Impacts of N-P-K-Mg Fertilizer Combinations on Tree Parameters and Fungal Disease Incidences in Apple Cultivars with Varying Disease Susceptibility.

Adequate mineral fertilization helps to ensure optimal tree growth, fruit development, and predictable yield of apple trees. This 7-year study (2016-2022) aims to investigate the effect of nitrogen (N), phosphorus (P), potassium (K), and magnesium (Mg) fertilizer combinations (NP, NPK, NPKMg, and control) on eight parameters (trunk cross-sectional area-TCSA; fruit yield-FY; number of fruit per tree-FNT; crop load-CL; fruit diameter-FD; fruit weight-FW; fruit scab incidence-FSI; and powdery mildew incidence on shoot-PMIS) on the cultivars (cvs) 'Golden Reinders' (disease susceptible) and 'Pinova' (scab and mildew tolerant). In the 7-year period, TCSA values continuously increased for both cultivars over the years. Fertilizer treatments showed significant differences on TCSA but the effect varied greatly annually among fertilizer treatments. Fertilizer treatments had increasing effects on FY and FNT in 2018 and 2022, on CL in 2018, on FD in 2018 and 2019, and on FW in 2016 and 2018 in both cultivars compared to the control treatment. FSI values were the lowest in the NPKMg treatment for cv. 'Golden Reinders' in 2016, 2017, and 2022; for cv. 'Pinova' in 2016; PMIS values for cv. 'Golden Reinders' in 2017, 2018, 2021, and 2022; and for cv. 'Pinova' in 2018. Correlation and regression analyses revealed strong and significant (p = 0.05) relationships between FNT versus (vs.) TCSA, FNT vs. FY, FW vs. TCSA, CL vs. FY, FW vs. FD, and FSI vs. FW. In conclusion, our study showed that multiyear application of fertilizer combinations can successfully increase TCSA and yield parameters as well as reduce fungal disease incidences, especially on the disease-susceptible cultivar in sandy soil with moderate fertility, under Central-European continental climate conditions.

Exogenous dsRNA trigger RNAi in Venturia inaequalis resulting in down regulation of target genes and growth reduction.

BACKGROUND: Venturia inaequalis is an apple scab causing fungal pathogen. It is a highly contagious and destructive pathogen which rapidly spreads infection in the surrounding orchards if not managed. The management and control of disease require multiple fungicides to be sprayed at different development stages of the apple. Persistent applications of fungicides also raises environmental concerns. Here, we demonstrate the potential of using spray induced gene silencing (SIGS) by developing target specific gene constructs for the synthesis of corresponding double-stranded RNA (dsRNA). METHODS AND RESULTS: The exogenous application of dsRNAs was found to reduce mycelial growth and spore formation of V. inaequalis on culture plates. Four genes of V. inaequalis viz. CIN1, CE5, VICE12 and VICE16 which get upregulated during infection, were selected as targets for the development of gene construct expressing the corresponding dsRNA. The effect of exogenously supplied in vitro synthesized dsRNA on V. inaequalis was assessed in culture bioassay experiments with respect to growth, and spore formation. The expression level of the target genes in treated and control fungus was evaluated using quantitative PCR. Fungus treated with VICE12 targeted dsRNA showed maximum reduction in colony size (~ 55%), conidia formation (~ 93%) and expression level of the corresponding gene (2.2 fold), which was followed by CIN1-dsRNA. VICE16-dsRNA treatment was least effective with 32% reduction in growth, the non-significant effect of conidial spore formation and 1.13 fold down regulation of corresponding target gene expression level. CONCLUSION: The result of this investigation validates the hypothesis that RNAi is evoked in V. inaequalis by exogenously supplied dsRNA and spray induced gene silencing (SIGS) based solutions may reduce burden of fungicide usage on apple crop against apple scab disease in future.

Cell Wall Carbohydrate Dynamics during the Differentiation of Infection Structures by the Apple Scab Fungus, Venturia inaequalis.

