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Venturia effusa (syn. Fusicladium effusum), causal agent of pecan scab, is the most prevalent pathogen of pecan (Carya illinoinensis), causing severe yield losses in the southeastern United States. V. effusa is currently known only by its asexual (conidial) stage. However, the degree and distribution of genetic diversity observed within and among populations of V. effusa are typical of a sexually reproducing fungal pathogen, and comparable with other dothideomycetes with a known sexual stage, including the closely related apple scab pathogen, V. inaequalis. Using the mating type (MAT) idiomorphs from V. inaequalis, we identified a single MAT gene, MAT1-1-1, in a draft genome of V. effusa. The MAT1-1-1 locus is flanked by two conserved genes encoding a DNA lyase (APN2) and a hypothetical protein. The MAT locus spanning the flanking genes was amplified and sequenced from a subset of 14 isolates, of which 7 contained MAT1-1-1 and the remaining samples contained MAT1-2-1. A multiplex polymerase chain reaction screen was developed to amplify MAT1-1-1, MAT1-2-1, and a conserved reference gene encoding β-tubulin, and used to screen 784 monoconidial isolates of V. effusa collected from 11 populations of pecan across the southeastern United States. A hierarchical sampling protocol representing region, orchard, and tree allowed for analysis of MAT structure at different spatial scales. Analysis of this collection revealed the frequency of the MAT idiomorphs is in a 1:1 equilibrium of MAT1-1:MAT1-2. The apparent equilibrium of the MAT idiomorphs provides impetus for a renewed effort to search for the sexual stage of V. effusa. [Formula: see text] Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Apple scab is one of the most economically important diseases of apple in temperate production regions. In the absence of durable host resistance in commercially preferred cultivars, considerable applications of fungicides are needed to manage this disease. With the sequential development of resistance to nearly all classes of single-site fungicides in the apple scab pathogen Venturia inaequalis, synthetic multisite fungicides, such as mancozeb and captan, often comprise the core of chemical management programs for apple scab. Although these fungicides have demonstrable benefits for both disease and fungicide resistance management, the sustainability movement within agriculture aims to reduce reliance on such fungicides because of their broader environmental impacts. In this study, we establish a framework to enhance the feasibility of chemical management programs that do not rely on use of synthetic multisite protectant fungicides to manage apple scab. Specifically, we wish to evaluate chemical programs that integrate the biopesticide Bacillus subtilis QST 713 (Serenade Opti) in rotation with benzovindiflupyr (Aprovia), a single-site fungicide belonging to the class of succinate dehydrogenase inhibitors (SDHI), to circumvent the need for applications of synthetic multisite fungicides. During implementation of these programs, disease incidence data were taken at biweekly intervals. Regardless of the seasonal challenges presented in the 2 years of this study, when Bacillus subtilis QST 713 was used in place of captan and mancozeb mixtures, we did not observe any significant differences (P > 0.05) in development of apple scab symptoms between any of the management programs for the vertical axis or super spindle orchards in either year. This potential for substituting synthetic multisite fungicides with biopesticides is best realized when the programs are used with a decision support system in a super spindle planting system, where trees have reduced canopy densities. This 2-year study shows the potential to achieve adequate disease control using the integration of SDHI fungicides and biological controls without the use of synthetic multisite fungicides.

The feasibility of resuming the use of dodine for post-infection spraying in Uruguay was evaluated. For this purpose, the current status of sensitivity of Venturia inaequalis populations in commercial apple orchards was determined. V. inaequalis populations collected from six commercial orchards, with different histories of dodine use in the last 10 years, were assessed by measuring mycelial relative growth (RG) at a single assessment concentration (SAC) of 0.2 mg a.i. L−1 of dodine. The SAC was previously determined by fitting linear regressions between log10 EC50 and RG of a subset of isolates at seven increasing concentrations of dodine, from 0.05 to 5 mg a.i. L−1. In addition, the post-infection activity of dodine was evaluated in the field. For this, 2-year-old potted apple trees were exposed during pre-bloom stage to two natural rainfall events in an apple orchard with history of dodine use. Forty hours after the start of each rainfall, eight potted apple trees were hand sprayed with dodine at label concentration and eight were sprayed with water and used as controls. The in vitro assay indicates the presence of resistant isolates (RG > 90%) to dodine in all V. inaequalis populations assessed. Highly-resistant isolates (RG ≥ 95%) also were found in all populations, ranging from 3.4 to 28.6%, and showed statistical differences among orchards (P ≤ 0.10). In the field assay, dodine-sprays showed an incomplete control. Typical apple scab symptoms developed in dodine-treated (27.6% of the leaves) and control apple trees (60.6% of the leaves). These results show that dodine resistance is present in V. inaequalis populations of commercial apple orchards in Uruguay and confirms the occurrence of practical resistance in the field. Therefore, the resumption of dodine use should be cautious for apple scab management in Uruguay.

Apple scab caused by Venturia inaequalis is the most destructive apple disease worldwide. To manage apple scab in Uruguay, protectant fungicides are applied in anticipation of each rain event during ascospore discharge period. To optimize fungicide application, we studied the beginning and the end of ascospore discharge period and its behavior during nighttime and successive rain days, in natural and artificial conditions. Results showed that V. inaequalis ascospore discharge occurs in a period of 2 months, starting in the green tip stage and ending in early November, around 40 days before the usual warning system communication. Mature ascospores are released immediately after a rain event begins, regardless whether it is day- or nighttime. During consecutive days or nights of rain, successive discharges occur during 3 and eventually 4 days, with the greatest discharge occurring in the first 2 days. This finding suggests that the fungicide applications could be suspended earlier than usual and that strategic irrigations previously to forecast could be implemented to manage apple scab. However, field trials should be conducted to further validate this finding.

Apple scab caused by Venturia inaequalis (Cke.) Wint. is the most important disease of apple trees worldwide and requires a high number of fungicide applications. The G143A substitution in the inhibitor binding site of cytochrome b of V. inaequalis confers a high level of resistance to strobilurins targeting the bc1 complex. The aim of this work was to substitute the labor intensive in vitro assays, with the faster quantitative PCR. An allele-specific qPCR method with a newly designed primer set was successfully developed to quantitatively determine the frequency of QoI-resistant A143 allele in populations of V. inaequalis. To be able to suggest that the molecular method could be applied as unique and robust technique, we carried out in vitro sensitivity test to trifloxystrobin; first testing the relative germination and subsequently confirmed with the quantification of mutated allele frequencies by qPCR on forty-nine Italian V. inaequalis populations. qPCR gave a similar pattern to that obtained using in vitro conidial germination test in predominantly sensitive and resistant populations, the variability between these two tests was observed in some heterogeneous populations. The qPCR assay developed in this study efficiently quantifies the A143 allele and we can conclude that this method could be useful for the study of the fungicide resistance at population level in the fields, giving a quick response also with a large amount of samples.

Frequentes falhas de manejo da Sarna da Macieira (SDM) nos pomares, associadas aos problemas ambientais devido a utilização incorreta dos fungicidas, impulsionam o desenvolvimento de medidas alternativas. Assim, o objetivo do presente estudo foi verificar a possibilidade de uso dos bioestimulantes de forma isolada ou em mistura com fungicidas para o manejo da SDM. Para isto plantas de macieira ‘Gala’ foram pulverizadas de setembro a novembro dos anos de 2014 a 2018, entre um e dois dias antes de um período chuvoso com diferentes classes de bioestimulantes (aminoácidos, fosfitos, regulador de crescimento, fertilizantes foliares a base de extrato vegetal, algas e minerais). Alguns bioestimulantes testados reduziram a incidência da SDM em folhas e/ou frutos, bem como melhoraram os parâmetros de qualidade, no entanto este efeito foi dependente da classe de bioestimulante utilizado, forma que foi aplicado (isolado ou mistura), e ciclo da macieira avaliado.

The aim of the study was to assess the suitability of several apple cultivars for breeding of new genotypes resistant/low susceptible to apple scab and powdery mildew based on their general (GCA) and specific (SCA) combining abilities. Infection degree of the apple seedlings, being the progeny of 11 cultivars crossed in the model: ♀4 x ♂7 – 4 maternal cultivars (‘Alwa’, ‘Golden Delicious’, ‘Free Redstar’, ‘Gold Milenium’) and 7 paternal cultivars (‘Glogierowka’, ‘Kronselka’, ‘Kosztela’, ‘McIntosh’, ‘Oliwka Żolta’, ‘Malinowa Oberlandzka’, ‘Koksa Pomaranczowa’) by both apple pathogens was evaluated in July 2019. Significantly negative value for the GCA effect for the infection degree by apple scab was estimated for ‘Free Redstar’. This indicates that this cultivar is a good donor of genes responsible for resistance to apple scab in the progeny. Negative value of GCA effect for susceptibility to powdery mildew was also stated for ‘Free Redstar’. This means that this cultivar may serve as good donor of genes determining resistance/low susceptibility to powdery mildew in its progeny. Negative SCA effects for resistance/low susceptibility to apple scab, moreover, were indicated for 3 cross combinations: ‘Free Redstar’ x ‘McIntosh’, ‘Alwa’ x ‘Malinowa Oberlandzka’ and ‘Alwa’ x ‘McIntosh’. This indicates that genetic interaction of both parental genotypes results in genetic determination of resistance or high tolerance to apple scab in their progeny.

Of the different physical characteristics of fruits, one of the most essential qualities is flesh firmness, as is an important parameter when selling fresh apples, processing the fruit (processing industry) and during its storage. In the scope of our research, we examined five apple varieties originating from the Derecske horticultural site of KITE cPlc. in 2016. The plantation was planted to be suitable for intensive apple production. Samples were collected at the time of maturity (August-October) of the given variety. The five examined apple varieties (Gaia, Isaaq, Modí, Smeralda and Fujion) are all resistant to apple scab (fungal disease caused by Venturia inaequalis). The two-month storage experiment was conducted at 16-17 °C, which had an aggressive effect on our stored apples. Flesh firmness, weight and diameter were measured on three dates for each of the five apple varieties. Our results were evaluated using IBM SPSS Statistics 20 software. We aimed to compare flesh firmness, weight and diameter of the five apple varieties and their changes during storage. Isaaq (86.34 N/cm2), Modí (94.06 N/cm2) and Fujion (84.90 N/cm2) had outstanding initial flesh firmness results. The Modí apple variety provided the best storage parameters.

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 analysed 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.

Background Breeding for resistance to apple scab (caused by Venturia inaequalis), the most devastating fungal disease of apples, relies on genetic resources maintained in germplasm collections. Methods To identify new sources of scab resistance, we evaluated 177 Malus accessions, including 27 primary and 13 hybrid Malus species from diverse geographical origins, in an orchard at Geneva, New York. We also screened a differential host set for 2 years to monitor for changes in the effectiveness of ten known scab resistance genes, which allowed us to confirm the presence of virulent pathogen races in the orchard. Results We found that ~ 37% of the wild Malus accessions and domesticated cultivars were resistant to apple scab in the field. Several of these accessions were unrelated to sources of previously known resistance genes and are promising for apple scab genetic research and resistance breeding. Cultivars carrying the Rvi6 (Vf) gene from Malus floribunda clone 821, e.g. ‘Liberty’ or ‘Florina’, remained resistant despite the breakdown of Rvi6. ‘Demir’, a Malus hybrid from Turkey, and ‘Chisel Jersey’, a traditional English hard cider cultivar, showed fewer symptoms than the Rvi6 resistant cultivar ‘Prima’. Races 1 to 7 and 9 of V. inaequalis were present in the orchard, but no scab was observed on the indicator host accessions for races 11 and 12. Conclusions Detailed and systematic screening of Malus germplasm identified resistant and moderately resistant donor accessions based on resistance reaction types. These accessions are promising for use in future genetic studies to identify novel sources of scab resistance alleles for apple breeding to develop cultivars with durable apple scab resistance.

The present investigation was carried out to evaluate the bio-efficacy of Pydiflumetofen 7.5% + Difenoconazole 12.5% w/v (200 SC) at different doses along with standard checks viz., Pydiflumetofen 20% SC, Difenoconazole 25% EC, Metiram 55% + Pyraclostrobin 5% WG, Mencozeb 75% WP at recommended doses and untreated check (Control) against apple scab, premature leaf fall and powdery mildew diseases of apple at Harsil and Jhala fruit belt (District- Uttarkashi) locations over two consecutive growing seasons. The field experiment was arranged in randomized complete block design with three replications and nine treatments. Data on different apple diseases for two crop seasons (2017 and 2018) revealed that all the fungicides tested were found effective for the management of scab, premature leaf fall and powdery mildew diseases of apple as compared to untreated check (Control). However, the treatment T4 [Pydiflumetofen 7.5% + Difenoconazole 12.5% w/v (200 SC) @ 600 ml/ha] was found most effective followed by T3 [Pydiflumetofen 7.5% + Difenoconazole 12.5% w/v (200 SC) @ 500ml/ha] for all the diseases studied under Harshil and Jhala locations during both the studied years under present materials and conditions. However, the highest disease intensity for all the studied diseases was recorded in untreated control. The data of two consecutive crop seasons indicated maximum yield (96.07 q/ha & 93.69 q/ha in 2017 and 2018, respectively from Harshil location and 100.37 q/ha & 98.13 q/ha in 2017 and 2018, respectively from Jhala location) when the crop was sprayed with Pydiflumetofen 7.5% + Difenoconazole 12.5% w/v (200 SC) @ 600 ml/ha followed by when sprayed with the same treatment @ 500 ml/ha (92.25 q/ha in 2017 and 89.82 q/ha in 2018 from Harshil location and 97.64 q/ha in 2017 and 95.41 q/ha in 2018 from Jhala location). Whereas, minimum yield (66.70 q/ha in 2017 and 56.17 q/ha in 2018 from Harshil location and 74.25 q/ha in 2017 and 63.01 q/ha in 2018 from Jhala location) was recorded when tress were left unsprayed (Control) from both the locations during both the studied years.

In agriculture farming business, plant diseases are one of the reasons for the financial deficits around the globe. It is the fundamental factor, as it causes significant abatement in both capacity and quality of the growing crops. In plants, fruits are amongst the major sources of nutrients, however, there exists a wide range of diseases which adversely affect both quality and production of the fruits. To overcome such predicament, computer vision (CV) based methods are introduced. These methods are quite effective, which not only detect the diseases/infections at the early stages but also assign them a label. In this article, we propose a deep convolutional neural network-based method for the diseases classification of different fruits’ leaves. Initially, the deep features are extracted by utilizing pre-trained deep models including VGG-s and AlexNet, which are later fine-tuned by employing a concept of transfer learning. A multi-level fusion methodology is also proposed, prior to the selection step, based on an entropy-controlled threshold value - calculated by averaging the selected features. The resultant final feature vector is later fed into a host classifier, multi-SVM. Five different diseases are considered for experiments including apple black rot, apple scab, apple rust, cherry powdery mildew, and peach bacterial spots, which are collected from a plant village dataset. Classification results clearly reveal the improved performance of proposed method in terms of sensitivity (97.6%), accuracy (97.8%), precision (97.6%), and G-measure (97.6%).

A pattern classifier (PC) is used to solve a variety of non-separable and complex computing problems. One of the key problems is to efficiently predict a type of disease in a typical fruit tree. The timely and accurately predicted disease in an apple tree may help a farmer to take appropriate preventive measures in advance. In this article, an apple disease diagnosis system is developed to predict the apple scab and leaf/spot blight diseases. In this article, low level and shape-based features are used for the development of an intelligent apple disease prediction system. First, the key image features like entropy, energy, inverse difference moment (IDM), mean, standard deviation (SD), perimeter, etc., are extracted from the apple leaf images. The model for the proposed system is trained by using multi-layer perceptron (MLP) pattern classifier and eleven apple leaves image features. The Gradient descent back-propagation algorithm is used for building the intelligent system to carry out the pattern classification. The proposed system is tested using some random samples and exhibits excellent diagnosis accuracy of 99.1%. The sensitivity of the proposed prediction model is 98.1% and specificity of ~99.9%.

One of the most important groups of bioactive substances in apples are antioxidants, which have a health-preserving effect. The amount of these compounds varies not only during the ripening of the apple fruit, but also continuously during post-harvest storage. A growing group of health-conscious consumers are looking for apple varieties with a nutritional value that satisfies their needs. In the scope of our research, we examined 5 apple varieties originating from the Derecske horticultural site of KITE cPlc. in 2016. The plantation was planted to be suitable for intensive apple production. Samples were collected at the time of maturity (August-October) of the given variety. The 5 examined apple varieties (Gaia, Isaaq, Modí, Smeralda and Fujion) are all resistant to apple scab (fungal disease caused by Venturia inaequalis). The 2-month storage experiment was conducted at 16–17 °C, which had an aggressive effect on our stored apples. Total polyphenol (TPC) and flavonoid (TFC) content as well as FRAP values of the apples were measured. Measurements were taken immediately after harvest and after 1 and 2 months of storage. Our results were evaluated by using the IBM SPSS Statistics 20 software. Our aim was to compare the antioxidant results of 5 scab-resistant apple varieties, which change continuously during storage. The antioxidant content of our apple varieties increased during 2 months storage. The best result was achieved by the Modí apple variety (FRAP: 467.32 mg/100g; TFC: 317.76 mg/100g; TPC: 1771.97 mg/100g). Consumers may want to consume apples stored for a longer period of time if they are to absorb large amounts of antioxidants.

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.

Apple scab is one of the most economically important diseases of apples worldwide. The disease is caused by the haploid ascomycete Venturia inaequalis. Growing apples in cultivar mixtures may reduce disease severity. To determine how the pathogen population structure is affected by host mixtures we studied 24 V. inaequalis isolates sampled from three different apple cultivars (“Bramley”, “Cox” and “Worcester”) growing in a mixed orchard approximately 50 years old. The isolates were aligned against a reference genome and single nucleotide polymorphisms (SNPs) were called between the isolates. The populations isolated from Bramley and Worcester were distinct, while Cox isolates were an admixture. This supports previous tests of the ability of isolates to cross-infect hosts, and molecular comparisons using simple sequence repeats (SSRs). Genotype specific allele (GSA) loci were not distributed randomly across contigs in proportion to contig length, but were clustered. Clustered GSA were observed in almost all contigs. This indicates population differentiation across the whole genome, presumably due to lack of crossing-over events between Bramley and Worcester isolates. This lack is probably due to physical separation effects: sexual mating is more likely to take place and succeed between isolates from lesions on the same leaf than from contact between independently infected leaves in leaf litter on the orchard floor. This would especially be the case if sexual reproduction is initiated before leaf-fall.

Apple scab, caused by Venturia inaequalis, is the most common fruit and foliar disease in commercial apple production worldwide. Early in the production season, preventative contact fungicide sprays are essential for protecting highly susceptible continuously unfolding and expanding young leaves. In South Africa, mancozeb is a key contact fungicide used for controlling apple scab early in the season. The current study developed deposition benchmarks indicative of the biological efficacy of mancozeb against apple scab, using a laboratory-based apple seedling model system. The model system employed a yellow fluorescent pigment that is known to be an effective tracer of mancozeb deposition. A concentration range of mancozeb (0.15 to 1 times the registered dosage) and fluorescent pigment concentrations was sprayed onto seedling leaves, which yielded various fluorescent particle coverage (FPC%) levels. Modeling of the FPC% values versus percent disease control yielded different benchmark values when disease quantification was conducted using two different methods. Thermal infrared imaging (TIRI) disease quantification resulted in a benchmark model where 0.40%, 0.79%, and 1.35 FPC% yielded 50, 75, and 90% apple scab control, respectively. These FPC% values were higher than the benchmarks (0.10, 0.20, and 0.34 FPC%, respectively) obtained with quantitative real-time PCR (qPCR) disease quantification. The qPCR benchmark model is recommended as a guideline for evaluating the efficacy of mancozeb sprays on leaves in apple orchards since the TIRI benchmark model underestimated disease control. The TIRI benchmark model yielded 68% disease control at the lowest mancozeb dosage, yet no visible lesion developed at this dosage. Both benchmark models showed that mancozeb yielded high levels of disease control at very low concentrations; for the qPCR benchmark model the FPC% value of the FPC90 (90% control) corresponded to 0.15 times that of the registered mancozeb concentration in South Africa, i.e., 85% lower than the registered dosage.

Pseudothecia development stages of Venturia inaequalis (apple scab) were investigated in two climatically different regions in the Western Cape of South Africa. The aim was to determine the pseudothecial density (PD; pseudothecia per fertile lesion [p/f]) and ascal density (AD; asci per pseudothecium [a/p]) that contributes to defining the potential ascospore dose in a common prediction model of the apple scab infection risk. The PD and AD were compared between Elgin (EL), now considered a warm winter apple-growing region because of climate warming, and Koue Bokkeveld (KB), a cold winter region. In 2012 and 2013, scabbed apple leaves were collected during leaf-drop in KB and EL and overwintered either in their region of origin or in the other region. PD was significantly higher in scabbed leaves collected and overwintered in KB (mean, 24.11 p/f) than in leaves collected in KB and overwintered in EL (mean, 17.11 p/f; P < 0.001). PD of scabbed leaves collected and overwintered in EL (mean, 15.27 p/f) or collected in EL and overwintered in KB (mean, 16.07 p/f) did not differ significantly. Ascal density did not differ significantly in any treatment or season. We concluded that the significantly higher PD of scabbed leaves collected from the cooler region of KB and overwintered in KB compared with scabbed leaves collected in EL or KB and overwintered in EL could be caused by adaptations of V. inaequalis populations to the respective climates. This implied long-term effects of climate warming on apple scab epidemiology and management.

Asian pear scab is a fungal disease caused by Venturia nashicola. The identification of genes conferring scab resistance could facilitate the breeding of disease-resistant cultivars. Therefore, the present study aimed to identify a scab-resistance gene using an interspecific hybrid population ((Pyrus pyrifolia × P. communis) × P. pyrifolia). Artificial inoculation of V. nashicola was carried out for two years. The segregation ratio (1:1) of resistant to susceptible individuals indicated that resistance to V. nashicola was inherited from P. communis and controlled by a single dominant gene. Based on two years phenotypic data with the Kruskal–Wallis test and interval mapping, 12 common markers were significantly associated with scab resistance. A novel scab resistance gene, Rvn3, was mapped in linkage group 6 of the interspecific hybrid pear, and co-linearity between Rvn3 and one of the apple scab resistance genes, Rvi14, was confirmed. Notably, an insertion in pseudo-chromosome 6 of the interspecific hybrid cultivar showed homology with apple scab resistance genes. Hence, the newly discovered Rvn3 was considered an ortholog of the apple scab resistance gene. Since the mapping population used in the present study is a pseudo-BC1 population, pyramiding of multiple resistance genes to pseudo-BC1 could facilitate the breeding of pear cultivars with durable resistance.

Apples are one of the most productive varieties of fruit in the world, with a high nutritional and medicinal value. However, numerous diseases affect apple production on a wide scale, resulting in significant economic losses. These diseases often go overlooked until just before, after, or after fruit has been processed. Many pathogens can be avoided with cultural traditions and (optional) fungicides, even if there are no cures for tainted fruit. However, accurate diagnosis is essential for determining the right management practices and preventing further losses. Apple scab, apple rot, and apple blotch are some of the most prevalent diseases that affect apples. The proposed approach will greatly aid in the automated identification and classification of apple diseases, according to our test results. We discovered that normal apples were easy to discern from diseased apples in our trial, and that the texture-based GLCM function produced more reliable results for apple disease classification, with a classification accuracy of more than 96.43 percent. This demonstrates that combining the GLCM extraction function with naive bayes classification will greatly improve accuracy.

In order to understand mechanisms of ontogenic resistance to apple scab, we analyzed various aspects of young and old leaves. We have introduced an apple plants cultivation system where in vitro propagated and rooting explants produce a genetically uniform population of apple (Malus domestica cv. Idared) plants. In this work, we demonstrate that apple plants produced in our cultivation system showed susceptibility to Venturia inaequalis, the cause of apple scab disease in young leaves and resistance in old leaves, which is similar to orchard situation. Our analysis shows that the cessation of epidermal cell expansion and shape formation coincided with the onset of ontogenic resistance in older leaves. Formation of specific cuticular lamellar structures did not coincide with ontogenic resistance onset. Further, chemical composition analysis of wax from young susceptible and old resistant leaves did not reveal specific compounds involved in ontogenic resistance. Differences in homogalacturonan content in cell walls in susceptible and resistant cells as well as decreased methylesterification of pectin in resistant leaves suggest that polysaccharide composition of the cell wall may play a role in mycelium growth and nutrition.

Site-specific high throughput monitoring of airborne ascospores of Venturia inaequalis, the causal agent of apple scab, can improve existing warning systems. A new qPCR assay was developed to quantify ascospores collected by a simple rotating-arm spore sampler. The qPCR assay was highly specific and sensitive, with a limit of quantification of 20 ascospores per sample. The new detection system was compared to sampling with a traditional Burkard volumetric spore trap and to microscopic quantification. During controlled ascospore release experiments in a closed environment, strong correlations (ρ: 0.96 to 0.99) were observed between the two types of samplers and the two methods of quantification but significantly larger numbers of spores (log difference: 0.43 to 0.69) were obtained when using the rotating-arm sampler and when using molecular quantification. During comparisons under outdoor conditions over a three-year period, reasonable correlations between the techniques (average ρ = 0.61) were observed. When rotating-arm samplers operate continuously they can get saturated but their counts still correlated better with those from the Burkard sampler than when they only operate during rain and until two hours after. This suggests that ascospores were also captured outside of rain events. Based on these comparisons, molecular quantification of spores captured with the rotating-arm sampler appears to be a sensitive and reliable method to determine airborne ascospores of V. inaequalis and holds promise as a tool to guide targeted fungicide applications in commercial orchards as well as to increase our knowledge of the aerobiology of this pathogen.

Apple Scab, caused by Venturia inaequalis (Cke.) Wint. (anamorph Spilocea pomi Fr.), is considered to be one of the most important fungal diseases of apple, which occurs almost every year in severe epidemic form under favourable environmental conditions wherever apple is cultivated. The disease has two distinct phases in its life cycle i.e. the primary saprophytic stage on the fallen dead leaves and the secondary parasitic phase on the trees. The intensity of the disease varied with the environmental conditions, cultivar and orchard management practices. Yield losses during epidemic years in 1996, 2008 and 2013 in the Gangotri fruit valley of Dist. Uttarkashi went up to 84%. Among the environmental parameters, temperature, moisture and relative humidity have the highest influence on the epidemic development. The formations of pseudothecia and ascospore discharge were associated mainly with late autumn (October) infection. Urea @ 5% concentration had a significant effect on breaking of the life cycle of V. inaequalis in overwintered leaves. The pseudothecia formation takes 32–48 days more after sexual reproduction at temperature around 8–10 °C and this process completes between February and March every year. The maximum ascospores productivity was recorded at fruit development stage (pea size to walnut size) of apple with ascospore productivity of 1, 23,000/ml and thereafter declined gradually. The ascospore emission period was 64–78 days and mean numbers of cumulative degree days for 50 and 95% spore release were 456 and 960, respectively. Data accumulated over 17 years were analyzed for validation of Mills criteria to establish its relevance in rescheduling fungicide applications under monitored spray programme. On examination of the primary infection period from Gangotri valley, some differences were observed between our results and Mills table for ascospores infection. Potential ascospore dose is a useful tool for predicting the total amount of inoculums in an orchard and has been shown to effectively improve apple scab management. PAD values were 50 times higher in the poorly managed orchards than in the integrated orchards. However, integrated orchards were considered well-managed in the investigated years and showed that the epidemic risk was low and medium, while it was very high in the poorly managed orchards on the basis of PAD. Based on the long term studies carried out at different locations from 1993 to 2018, a computer based warning system was developed to forewarn the farmers for adoption of suitable management practices. The information generated are passed on to the growers by blowing a characteristic signaling, local news paper, SMS, telephonic communication, Govt. organization and through personal contacts or messages flashed 4–5 times through radio stations. In addition, growing season kisan mela, press releases and newsletters are issued via the extension workers of the University and Govt. department.

In the Krasnodar Territory, on the Renet Simirenko apple variety, symptoms corresponding to the manifestation of viral diseases were found: chlorotic leaf spot, dark green depressed spots on the fruit, which manifest themselves at the end of ripening before harvesting and during storage. Identification of pathogens was performed by Enzyme Immunoassay (ELISA). Samples were tested for the presence of apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), and apple mosaic virus (ApMV). As a result of the analysis, viruses of chlorotic leaf spot of the apple tree (ACLSV) and pitted of apple tree wood (ASPV) were detected. To protect the apple tree from viruses, the drug Farmaiod, GS, whose active ingredient is iodine, was tested. The first treatment was carried out during the 2014 leaf fall period with a consumption rate of 1.0 l/ha and continued in the following 2015 in the phenophase "green cap" and "fruit diameter 10-20 mm" in two versions with consumption rates of 0.5 and 1,0 l/ha. With the onset of leaf fall in the same areas, the treatment cycle was repeated according to the same scheme. Accounting was carried out by the method of visual diagnostics, in addition, at the end of the first and second seasons, virological analyzes were performed by ELISA. As a result of research, it was noted that 3-fold use of the drug Farmaiod, GS in apple trees of the variety Renet Simirenko restrained the development of the apple tree acle virus (ASPV) and completely suppressed the apple leaf chlorophyll virus (ACLSV). The weather conditions of 2016 test triggered a strong manifestation of scab on leaves and fruits. In the experimental variants, it was noted that the drug at 78.3-83.1 % inhibits the development of apple scab. Treatment with Farmaiod, GS also fostered harvest increase and premium output. The maximum results regarding restraints of infectious processes as well as harvest increase were received when applying the preparation with consumption rate of 1,0 l/ha.

Apple scab prediction is based on the model of Mills which describes the risk of Venturia inaequalis infection using leaf wetness duration and air temperature data. The quality of prognosis relies on sensor quality. Six brands of commercially available leaf wetness sensors were compared at the Julius Kuehn-Institute (Germany) apple orchard from 2011 to 2013. Also a new wetness sensor was developed and optimized from 2009 to 2015 in cooperation with Thies Clima company. The Thies-wetness sensor was equipped with a glass–ceramic surface, an adjustable heating system to avoid dew and an adjustable cooling system to prolong wetness duration of sensor surface. Prototypes were tested in climate chamber before exposition in the orchard to achieve sensor settings with drying properties broadly similar to apple leaves. The market-ready prototype showed suitability for prognosis in field assays in primary seasons of 2015–2017 and maybe integrated into prognosis of apple scab without further modeling. The Thies-sensor was shown to be more reliable, uniform, sensitive and more suitable for forecasting than the purchased models.

Succinate dehydrogenase inhibitors (SDHI) are an important class of fungicides for management of apple scab, especially as resistance to other classes of fungicides has become prevalent in the northeastern United States. Considering their single-site mode of action, there is high risk of resistance development to SDHI fungicides. Such risk mandates the need for proper monitoring of shifts in population sensitivity. This study aims to provide a means for phenotypic and genotypic characterization of SDHI fungicide resistance for Venturia inaequalis, the causal agent of apple scab. To complement the published sequence of VisdhB, target genes VisdhC and VisdhD were identified using sequences of homologous genes in other fungal organisms and a draft genome of V. inaequalis. Using mycelial growth and conidial germination assays, baseline sensitivities and cross sensitivities of V. inaequalis were determined for several SDHI fungicides. Mean baseline EC50 values for conidial germination of benzovindiflupyr, fluxapyroxad, pydiflumetofen, and inpyrfluxam were found to be 0.0021, 0.0284, 0.014, and 0.0137 μg ml−1, respectively. Mean baseline EC50 values for mycelial growth of benzovindiflupyr, fluxapyroxad, pydiflumetofen, and inpyrfluxam were found to be 0.0575, 0.228, 0.062, and 0.0291 μg ml−1, respectively. A significant and positive correlation in sensitivity was found between benzovindiflupyr, fluxapyroxad, pydiflumetofen, and inpyrfluxam as well as penthiopyrad and fluopyram, with the highest correlation between benzovindiflupyr and penthiopyrad for mycelial inhibition of V. inaequalis (r = 0.950, P < 0.001). For inhibition of conidial germination, the highest correlation was observed between penthiopyrad and fluopyram (r = 0.775, P < 0.001). Furthermore, the sequences of the VisdhC and VisdhD genes were identified and characterized for baseline isolates of V. inaequalis. Residues of similar position to mutations found in other systems that confer resistance to SDHI fungicides were identified in baseline isolates, but no mutations were identified in baseline isolates or those previously exposed to SDHI fungicides. This study will serve as a reference for future monitoring of resistance to SDHI fungicides in V. inaequalis at both a phenotypic and genotypic level.

Apple scab is an economically important pest for apple production. It is controlled by applying fungicides when conditions are ripe for the development of its spores. This occurs when leaves are wet for a long enough time at a given temperature. However, leaf wetness is not a sufficiently well-defined agro-meteorological variable. Moreover, the readings of leaf wetness sensors depend to a large extent on their location within the tree canopy. Here we show that virtual wetness sensors, which are based on the easily obtained meteorological parameters such as temperature, relative humidity and wind speed, can be used in place of physical sensors. To this end, we have first collected data for two growing seasons from two types of wetness sensors planted in four locations in the tree canopy. Then, for each sensor we have built a machine-learning model of leaf wetness using the aforementioned meteorological variables. These models were further used as virtual sensors. Finally, Mills models of apple scab infection were built using both real and virtual sensors and their results were compared. The comparison of apple scab models based on real sensors shows significant variability. In particular, the results of a model depend on the location of the sensor within the canopy. The models based on data obtained from virtual sensors, are similar to the models based on physical sensors. Both types of models generate results within the same range of variability. The outcome of the study shows that the control of apple scab can be based on machine learning models based on standard meteorological variables. These variables can be readily obtained using inexpensive meteorological stations equipped with basic sensors. These results open the way to a widespread application of precise control of apple scab and consequently significant reduction of the use of pesticides in apple production with benefits for environment, human health and economics of production.

Apple scab (Venturia inaequalis) (AS) and frogeye leaf spot or black rot (Botryosphaeria obtusa) (FLS or BR) are two diseases of apples that affect developing foliage and fruit during summer and ca...

Trunk injections reduce pesticide inputs and environmental contamination, and recent work has addressed apple production systems. Insecticides and fungicides have been demonstrated for apple tree injection, however combinations of the two have not yet been tested. Trunk injections of the systemic insecticide emamectin benzoate and systemic acquired resistance (SAR) fungicide phosphorous acid were performed on mature apple trees to combine management strategies for foliar insect pests and apple scab (Venturia inaequalis). Injections of emamectin benzoate followed by phosphorous acid into the same set of injection ports resulted in higher mortality of Choristoneura rosaceana larvae and lower incidence of apple scab compared to the control. Scab incidence on trees in which phosphorous acid was injected into the same set of ports before emamectin benzoate were not different from control trees early in the growing season. Injections of emamectin benzoate and phosphorous acid into the same holes in either order showed higher mortality and reduced larval feeding in C. rosaceana bioassays compared with products injected into separate holes. This study demonstrates that two pesticides can interact dynamically within the vascular system of a tree, which has important implications for expanding the utility of trunk injection for fruit tree management.

Apple (Malus domestica) is a widely grown fruit in various climate types of the world and is exposed to the attack of many fungal disease agents during the growing period. One of them is Venturia inaequalis, which is probably the most important in terms of economic loss worldwide, which makes apple scab disease important in apple growing areas. In this study, the genetic diversity of apple scab in Bingol and Isparta provinces has been examined, which represent the two extremes of Turkey in terms of apple production. The 18 isolates obtained from these regions and were amplified by PCR using 7 RAPD primers. Polymorphic bands of the isolates were obtained by subjecting to agarose gel imaging. The average number of bands per primer was determined to be 3.91, with the highest polymorphic bands obtained from OPF04 encoded primer and the least polymorphic bands from OPG15 encoded primer. According to the clustering analysis, the total isolate population belongs to 5 different groups. Significant privileges were not observed between the two regions’s isolates It is thought that RAPD primers have a certain level to determine the diversity but not high levels of intra- and interregional variation in the isolates of V. inaequalis.

A 2-year study on the use of organic and conventional adjuvants alone, or mixed with urea, was conducted for management of overwintering inoculum of the apple scab pathogen, Venturia inaequalis. Select adjuvants (LI 700, Bond Max, Latron B-1956, and Organic Wet Betty [OWB]) have the potential to hasten urea-driven leaf litter decomposition and reduce V. inaequalis overwintering inoculum comparable to urea, and that one organic surfactant could perform the same level of leaf decomposition as urea. Combinations of adjuvants with urea significantly improved leaf litter degradation compared with urea alone, concomitant with reducing the number of pseudothecia present and pseudothecium fertility. We demonstrate that the combination of urea with Bond Max or OWB reduced pseudothecia fertility and ascospore production to less than 5% in the remaining pseudothecia, a significantly greater reduction than with urea alone. These results suggest that conventional growers combine urea with Bond Max or OWB to more effectively reduce overwintering inoculum, and that the adjuvant OWB can provide organic growers with comparable performance to urea used in conventional orchards for improved sanitation.

