Field-scale gene flow of fungicide resistance in Pyrenophora teres f. teres and the effect of selection pressure on the population structure.

The effectiveness of fungicides to control foliar fungal crop diseases is being diminished by the increasing spread of resistances to fungicides. One approach that may help to maintain efficacy is remediation of resistant populations by sensitive ones. However, the success of such approaches can be compromised by re-incursion of resistance through aerial spore dispersal; although, knowledge of localized gene flow is lacking. Here, we report on a replicated mark-release-recapture field experiment with several treatments set up to study spore-dispersal-mediated gene flow of a mutated allele that confers demethylase inhibitor resistance in Pyrenophora teres f. teres (Ptt). Artificial inoculation of the host, barley (Hordeum vulgare), was successful across the 12-ha trial, where the introduced sensitive- and resistant-populations were, respectively, 6- and 13-fold the DNA concentration of the native Ptt population. Subsequent disease pressure remained low which hampered spread of the epidemic to such extent that gene flow was not detected at, or beyond 2.5 m from source points. In the absence of gene flow, plots were assessed for treatment effects; fungicide applied to populations that contained 14.3% of allele mutation increased in frequency to 24.5%, whereas sensitive populations had no change in structure. Untreated controls of native Ptt population remained genetically stable, yet untreated controls that were inoculated with sensitive Ptt had half the resistance frequency of the native population structure. The trial demonstrates the potential for management to remediate fungicide resistant pathogen populations, where localized gene flow is minimal; to safeguard chemical crop protection into the future.

Exploring marine compounds as potential biocontrol agents against powdery mildew for agricultural sustainability: a computer-based approach.

Powdery mildew is a pervasive fungal disease causing significant economic losses globally. Continuous use of synthetic fungicides has led to environmental concerns and resistant fungal strains. This study explores marine-derived cephalostatins from the South African Natural Compounds Database as novel fungicidal agents for managing powdery mildew. Using molecular docking techniques, we investigated the interaction between selected cephalostatins and critical proteins involved in powdery mildew pathogenesis. Compounds were selected based on drug-likeness and bioactivity, adhering to Lipinski's Rule of Five. Molecular interactions, binding affinities, and stability were analysed using AutoDock Vina, PyMOL, and Discovery Studio. Cephalostatin 17 exhibited the highest binding affinity (-10.4 kcal/mol), indicating strong potential for inhibiting fungal growth through significant hydrogen bonding and hydrophobic interactions. The study's primary limitation is the reliance on computational predictions, necessitating experimental validation. Cephalostatin 17 stands out as a promising candidate for sustainable agricultural practices.

Multiple routes to fungicide resistance: Interaction of Cyp51 gene sequences, copy number and expression.

We examined the molecular basis of triazole resistance in Blumeria graminis f. sp. tritici (wheat mildew, Bgt), a model organism among powdery mildews. Four genetic models for responses to triazole fungicides were identified among US and UK isolates, involving multiple genetic mechanisms. Firstly, only two amino acid substitutions in CYP51B lanosterol demethylase, the target of triazoles, were associated with resistance, Y136F and S509T (homologous to Y137F and S524T in the reference fungus Zymoseptoria tritici). As sequence variation did not explain the wide range of resistance, we also investigated Cyp51B copy number and expression, the latter using both reverse transcription-quantitative PCR and RNA-seq. The second model for resistance involved higher copy number and expression in isolates with a resistance allele; thirdly, however, moderate resistance was associated with higher copy number of wild-type Cyp51B in some US isolates. A fourth mechanism was heteroallelism with multiple alleles of Cyp51B. UK isolates, with significantly higher mean resistance than their US counterparts, had higher mean copy number, a high frequency of the S509T substitution, which was absent from the United States, and in the most resistant isolates, heteroallelism involving both sensitivity residues Y136+S509 and resistance residues F136+T509. Some US isolates were heteroallelic for Y136+S509 and F136+S509, but this was not associated with higher resistance. The obligate biotrophy of Bgt may constrain the tertiary structure and thus the sequence of CYP51B, so other variation that increases resistance may have a selective advantage. We describe a process by which heteroallelism may be adaptive when Bgt is intermittently exposed to triazoles.

Fungicide Sensitivity and Non-Target Site Resistance in Rhizoctonia zeae Isolates Collected from Corn and Soybean Fields in Nebraska.