Scab, caused by the biotrophic fungal pathogen Venturia inaequalis, is the most economically important disease of apples. During infection, V. inaequalis colonizes the subcuticular host environment, where it develops specialized infection structures called runner hyphae and stromata. These structures are thought to be involved in nutrient acquisition and effector (virulence factor) delivery, but also give rise to conidia that further the infection cycle. Despite their importance, very little is known about how these structures are differentiated. Likewise, nothing is known about how these structures are protected from host defenses or recognition by the host immune system. To better understand these processes, we first performed a glycosidic linkage analysis of sporulating tubular hyphae from V. inaequalis developed in culture. This analysis revealed that the V. inaequalis cell wall is mostly composed of glucans (44%) and mannans (37%), whereas chitin represents a much smaller proportion (4%). Next, we used transcriptomics and confocal laser scanning microscopy to provide insights into the cell wall carbohydrate composition of runner hyphae and stromata. These analyses revealed that, during subcuticular host colonization, genes of V. inaequalis putatively associated with the biosynthesis of immunogenic carbohydrates, such as chitin and ß-1,6-glucan, are downregulated relative to growth in culture, while on the surface of runner hyphae and stromata, chitin is deacetylated to the less-immunogenic carbohydrate chitosan. These changes are anticipated to enable the subcuticular differentiation of runner hyphae and stromata by V. inaequalis, as well as to protect these structures from host defenses and recognition by the host immune system. IMPORTANCE Plant-pathogenic fungi are a major threat to food security. Among these are subcuticular pathogens, which often cause latent asymptomatic infections, making them difficult to control. A key feature of these pathogens is their ability to differentiate specialized subcuticular infection structures that, to date, remain largely understudied. This is typified by Venturia inaequalis, which causes scab, the most economically important disease of apples. In this study, we show that, during subcuticular host colonization, V. inaequalis downregulates genes associated with the biosynthesis of two immunogenic cell wall carbohydrates, chitin and ß-1,6-glucan, and coats its subcuticular infection structures with a less-immunogenic carbohydrate, chitosan. These changes are anticipated to enable host colonization by V. inaequalis and provide a foundation for understanding subcuticular host colonization by other plant-pathogenic fungi. Such an understanding is important, as it may inform the development of novel control strategies against subcuticular plant-pathogenic fungi.

Quantitative Traits of Interest in Apple Breeding and Their Implications for Selection.

Apple breeding is a laborious and long-lasting process that requires qualified resources, land, time, and funds. In this study, more than 5000 F1 apple hybrids from direct and testcrosses were analyzed. The results revealed how the phenotypic expression of the main quantitative traits of interest assessed in five half-sib families was controlled by the additive genetic effects and by non-additive effects of dominance and epistasis. The statistical number of hybrids required to ensure efficient selection increased exponentially with the number of desirable traits. The minimum number of progenies required to obtain a hybrid with associated quantitative traits of agronomic interest was highly variable. For two independent traits essential in selection (fruit size and quality), but incorporated together in the same hybrid, the statistical number was between about 30 and 300. If three more cumulative traits were added (a large number of fruits per tree, resistance/tolerance to apple scab, and powdery mildew attack), the limits increased to between 1500 and 18,000. The study highlighted the need for new apple varieties due to the narrowing of the genetic diversity of the cultivated species and how the choice of parents used in hybridizations (as well as the objectives pursued in the selection) can increase the efficiency of apple breeding.

Implementing wood ants in biocontrol: Suppression of apple scab and reduced aphid tending.

BACKGROUND: Ants can become efficient biocontrol agents in plantation crops as they prey on pest insects and may inhibit plant pathogens by excreting broad-spectrum antibiotics. However, ants also provide a disservice by augmenting attended honeydew producing homopterans. This disservice may be avoided by offering ants artificial sugar as an alternative to honeydew. Here we tested the effect of artificial sugar feeding on aphid abundance in an apple plot with wood ants (Formica polyctena, Förster), and tested the effect of ant presence on apple scab (Venturia inaequalis, Cooke) disease incidence. RESULTS: Over a 2-year period, sugar feeding eliminated ant-attended aphid populations on the apple trees. Furthermore, scab symptoms on both leaves and apples were reduced considerably on ant trees compared to control trees without ants. The presence of ants on the trees reduced leaf scab infections by 34%, whereas spot numbers on fruits were reduced by between 53 and 81%, depending on apple variety. In addition, the spots were 56% smaller. CONCLUSION: This shows that problems with wood ant-attended homopterans can be solved and that ants can control both insect pests and plant pathogens. We therefore propose wood ants as a new effective biocontrol agent suitable for implementation in apple orchards and possibly other plantation crops. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The Overall Environmental Load and Resistance Risk Caused by Long-Term Fungicide Use to Control Venturia inaequalis in Apple Orchards in Latvia.

Apple orchards are perennially planted where pesticides are applied to control numerous pests and diseases. The extensive long-term use of fungicides can lead to overall environmental load and resistance risk. This study aims to assess which fungicide-active substances have been used more intensively in the last decade in Latvia, evaluating the overall environmental load using the Pesticide Load Indicator (PLI). It was essential to see whether the amount of active substance usage rises, how it correlates with the total changes of the PLI and which substances are with the highest scores. The other issue was to test the sensitivity of Venturia inaequalis populations to systemic fungicides. Six full-bearing apple orchards that reflected local plant protection practices were selected from the different growing regions of Latvia to analyze fungicide use from 2012 to 2021 and test V. inaequalis populations' sensitivity to systemic substances difenoconazole and cyprodinil. The PLI demonstrated that the protective fungicides were the most crucial group overall, with the highest potential impact on the environment and human health. Systemic fungicides had a relatively lower environmental impact, but after long-term use, the pathogen population's sensitivity to difenoconazole and cyprodinil was reduced. Introducing new fungicide classes and biological control agents could help growers improve plant protection strategies against V. inaequalis, reducing the risk of resistance and environmental load.

Plant Disease Models and Forecasting: Changes in Principles and Applications over the Last 50 Years.

This review gives a perspective of selected advances made since the middle of the 20th century in plant disease modeling, and the associated increase in the number of models published during that time frame. This progress can be mainly attributed to advances in (i) sensors and automatic environmental data collection technology, (ii) instrumentation and methods for studying botanical epidemiology, and (iii) data analytics and computer science. We review the evolution of techniques for developing data-based (empirical) models and process-based (mechanistic) models using the wheat rusts as a case study. We also describe the increased importance of knowledge about biological processes for plant disease modeling by using apple scab as a second case study. For both wheat rusts and apple scab, we describe how the models have evolved over the last 50 years by considering certain milestones that have been achieved in disease modeling. Finally, we describe how plant disease models are used as part of a multi-modeling approach to develop decision-making tools in the application of integrated pest management.

Formulated hydroxy fatty acids from fruit pomaces reduce apple scab development caused by Venturia inaequalis through a dual mode of action.

The outermost hydrophobic layer of plants, i.e. the cuticle, is mainly composed of cutin, a polyester of hydroxy fatty acids with reported eliciting and/or antimicrobial activities for some of them. By-products of the fruit processing industry (fruit pomaces), often strongly enriched in cuticular material, are therefore a potential source of bioactive compounds for crop protection against pathogen attack. We investigated the utilization of tomato and apple pomaces in the development of a cutin-based biocontrol solution against apple scab, a major apple disease caused by Venturia inaequalis. Several cutin monomer extracts obtained through different strategies of depolymerization and purification were first compared for their ability to induce a targeted set of defense genes in apple seedlings after foliar application. After a step of formulation, some extracts were chosen for further investigation in planta and in vitro. Our results show that formulated cutin monomers could trigger a significant transcriptome reprogramming in apple plants and exhibit an antifungal effect on V. inaequalis. Cutin monomers-treated apple seedlings were significantly protected against infection by the apple scab agent. Altogether, our findings suggest that water-dispersed cutin monomers extracted from pomaces are potential new bio-based solutions for the control of apple scab.

The Venturia inaequalis effector repertoire is dominated by expanded families with predicted structural similarity, but unrelated sequence, to avirulence proteins from other plant-pathogenic fungi.

BACKGROUND: Scab, caused by the biotrophic fungus Venturia inaequalis, is the most economically important disease of apples worldwide. During infection, V. inaequalis occupies the subcuticular environment, where it secretes virulence factors, termed effectors, to promote host colonization. Consistent with other plant-pathogenic fungi, many of these effectors are expected to be non-enzymatic proteins, some of which can be recognized by corresponding host resistance proteins to activate plant defences, thus acting as avirulence determinants. To develop durable control strategies against scab, a better understanding of the roles that these effector proteins play in promoting subcuticular growth by V. inaequalis, as well as in activating, suppressing, or circumventing resistance protein-mediated defences in apple, is required. RESULTS: We generated the first comprehensive RNA-seq transcriptome of V. inaequalis during colonization of apple. Analysis of this transcriptome revealed five temporal waves of gene expression that peaked during early, mid, or mid-late infection. While the number of genes encoding secreted, non-enzymatic proteinaceous effector candidates (ECs) varied in each wave, most belonged to waves that peaked in expression during mid-late infection. Spectral clustering based on sequence similarity determined that the majority of ECs belonged to expanded protein families. To gain insights into function, the tertiary structures of ECs were predicted using AlphaFold2. Strikingly, despite an absence of sequence similarity, many ECs were predicted to have structural similarity to avirulence proteins from other plant-pathogenic fungi, including members of the MAX, LARS, ToxA and FOLD effector families. In addition, several other ECs, including an EC family with sequence similarity to the AvrLm6 avirulence effector from Leptosphaeria maculans, were predicted to adopt a KP6-like fold. Thus, proteins with a KP6-like fold represent another structural family of effectors shared among plant-pathogenic fungi. CONCLUSIONS: Our study reveals the transcriptomic profile underpinning subcuticular growth by V. inaequalis and provides an enriched list of ECs that can be investigated for roles in virulence and avirulence. Furthermore, our study supports the idea that numerous sequence-unrelated effectors across plant-pathogenic fungi share common structural folds. In doing so, our study gives weight to the hypothesis that many fungal effectors evolved from ancestral genes through duplication, followed by sequence diversification, to produce sequence-unrelated but structurally similar proteins.

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.

Production and Biological Assessment of VNIISPK Cultivars of Various Ploidy for the Zone of Temperate Continental Climate.

Increasing the reliability of fruit crops in unstable weather conditions of a temperate continental climate has become particularly relevant. This research was carried out based on a bioresource collection from the Russian Research Institute of Fruit Crop Breeding (VNIISPK BRC). Apple cultivars (diploids and triploids) of different maturation periods were studied. Triploid cultivars (3×) of summer maturation were developed using diploid gametes of the 'Papirovka tetraploid' cultivar as a donor (2-4-4-4×), triploids of winter maturation were developed using 13-6-106 (Suvorovetz: open pollination), and 'Wealthy tetraploid' (2-4-4-4×) or 'Giant Spy' (2-4-4-4×) were used as donors of diploid gametes. 'Antonovka' and 'Melba' were taken as control cultivars. The aim of this work was to evaluate the apple breeding cultivars according to the main economic and biological indicators and to determine the threshold of resistance to unfavorable conditions of the winter period for possible cultivation in specific conditions of a temperate continental climate. As a result of using the method of modeling damaging factors, apple cultivars that withstood not only the critical temperatures of January, but also especially dangerous frosts after thaws at the end of winter were identified, with the stability of vital tissues at the level of 'Antonovka' (a control cultivar), scab immune cultivars (RVi6) 'Ivanovskoye', 'Zdorovie' and 'Pamyati Hitrovo', and immune triploids (3×) 'Vavilovskoye', 'Aleksandr Boyko', Rozhdestvenskoye' and 'Academic Saveliev'. During the whole winter, the winter resistance of summer scab immune triploids, 'Zhilinskoye', 'Maslovskoye', 'Yablochny Spas' and 'Spasskoye', was at the level of 'Antonovka', but was higher than 'Melba'. The high precocity of scab-immune cultivars, 'Afrodita', 'Ivanovskoye', 'Veniaminovskoye', and 'Yubiley Moskvy', and triploids 'Patriot' and 'Rozhdestvenskoye', have been identified. Triploid cultivars are superior to diploid cultivars and control 'Antonovka' in fruit size. According to the complex of characteristics, promising apple cultivars were identified for planting in intensive orchards of the temperate continental climate zones, namely 'Avgusta' and 'Solnyshko' (summer cultivars), and 'Aleksandr Boyko', 'Vavilovskoye', 'Venyaminovskoye', 'Ivanovskoye' and 'Rozhdestvenskoye' (winter cultivars).

Assessment of Lipopeptide Mixtures Produced by Bacillus subtilis as Biocontrol Products against Apple Scab (Venturia inaequalis).

Apple scab is an important disease conventionally controlled by chemical fungicides, which should be replaced by more environmentally friendly alternatives. One of these alternatives could be the use of lipopeptides produced by Bacillus subtilis. The objective of this work is to study the action of the three families of lipopeptides and different mixtures of them in vitro and in vivo against Venturia inaequalis. Firstly, the antifungal activity of mycosubtilin/surfactin and fengycin/surfactin mixtures was determined in vitro by measuring the median inhibitory concentration. Then, the best lipopeptide mixture ratio was produced using Design of Experiment (DoE) to optimize the composition of the culture medium. Finally, the lipopeptides mixtures efficiency against V. inaequalis was assessed in orchards as well as the evaluation of the persistence of lipopeptides on apple. In vitro tests show that the use of fengycin or mycosubtilin alone is as effective as a mixture, with the 50-50% fengycin/surfactin mixture being the most effective. Optimization of culture medium for the production of fengycin/surfactin mixture shows that the best composition is glycerol coupled with glutamic acid. Finally, lipopeptides showed in vivo antifungal efficiency against V. inaequalis regardless of the mixture used with a 70% reduction in the incidence of scab for both mixtures (fengycin/surfactin or mycosubtilin/surfactin). The reproducibility of the results over the two trial campaigns was significantly better with the mycosubtilin/surfactin mixture. The use of B. subtilis lipopeptides to control this disease is very promising.

Comparative transcriptomics unravels new genes imparting scab resistance in apple (Malus x domestica Borkh.).

Apple scab is caused by an ascomycete fungus, Venturia inaequalis (Cke.) Wint., which is one of the most severe disease of apple (Malus × Domestica Borkh.) worldwide. The disease results in 30-40% fruit loss annually and even complete loss in some places. Owing to the evolving susceptibility of resistant apple genotypes harboring R-genes to new variants of V. inaequalis, a comparative transcriptome analysis using Illumina (HiSeq) platform of three scab-resistant (Florina, Prima, and White Dotted Red) and three susceptible (Ambri, Vista Bella, and Red Delicious) apple genotypes was carried out to mine new scab resistance genes. The study led to the identification of 822 differentially expressed genes in the tested scab-resistant and scab-susceptible apple genotypes. The most upregulated genes uniformly expressed in resistant varieties compared to susceptible ones were those coding for 17.3 kDa class II heat shock protein-like, chaperone protein ClpB1, glutathione S-transferase L3-like protein, B3 domain-containing protein At3g18960-like, transcription factor bHLH7, zinc finger MYM-type protein 1-like, and nine uncharacterized proteins, besides three lncRNAs. The genes that were downregulated in susceptible and upregulated in resistant cultivars were those coding for non-specific lipid transfer protein GPI-anchored 1, rust resistance kinase Lr10-like, disease resistance protein RPS6-like, and many uncharacterized proteins. DESeq2 analysis too revealed 20 DEGs that were upregulated in scab-resistant cultivars. Furthermore, a total of 361 genes were significantly upregulated in scab-susceptible variety, while 461 were found downregulated (P value < 0.05 and Log2 (FC) > 1). The differentially expressed genes (DEGs) were related to various pathways, i.e., metabolic, protein processing, biosynthesis of secondary metabolites, plant hormone signal transduction, autophagy, ubiquitin-mediated proteolysis, plant-pathogen interaction, lipid metabolism, and protein modification pathways. Real-time expression of a set of selected twelve DEGs further validated the results obtained from RNA-seq. Overall, these findings lay the foundation for investigating the genetic basis of apple scab resistance and defense pathways that might have a plausible role in governing scab resistance in apple against V. inaequalis.

The Use of Potassium Phosphonate (KHP) for the Control of Major Apple Pests.

Phosphonate-based products have demonstrated diverse abilities to protect crops against pests, with various modes of action proposed. In this article, we specifically investigated potassium phosphonate (KHP) on apple crops. Its performance to control three major apple bioagressors (Venturia inaequalis, Erwinia amylovora, and Dysaphis plantaginea) was evaluated under semicontrolled conditions. The product was able to confer significant protection rates (40 to 75% for apple scab, 40% for fire blight, and 30% for rosy aphid), which can be explained by its more or less efficient biocidal activity against the three pests, and by its ability to induce apple immunity (pathogenesis-related proteins and secondary metabolites genes). A cumulative effect of treatments as well as the systemic behavior of the product was demonstrated. Fields trials against apple scab and the postharvest disease bull's eyes rot (Neofabraea vagabunda) were performed on different apple varieties by applying KHP combined with light pest management programs either reducing (dessert orchards) or suppressing (cider orchards) fungicide applications. KHP was able to reduce apple scab by 70 to 90% on shoots and young and harvested fruit, and bull's eyes rot by 70 to 90% on harvested fruit. Overall, our results indicate that KHP is useful for the protection of apple trees against its major pests by direct effect and by triggering the host defense system.

Mapping of quantitative trait loci for scab resistance in apple (Malus × domestica) variety, Shireen.

BACKGROUND: Scab caused by Venturia inaequalis (Cke.) Wint. is the most important fungal disease of apple. Fungicide application is a widely practiced method of disease control. However, the use of chemicals is costintensive, tedious, and ecologically unsafe. The development of genetic resistance and the breeding of resistant cultivars is the most reliable and safest option. One such source of scab resistance happens to be the variety 'Shireen', released from SKUAST-Kashmir. However, to date, the nature of resistance and its genetic control have not been characterized. Objective This research aimed to elucidate the genetic basis of scab resistance in Shireen. METHODS: Genetic mapping of quantitative trait loci (QTL) for resistance to apple scab disease was performed using an F1 cross developed between the susceptible cultivar 'StarKrimson' and the resistant cultivar 'Shireen'. The population was evaluated for two consecutive years. Further, six candidate genes were analyzed via quantitative real-time PCR, to determine their expression level in response to the pathogen infestation. RESULTS: Genotyping and disease phenotyping of populations led us to identify two quantitative trait loci (QTLs), namely qRVI.SS-LG2.2019 and qRVI.SS-LG8.2019 on chromosomes 2 and 8 with LOD-values of 7.67 and 4.99 respectively, and six potential CDGs for the polygenic resistance in 'Shireen'. The genomic region corresponding to the mapped QTLs in LG 2 and LG 8 of 'Shireen' was examined for candidate genes possibly related to scab resistance using in silico analysis. CONCLUSION: The QTLs mapped in the genetic background of Shireen are the novel QTLs and may be transferred to desirable genetic backgrounds and provide opportunities for isolation and cloning of genes apart from their utility to achieve durable resistance to scab.

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 0.0078 mg 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.

CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis.

Apple scab, caused by the fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Using the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN) as a target for inactivation, which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference, we show, for the first time, that the CRISPR-Cas9 gene editing system can be successfully applied to the apple scab fungus. More specifically, using a CRISPR-Cas9 single guide RNA (sgRNA) targeted to the THN gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ∼16.7% gene inactivation efficiency. Notably, of the six THN mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of V. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening. In establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher gene function in this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.

Cross-resistance between myclobutanil and tebuconazole and the genetic basis of tebuconazole resistance in Venturia inaequalis.

BACKGROUND: Myclobutanil is one of the most widely used demethylation inhibitor (DMI) fungicides for the management of apple scab, caused by Venturia inaequalis. Strains of V. inaequalis resistant to myclobutanil have been reported across the world. Tebuconazole, another DMI fungicide, has been proposed as an alternative to myclobutanil, and the extent of cross-resistance with myclobutanil therefore needs to be evaluated. The sensitivity to tebuconazole and myclobutanil of a total of 40 isolates was determined. Half the isolates came from an isolated orchard which had never been sprayed with fungicides and half from orchards sprayed regularly with myclobutanil, but still with disease control problems. The progeny of a tebuconazole resistant (R) × sensitive (S) V. inaequalis cross were analyzed in order to improve understanding of the genetic control of tebuconazole sensitivity. RESULTS: There is cross-resistance between myclobutanil and tebuconazole (r = 0.91; P < 0.001). Sensitivity to tebuconazole of the progeny of a R × S cross varied quantitatively in a pattern which implied at least two gene loci differing between the parental strains. In addition, the asymmetric distribution of the sensitivity in the progeny implied possible epistatic effects. CONCLUSION: Resistance to myclobutanil and tebuconazole is strongly correlated. At least two genes are involved in the control of tebuconazole resistance in V. inaequalis.