Gametophytic self-incompatibility is generally considered a hurdle for breeding programs, as it reduces the number of possible cross combinations. The wide variety of S-haplotypes in apple (Malus × domestica) reduces the probability of fully incompatible crosses, while the occurrence of semi-compatible combinations is more frequent. The yield in such combinations does not vary much with respect to full compatibility, but noteworthy a single S-haplotype from the male parent (pollen donor) will be transmitted to the progeny. This aspect should be carefully taken into account in breeding programs when one or more genes of interest are placed in proximity to the S-locus, as the segregation of the paternal alleles in the progeny will skew from the expected 1:1 ratio, reducing the frequency of the allele linked to the rejected S-haplotype. Among the genes linked to the S-locus in the rosaceous fruit trees, one of the most important is the apple scab resistance gene Rvi5. This gene, introduced in Malus × domestica from M. micromalus and × M. atrosanguinea 804, was fine-mapped in two large segregating populations and proved to be placed in close proximity to the S-locus, on the bottom of chromosome 17. Therefore, knowledge of the S-genotype of parental cultivars can be exploited to plan semi-compatible crosses that dramatically increase the frequency of the resistance gene in the progeny, and to avoid those combinations that conversely would reduce it.

In the agriculture industry one of the major causes for economic loss is plant diseases. For sustaining agriculture, it is necessary to monitor the health of plants and detect the diseases in plants at an early stage. Constant monitoring is necessary in the case of Apple trees. Early disease detection and crop health monitoring can reduce losses in agriculture. In the case of Apple trees, common diseases that affect the trees and the symptoms which are visible in the leaves are Apple scab, Black rot, Cedar apple rust. YOLO (You Only Look Only Onces) networks are used to detect the diseases and find the remaining healthy part on the leaf. The hardware part involves a self designed drone to take the video image of all the trees in the farm. The paper compares the limitations and benefits of these potential methods

BackgroundScab is the most important fungal disease of apple and pear. Apple (Malus x domesticaBorkh.) and European pear (Pyrus communisL.) are genetically related but they are hosts of two different fungal species:Venturia inaequalisfor apple andV. pyrinafor European pear. The apple/V. inaequalispathosystem is quite well known, whereas knowledge about the pear/V. pyrinapathosystem is still limited. The aim of our study was to analyse the mode of action of a major resistance gene of apple (Rvi6) in transgenic apple and pear plants interacting with the two scab species (V. inaequalisandV. pyrina), in order to determine the degree of functional transferability between the two pathosystems.ResultsTransgenic pear clones constitutively expressing theRvi6gene from apple were compared to a scab transgenic apple clone carrying the same construct. After inoculation in greenhouse withV. pyrina, strong defense reactions and very limited sporulation were observed on all transgenic pear clones tested. Microscopic observations revealed frequent aborted conidiophores in theRvi6transgenic pear /V. pyrinainteraction. The macro- and microscopic observations were very comparable to theRvi6apple /V. inaequalisinteraction. However, this resistance in pear proved variable according to the strain ofV. pyrina, and one of the strains tested overcame the resistance of most of the transgenic pear clones. Comparative transcriptomic analyses of apple and pear resistant interactions withV. inaequalisandV. pyrina, respectively, revealed different cascades of molecular mechanisms downstream of the pathogen recognition byRvi6in the two species. Signal transduction was triggered in both species with calcium (and G-proteins in pear) and interconnected hormonal signaling (jasmonic acid in pear, auxins in apple and brassinosteroids in both species), without involvement of salicylic acid. This led to the induction of defense responses such as a remodeling of primary and secondary cell wall, lipids biosynthesis (galactolipids in apple and cutin and cuticular waxes in pear), systemic acquired resistance signal generation (in apple) or perception in distal tissues (in pear), and the biosynthesis of phenylpropanoids (flavonoids in apple but also lignin in pear).ConclusionThis study is the first example of a successful intergeneric transfer of a resistance gene amongRosaceae, with a resistance gene functioning towards another species of pathogen.

Although human-imposed selection is known to have altered plant traits during crop domestication, the effect of selection on host susceptibility or resistance is not well understood. Moreover, the domestication of perennial tree fruit crops, unlike annual crops, was driven by hybridization, clonal propagation, and selection of desirable phenotypes, which could confound the accurate assessment of domestication-associated effects on host resistance or susceptibility genes. We studied the effects of domestication on disease-related nucleotide-binding leucine-rich repeat (NLR) genes by combining phylogenetic, haplotype, and selection signature analysis using sequence data from Golden Delicious double haploid (GDDH13 v.1.1) apple genome and 80 resequenced domesticated and wild Malus accessions. The NLR gene family in the GDDH13 v.1.1 apple genome constituted 546 genes that showed expansion mainly through proximal (39.1%) and dispersed (29.5%) duplications. The genome duplication (WGD) within Rosaceae, affecting the Malus lineage, is also evident in the NLR genes. The NLR genes are found in genomic regions associated with previously detected disease resistance-related quantitative trait loci (QTL) for apple scab, fire blight, powdery mildew, and blue mold. A genomic diversity analysis identified that the NLR genes in domesticated apples (Malus domestica) have more variation (average πdom=4.01 × 10−3) than their main progenitor, wild M. sieversii (average πsie=2.95 × 10−3) and M. sylvestris (average πsyl=2.11 × 10−3), which hybridized with M. domestica during the domestication process. These results suggest evidence of selection on disease-related genes associated with domestication of apple. An increased diversity across NLR genes in the domesticated germplasm may be attributed to their diverse geographical origins and distinct pedigrees, together with selection for disease resistance during domestication. The NLR genes under selection can provide opportunities to explore their role in disease resistance in apples.

The relationship between housekeeping and pathogenicity-related genes and virulence or avirulence towards the primary Malus resistance genes (R) has not been previously studied for Venturia inaequalis fungus, the causal agent of apple scab. In this study, the sequences of two housekeeping genes encoding elongation factor alpha (EF-1α) and β-tubulin and two previously unstudied effector genes of V. inaequalis from mannosidase and glucosidase families of 100 strains collected from apple cultivars with Rvi6, Rvi1, and Rvi17 and without known scab resistance genes were submitted to the analyses. Based on the phylogenetic and diversity data, as well as recombination analyses of the sequenced regions, we assessed the phylogenetic relationships and genetic structure of the pathogen within the species and the evolutionary forces that are currently acting upon this microorganism. The topology of the obtained phylograms demonstrates the lack of a relationship between the phylogenetic position of the strain and the host cultivar and the geographical origin or race of the strain. The isolates from different hosts were differentiated but did not form diagnosable, distinct phylogenetic groups. These results suggest that the analyzed genes may be too conserved to reflect the adaptation of pathogens to apple genotypes with different R genes, thus, they do not adequately reflect race discrimination. In contrast, based on variation and gene flow estimation, genetic divergence was observed among strains virulent to apple trees containing Rvi6. The results of this study confirmed a lack of free recombination between strains and demonstrated that the analyzed regions are in linkage disequilibrium and contain non-random polymorphisms associated with the strain.