Rhizoctonia zeae was recently identified as the major Rhizoctonia species in corn and soybean fields in Nebraska and was shown to be pathogenic on corn and soybean seedlings. Fungicide seed treatments commonly used to manage seedling diseases include prothioconazole (demethylation inhibitor), fludioxonil (phenylpyrrole), sedaxane (succinate dehydrogenase inhibitor), and azoxystrobin (quinone outside inhibitor; QoI). To establish the sensitivity of R. zeae to these fungicides, we isolated this pathogen from corn and soybean fields in Nebraska during 2015 to 2017 and estimated the relative effective concentration for 50% inhibition (EC50) of a total of 91 R. zeae isolates from Nebraska and Illinois. Average EC50 for prothioconazole, fludioxonil, sedaxane, and azoxystrobin was 0.219, 0.099, 0.078, and > 100 µgml-1, respectively. In planta assays showed that azoxystrobin did not significantly reduce the disease severity on soybean (P > 0.05). The cytochrome b gene of R. zeae did not harbor any mutation known to confer QoI resistance and had a type-I intron directly after codon 143 suggesting that a G143A mutation is unlikely to evolve in this pathogen. For prothioconazole, fludioxonil, and sedaxane, EC50 of isolates did not differ significantly among years of collection (P > 0.05) and their single discriminatory concentrations were identified as 0.1 µgml-1. This is the first study to establish non-target site resistance of R. zeae to azoxystrobin and the sensitivity of R. zeae to commonly used seed treatment fungicides in Nebraska. This information will help to guide strategies for chemical control of R. zeae and monitor sensitivity shifts in future.

Transient Expression of Nicotiana tabacum Silicon-Induced Histidine-Rich Defensins in Nicotiana benthamiana Limits Necrotic Lesion Development Caused by Phytopathogenic Fungi.

Silicon (Si) supplementation permits plants to better deter infection. Supplementing hydroponically-propagated Nicotiana tabacum with 1 mM potassium silicate (K2SiO3) reduced necrotic lesion development on detached leaves by both Botrytis cinerea and Sclerotinia sclerotiorum. Previously, a family of Si-induced genes was identified in N. tabacum. These genes were members of the Solanaceous Histidine-Rich Defensin (HRD) superfamily and were termed NtHRD1s (the first identified family of Nicotiana tabacum Histidine-Rich Defensins). Defensins were originally identified to participate in innate immunity. Thus, the NtHRD1s were tested for antimicrobial effects on plant pathogens. Transient expression of NtHRD1 genes within Nicotiana benthamiana leaves restricted the development of necrotic lesions caused by B. cinerea and S. sclerotiorum. Thus, the NtHRD1s may be an additional Si-responsive factor conferring beneficial effects on plants.

Colletotrichum species causing Glomerella leaf spot and apple bitter rot in the southeastern United States exhibit disparities in relative frequency, morphological phenotype, and QoI sensitivity.

Glomerella leaf spot (GLS), Glomerella fruit rot (GFR) and apple bitter rot (ABR), caused by Colletotrichum spp. are amongst the most devastating apple diseases in the southeastern United States. While several species have been identified as causal pathogens of GLS, GFR, and ABR, their relative frequency and fungicide sensitivity status in the southeastern U.S. is unknown. In total, 381 Colletotrichum isolates were obtained from symptomatic leaves and fruit from 18 conventionally managed apple orchards and two baseline populations in western North Carolina and Georgia in 2016 and 2017. Multilocus DNA sequence analysis revealed that C. chrysophilum was the predominant cause of GLS and GFR, and C. fioriniae was the causal agent of ABR. Baseline and commercial populations of Colletotrichum spp. were evaluated for sensitivity to pyraclostrobin and trifloxystrobin and no statistical differences in sensitivity between the two species were observed for conidial germination. However, EC50 values were significantly lower for C. fioriniae compared to C. chrysophilum for both fungicides regarding mycelial inhibition. Isolates recovered from commercial orchards revealed that 5 populations of C. chrysophilum and 1 population of C. fioriniae had reduced sensitivity to trifloxystrobin, and 1 C. fioriniae population had reduced sensitivity to pyraclostrobin via conidial germination assays. The cytb gene for 27 isolates of C. fioriniae, C. chrysophilum, and C. fructicola with different QoI sensitivities revealed the G143A mutation in a single isolate of C. chrysophilum with insensitivity to both fungicides. Results of these studies suggest that two Colletotrichum spp. predominantly cause GLS and ABR in the southeastern U.S. and that a reduction in sensitivity to some QoI fungicides may be responsible for control failures. This study also provides basis for monitoring shifts in QoI sensitivity in Colletotrichum spp. causing disease on apple in the southeastern U.S.

Visual detection of fungicide resistance by combining RPA and CRISPR/Cas12a in peach Brown rot fungus Monilinia fructicola.

BACKGROUND: Peach brown rot caused by Monilinia fructicola severely affects the quality and yield of peach, resulting in large economic losses worldwide. Methyl benzimidazole carbamate (MBC) fungicides and sterol demethylation inhibitor (DMI) fungicides are among the most applied chemical classes used to control the disease but resistance in the target pathogen has made them risky choices. