 2016, a serious outbreak of apple scab (caused by Venturia inaequalis) in Aomori Prefecture, Japan, resulted from reduced fungal sensitivity to sterol demethylation inhibitors (DMIs). In this study, the lanosterol 14α-demethylase (CYP51A1) gene of V. inaequalis isolates from Aomori Prefecture was analyzed. A nonsynonymous nucleotide polymorphism causing an amino acid substitution from tyrosine to phenylalanine at position 133 (Y133F) in CYP51A1 was closely associated with low sensitivity to DMIs. This is first report of a point mutation in CYP51A1 associated with low sensitivity to DMIs in V. inaequalis.

New North American Isolates of Venturia inaequalis Can Overcome Apple Scab Resistance of Malus floribunda 821.

Apple scab, caused by Venturia inaequalis, is a destructive fungal disease of major apple cultivars worldwide, most of which are moderately to highly susceptible. Thus, development of scab resistant cultivars is one of the highest priorities of apple breeding programs. The principal source of resistance for breeding programs has been the scab resistance gene Rvi6 that originated from the Japanese crabapple Malus floribunda (Sieb.) sel. 821. Isolates of V. inaequalis able to overcome Rvi6 have been identified in Europe, but have not yet been reported on the American continents. We recently discovered scab infection on M. floribunda 821 trees in a research orchard at Geneva, NY, U.S.A., where approximately 10% of the leaves bore profusely sporulating apple scab lesions, many of which had coalesced to cover entire leaves. We observed both chlorosis, typical to Rvi6, and pinpoint pitting symptoms typical to failed infections by V. inaequalis on hosts bearing the Rvi7 gene. We assessed genetic diversity and population genetic structure of 11 V. inaequalis isolates in total, of North American and European origin, isolated from M. floribunda 821, 'Nova Easygro', 'Golden Delicious', TSR33T239, 'Schone van Boskoop', and 'Prima', using 16,321 genome-wide SNPs. Population genetic structure and PCA separated the isolates into distinct European and U.S. groups. The forgoing suggests that the new Rvi6 virulent isolates emerged within U.S. populations, rather than being transported from Europe. The complete resistance breakdown in M. floribunda 821 but not in descendant cultivars, which kept their field resistance, suggests that durable resistance to apple scab will require a more comprehensive understanding of Rvi6 mediated resistance in diverse genetic backgrounds.

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.

Elucidating genetic variability and population structure in Venturia inaequalis associated with apple scab diseaseusing SSR markers.

Apple scab caused by Venturia inaequalis Cooke (Wint.) is one the important diseases of trade and industrial significance in apple. In present study variability studies in pathogen isolates were studied, which is one of the most important factors for devising management studies of scab disease in apple. Genetic diversity of 30 Venturia inaequalis isolates from 12 districts of two geographical distinct regions of Jammu and Kashmir was calculated based on the allele frequencies of 28 SSR markers and the internal transcribed spacer (ITS) region of the ribosomal DNA. The ITS based characterized sequences were submitted to NCBI GenBank and accession numbers were sanctioned. Dendrogram showed that all the accessions formed 2 main clusters with various degree of sub clustering within the clusters. Analysis based on SSR study reveals that the heterozygosity ranged from 0.0 and 0.5, with an average value of 0.39. The expected heterozygosis or gene diversity (He) ranged from 0.0 to 0.50 with an average of 0.40. The Fst value ranges from 0 to 0.6 with an average of 0.194. Diversity within each population (HS) values ranging from 0.26 to 0.33. Average differentiation among populations (GST) was 0.11 and populations were isolated by significant distance (r 2 = 0.50, P < 0.01). From the AMOVA analysis, 25% of variation was observed among population, 9% among individuals and 66% within individuals observed in the population. Structure analysis grouped isolates into two populations. Principle coordinate analysis explained variation of 36.6% in population 1, 14.30% in population 2 and 13.10% in population 3(Admixture) with 64.07% as overall cumulative percentage of variation. This indicates that extensive short-distance gene flow occurs in Kashmir region that dispersal over longer distances also appears to occur frequently enough to prevent differentiation due to genetic drift. Also it is evident that Jammu and Kashmir most likely has V. inaequalis subpopulations linked to diverse climatic conditions of the Jammu region compared to the mountainous inland Kashmir region. The results of present study would help to understand the genetic diversity of V. inaequalis from Jammu and Kashmir that would lead in the development of more effective management strategies and development of new resistant cultivars through marker-assisted selection.

Genetic diversity of apple‐ and crabapple‐infecting isolates of Venturia inaequalis in Pennsylvania, the United States, determined by microsatellite markers

SummaryApple scab caused by Venturia inaequalis is the most destructive disease of apple worldwide. In this study, genetic diversity of 101 V. inaequalis isolates from cultivated apples and ornamental crabapples in Pennsylvania (PA, USA) was characterized using 14 microsatellite markers. A total of 157 alleles ranging from 5 (Vitg9/99) to 26 (Vica10/154) per locus were detected. Regardless of the host of origin, isolates were grouped into five clusters, which were largely supported by STRUCTURE and principal coordinate analysis. Cluster analyses based on genetic distances and population structure analysis suggest very small differentiation (PhiPT ranging from 0.016 to 0.103, depending on the population comparison) between apple and crabapple isolates of V. inaequalis. Pairwise comparisons among populations from different locations showed very low differentiation, and POPGENE analysis indicated frequent migration of alleles (Nm = 1.47). In pathogenicity tests using a detached leaf assay, isolates of V. inaequalis from crabapple caused characteristic scab symptoms on apple and were highly virulent. Results of the study indicate that scab lesions in crabapple trees in close vicinity to apple orchards could serve as reservoirs for spread of the pathogen. Movement of inoculum among locations and between hosts may be responsible for the limited population structure observed. Understanding the population structure of V. inaequalis isolates is significant for apple scab management as crabapples are often used as pollinizers and rootstock in apple orchards, and as ornamental trees.

Genetic Diversity and Gene Flow of Four South African Venturia inaequalis (Apple Scab) Populations.

Venturia inaequalis isolates were collected during the 2012/13 and 2013/14 seasons from the four principal apple growing regions of South Africa, Elgin (n = 114), Koue Bokkeveld (n = 126), Lower Langkloof (n = 92), and Upper Langkloof (n = 103). Sequence analysis of the ribosomal internal transcribed spacer (ITS) gene regions and genotyping with six (2012/13) and seven (2013/14) microsatellite (SSR) markers was conducted. A subset of 12 isolates from the individual ITS haplotype groups were sequenced for the translation elongation factor-1 alpha (TEF1) and the large subunit of the RNA polymerases II (RPB1) gene regions. Four haplotypes were found for ITS, whereas all isolates were identical for the TEF1 and RPB1 gene regions. The SSR markers revealed considerable variation with an average gene diversity (H) of 0.675. Multivariate analysis (discriminant analysis of principal components [DAPC]) revealed that the two Langkloof populations clustered together with the Koue Bokkeveld population. The population from the warmer winter region, Elgin, clustered separately from the rest of the populations (ΦPT = 0.076 to 0.116; P ≤ 0.05). Estimates of gene flow showed the highest migration rate from the Koue Bokkeveld, toward the Lower Langkloof (M = 151.1), and the least migration to and from the Elgin region (average M = 42.75). Occasionally, identical genotypes (clones) were detected across seasons in the Koue Bokkeveld and Elgin area, which might contribute to overwintering conidia. From this study, it is evident that South Africa most likely has V. inaequalis subpopulations linked to diverse climatic conditions of the coastal Elgin region compared with the mountainous inland regions of the Koue Bokkeveld and the Langkloof.

Population Genetic Structure of Apple Scab (Venturia inaequalis (Cooke) G. Winter) in Iran.

The population genetic structure of 278 Venturia inaequalis isolates, collected from different apple cultivars of eighteen different provinces in Iran, was investigated using 22 polymorphic microsatellite markers. Analysis of molecular variation, Bayesian clustering and Nei's genetic distance analyses based on 88 microsatellite alleles indicated substantial levels of gene flow among the collection sites. Ninety three percent of the variation was observed among the individuals within the populations and only 7% variation was observed among the populations. Structure analysis grouped the isolates into two populations. Maximum number of pathogen genotypes (44) was observed in the North of Iran that grows various different apple cultivars. Investigation on the variation of the pathogen on different cultivars in the North of Iran suggested a significant differentiation of the pathogen populations between wild apple and commercial cultivars. During sampling, varying ranges of scab infection were observed on various apple cultivars in forests, monoculture and mix orchards. Wild type apple (Malus orientalis) along the Caspian Sea Coast had the most infection in comparison with the Iranian endemic and commercial cultivars. Based on the genetic analysis and host tracking scenario of the pathogen, it was presumed that Iran could potentially be the center of origin of V. inaequalis, which requires further detailed studies with isolates collected from different parts of central Asia and world for confirmation.

Efficiency of pyramiding of three quantitative resistance loci to apple scab

Quantitative plant resistance is supposed to be more durable than qualitative resistance for the control of plant diseases. However, it has been experimentally shown that erosion of quantitative resistance can occur. Cumulation of quantitative resistance loci (QRLs) in the same cultivar is considered to improve the efficiency and durability of quantitative resistance, but the choice of QRLs to be combined is of crucial importance. This study investigated whether the combination of QRLs acting on different stages of pathogen development could improve the efficiency of resistance in the apple scab pathosystem. The efficiencies of three QRLs were evaluated against 10 isolates of Venturia inaequalis and the stages of pathogen development that were affected by the QRLs were defined microscopically. A gain in the efficiency of resistance was observed when QRLs were pyramided compared to when they acted alone. Thanks to the combined effects of the individual QRLs, the pyramiding of the three QRLs hindered fungal development at different stages: before the penetration of the plant cuticule, after the penetration with hypersensitivity reaction, and during the colonization and asexual reproduction. These effects were dependent on the V. inaequalis isolates. These results suggest that the gain in efficiency of resistance by pyramiding may derive from the combination of different and complementary molecular mechanisms underlying QRLs. Thus, the resistance achieved from pyramiding such a combination of QRLs should be durable.

Slow erosion of a quantitative apple resistance to Venturia inaequalis based on an isolate-specific Quantitative Trait Locus

Quantitative plant resistance affects the aggressiveness of pathogens and is usually considered more durable than qualitative resistance. However, the efficiency of a quantitative resistance based on an isolate-specific Quantitative Trait Locus (QTL) is expected to decrease over time due to the selection of isolates with a high level of aggressiveness on resistant plants. To test this hypothesis, we surveyed scab incidence over an eight-year period in an orchard planted with susceptible and quantitatively resistant apple genotypes. We sampled 79 Venturia inaequalis isolates from this orchard at three dates and we tested their level of aggressiveness under controlled conditions. Isolates sampled on resistant genotypes triggered higher lesion density and exhibited a higher sporulation rate on apple carrying the resistance allele of the QTL T1 compared to isolates sampled on susceptible genotypes. Due to this ability to select aggressive isolates, we expected the QTL T1 to be non-durable. However, our results showed that the quantitative resistance based on the QTL T1 remained efficient in orchard over an eight-year period, with only a slow decrease in efficiency and no detectable increase of the aggressiveness of fungal isolates over time. We conclude that knowledge on the specificity of a QTL is not sufficient to evaluate its durability. Deciphering molecular mechanisms associated with resistance QTLs, genetic determinants of aggressiveness and putative trade-offs within pathogen populations is needed to help in understanding the erosion processes.

The Effect of Delayed-Dormant Chemical Treatments on Demethylation Inhibitor (DMI) Sensitivity in a DMI-resistant Population of Venturia inaequalis.