Fire blight, caused by epiphytotic gram‐negative bacteria Erwinia amylovora, is the most destructive bacterial disease of apple (Malus spp.). Genetic mechanisms of fire blight resistance have mainly been studied using traditional biparental quantitative trait loci (QTL) mapping approaches. Here, we use large‐scale historic shoot and blossom fire blight data collected over multiple years and genotyping‐by‐sequencing (GBS) markers to identify significant marker–trait associations in a diverse set of 566 apple [Malus domestica (Suckow) Borkh.] accessions. There was large variation in fire blight resistance and susceptibility in these accessions. We identified 23 and 38 QTL significantly (p < .001) associated with shoot and blossom blight resistance, respectively. The QTL are distributed across all 17 chromosomes of apple. Four shoot blight and 19 blossom blight QTL identified in this study colocalized with previously identified QTL associated with resistance to fire blight or apple scab. Using transcriptomics data of two apple cultivars with contrasting fire blight responses, we also identified candidate genes for fire blight resistance that are differentially expressed between resistant and susceptible cultivars and located within QTL intervals for fire blight resistance. However, further experiments are needed to confirm and validate these marker–trait associations and develop diagnostic markers before use in marker‐assisted breeding to develop apple cultivars with decreased fire blight susceptibility.

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.

BackgroundScab, or black spot, caused by the filamentous fungal pathogenVenturia inaequalis, is the most economically important disease of apple (Malusxdomestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity ofV. inaequalisis required. A major bottleneck for the genetic characterization ofV. inaequalisis the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions inV. inaequalishave yet been published. Recently, CRISPR-Cas9 has emerged as an efficient tool for gene editing in filamentous fungi. With this in mind, we set out to establish CRISPR-Cas9 as a gene editing tool inV. inaequalis.ResultsWe showed that CRISPR-Cas9 can be used for gene inactivation in the apple scab fungus. As a proof of concept, we targeted the melanin biosynthesis pathway genetrihydroxynaphthalene reductase(THN), which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference. Using one of two CRISPR-Cas9 single guide RNAs (sgRNAs) targeted to theTHNgene, 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 these sixTHNmutants, 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 ofV. 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.ConclusionsIn establishing CRISPR-Cas9 as a tool for gene editing inV. inaequalis, we have provided a strong starting point for studies aiming to decipher the function of genes associated with the growth, reproduction, virulence and pathogenicity of 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.

Apple cv. 'Huangtaiping' (Malus pumila Mill.) is grown widely in northern China for the production of jellies, preserves, and cider. In 2018, atypical scab symptoms were observed on fruits of Huangtaiping in Heilongjiang Province of China. The disease incidence was estimated at approximately 0.4%. Symptoms were scab-like black spots (3 to 5 mm diam.) distinct from scab caused by Venturia inaequalis. Conidia were generally produced on lesions and using a modified microscope (Goh 1999), a single spore was picked up from each sample on water agar plate with a glass needle and then transferred to PDA amended with lactic acid (0.50 ml/L) and sulfate streptomycin (0.20 g/L). Fifteen isolates were obtained and incubated at 21°C for 6 weeks in darkness on PDA. The colonies on PDA were gray-black with circular morphology and floccose texture, which were similar with the characteristics of V. asperata described previously (Turan et al. 2019). The conidia were cylindrical to fusiform, 0 to 1 septate, yellowish and 19.7 (13.5 to 25.8) × 5.7 (3.6 to 6.9) μm (n = 10) in size, which were larger than previously described ones (Turan et al. 2019). DNA of three randomly selected isolates were extracted by a modified SDS method (Ping et al. 2004). The internal transcribed spacer (ITS) region of rDNA of the three selected isolates was amplified with the primers ITS4/ITS5 (White et al. 1990), sequenced and deposited in GenBank (MN958665, MN95866 and MN958667). BLAST analysis showed that the amplified sequences were identical and had 99.3% sequence identity with V. asperata (AF333447, MT459450 and MT459451), 95.4% sequence identity with V. cerasi (MK810963 and MK810964) and 94.3% sequence identity with V. carpophila (MN958609, MN958610 and MN958611). In addition, the complete large subunit ribosomal RNA gene (LSU) was amplified with the primers LROR/LR5 (Vilgalys and Hester 1990), sequenced and deposited in GenBank (MT845787, MT845788 and MT845789). BLAST analysis showed that the amplified sequences were identical and had 99.7% sequence identity with V. asperata (EF114711), 99.2% identity with V. carpophila (MT772296, MT845732 and MT845733 ) and 98% identity with V. cerasi (MK810848 and MK810849). Phylogenetic analysis based on concatenated ITS and LSU sequences showed that the tested isolates grouped with V. asperata strain 2349 in the same clade and the closest species with V. asperata was V. carpophila, followed by V. cerasi. In July 2019, pathogenicity of the isolate VAHLJ3-1-1 was evaluated on Huangtaiping. A conidia suspension with a concentration of 5×105/ml was sprayed evenly on the surface of six fruits. In order to maintain high humidity, inoculated fruits were wrapped with a plastic bag (a cotton ball with water was placed in the plastic bag) to maintain wetness for 3 days. Six fruits sprayed with water were used as a control. Four weeks after inoculation, similar symptom of atypical scab was observed on fruits of Huangtaiping, and V. asperata was isolated again from six inoculated fruits with reisolation frequency of 100% by the single spore isolation, while no symptom was observed on the control fruits. Based on the morphological and molecular identifications, the causal agent of atypical scab on Huangtaiping was identified as V. asperata. Apple scab is usually caused by V. inaequalis (Shen et al. 2020). However, apple scab has also been caused by V. asperata in Italy and France (Caffier et al. 2012; Turan et al. 2019). To the best of our knowledge, this is the first report of V. asperata associated with apple scab-like lesions in China. This information augments our knowledge of the spectrum of Venturia species associated with disease on apple fruit and will be a valuable foundation underpinning management strategies for this cultivar.