Development of a Sensitive TaqMan qPCR Assay for Detection and Quantification of Venturia inaequalis in Apple Leaves and Fruit and in Air Samples.

A TaqMan quantitative PCR (qPCR) assay based on the translation elongation factor 1-α gene was developed for the quantification of Venturia inaequalis in leaves and fruits of Malus × domestica and in spore trap samples. The designed primers and hydrolysis probe amplified a specific 86-bp fragment for V. inaequalis. The specificity of the assay was tested using 35 strains of V. inaequalis and 20 different fungal species, including common pathogens of apple and other species of Venturia. The limit of detection was 20 fg, which is lower than a single genome of V. inaequalis. The selectivity of the assay was tested using DNA from three cultivars of Malus × domestica, and no influence on pathogen amplification was found. The assay was also validated for repeatability and reproducibility. With this assay, it was possible to detect and quantify V. inaequalis in four cultivars (Ambrosia, Florina, Golden Delicious, and Mondial Gala) in both symptomatic and asymptomatic leaves and in symptomatic Golden Delicious apple fruit stored for 2 months. Furthermore, the assay was successfully tested on spore trap samples originating from apple orchards. The quantification of the molecular assay when compared with the estimated number of V. inaequalis cells, using an optical microscope, showed a correlation coefficient of 0.8186. The developed technique could be used to detect V. inaequalis in asymptomatic samples without any cross-reaction with other fungal species. Furthermore, to improve the efficacy of disease management with a timely application of fungicides, this assay could be used for the analysis of spore trap samples by using an implemented extraction method.

The Effects of Succinate Dehydrogenase Inhibitor Fungicide Dose and Mixture on Development of Resistance in Venturia inaequalis.

Understanding how fungicide application practices affect selection for fungicide resistance is imperative for continued sustainable agriculture. Here, we examined the effect of field applications of the succinate dehydrogenase inhibitor (SDHI) fluxapyroxad at different doses and mixtures on the SDHI sensitivity of Venturia inaequalis, the apple scab pathogen. Fungicide applications were part of selection programs involving different doses (high or low) and mixtures (with a second single-site fungicide or a multisite fungicide). These programs were tested in two apple orchards over 4 years to determine potential cumulative selection effects on resistance. Each year after program applications, apple scab lesions were collected, and relative growth assays were conducted to understand shifts in fluxapyroxad sensitivity. After 4 years, there was a trend toward a reduction in sensitivity to fluxapyroxad for most selection programs in comparison to that in the non-selective-pressure control. In most years, the selection program plots treated with low-dose fluxapyroxad applications resulted in a larger number of isolates with reduced sensitivity, supporting the use of higher doses for disease management. Few significant differences (P < 0.05) in fungicide sensitivity were observed between isolates collected from plots where fungicide mixtures were applied compared to that in untreated plots, supporting the use of multiple modes of action in field applications. In all, appropriate doses and mixtures may contribute to increased longevity of SDHI fungicides used on perennial crops like apples.IMPORTANCE Of much debate is the effect of fungicide application dose on resistance development, as fungicide resistance is a critical barrier to effective disease management in agricultural systems. Our field study in apples investigated the effect of fungicide application dose and mixture on the selection of succinate dehydrogenase inhibitor resistance in Venturia inaequalis, a fungal pathogen that causes the economically important disease apple scab. Understanding how to best delay the development of resistance can result in increased efficacy, fewer applications, and sustainable fungicide use. Results from this study may have relevance to other perennial crops that require multiple fungicide applications and that are impacted by the development of resistance.