Demethylation inhibitor (DMI) fungicides are an effective means to manage apple scab caused by Venturia inaequalis. Unfortunately, practical resistance to DMI fungicide chemistries is prevalent in populations in New York and the New England states. Management practices that delay the development of DMI resistance in V. inaequalis populations are highly desired by regional apple producers. Trials were conducted in a New York apple orchard during the 2011 and 2012 growing seasons to determine the impact of delayed-dormant (after bud break, but prior to green tissue) chemical treatments on the DMI sensitivity of a V. inaequalis population with stable resistance to DMI fungicides. Delayed-dormant treatment programs consisted of either an application of a copper fungicide, a manganese sanitation product, a DMI fungicide (myclobutanil), or no fungicide. Sensitivity to the DMI fungicide myclobutanil was evaluated for a minimum of 25 V. inaequalis single lesion conidial isolates from each of four replicated treatment blocks. In both years, mean percent relative growth on myclobutanil amended media for V. inaequalis isolates from the copper treatment program were significantly (P < 0.05) lower than isolates from blocks did not receive a delayed dormant fungicide treatment. The effect of the manganese treatment was inconsistent between years. V. inaequalis isolates collected from the myclobutanil treatment program were not significantly (P > 0.05) different in myclobutanil sensitivity from isolates collected from the blocks that did not receive a delayed dormant fungicide treatment. Overall, the results suggest that delayed dormant treatments of copper may favorably impact the myclobutanil sensitivity for a population of V. inaequalis with resistance to DMI fungicides, and should be considered as a standard management practice in apple production.

Uso de RFLP-PCR para a detecção de isolados de Venturia inaequalis resistentes ao cresoxim metílico do Sul do Brasil

O principal mecanismo que confere resistência do fungo Venturia inaequalis aos inibidores de oxidação (quinol QoIs) é a ocorrência de mutações no gene citocromo b CYTB (G143A). O objetivo do trabalho foi a implementação da metodologia RFLP-PCR para caracterizar a reação de isolados de V. inaequalis à presença de Qols. Foram utilizados sete isolados monospóricos de V. inaequalis, coletados em pomares comerciais de Santa Catarina e cedidos pela Estação Experimental da EPAGRI de São Joaquim, Santa Catarina. Os isolados foram classificados como sensíveis ou resistentes após crescimento em meio BDA contendo 10 µg.mL-1 de cresoxim metílico. Para determinação da presença da mutação G143A foram utilizados os marcadores específicos ViCytB-5F e ViCytB-6071R. O DNA dos isolados foi amplificado em reação de PCR e separado em gel de agarose 1,5%. Os fragmentos foram então digeridos com a enzima de restrição Fnu4HI identificadora da mutação e os produtos da digestão foram analisados por eletroforese num gel de agarose 1,0%. Os genótipos revelados estavam associados ao fenótipo estabelecido pelo crescimento dos isolados em presença do fungicida, mostrando que 6 isolados já apresentavam o alelo de resistência. O uso de técnicas de RFLP-PCR permitiram uma avaliação rápida e confiável na detecção da resistência de V. inaequalisao cresoxim metílico.

Prevalence of Myclobutanil Resistance and Difenoconazole Insensitivity in Populations of Venturia inaequalis.

Demethylation inhibitors (DMIs) are a class of single-site fungicides with high levels of protective and curative efficacy against Venturia inaequalis, the causal agent of apple scab. To determine the prevalence of resistance to the DMI fungicide myclobutanil, 3,987 single-lesion conidial V. inaequalis isolates from 141 commercial, research, and baseline orchard populations were examined throughout New England, the mid-Atlantic, and the Midwest from 2004 to 2013. Of these orchard populations, 63% had practical resistance, 13% had reduced sensitivity, and 24% were sensitive to myclobutanil. A sensitivity baseline for the recently introduced DMI fungicide difenoconazole was established to make comparisons with myclobutanil sensitivity in orchard populations. The mean effective concentration of difenoconazole at which mycelial growth was inhibited by 50% (EC50) was determined to be 0.002 μg ml-1 for 44 baseline isolates of V. inaequalis. From 2010 to 2013, 1,012 isolates of V. inaequalis from 37 of the 141 orchard populations above were screened for sensitivity to difenoconazole. In all, 1 orchard population had reduced sensitivity to difenoconazole, while the remaining 36 orchard populations were sensitive to the fungicide. In field experiments, difenoconazole demonstrated high levels of apple scab control on mature apple fruit, despite the fact that the population of V. inaequalis had practical resistance to difenoconazole. Although our results indicate widespread resistance to myclobutanil but not difenoconazole, due to the propensity for cross-sensitivity among DMI fungicides, growers with myclobutanil resistance should be cautious when using difenoconazole for disease management.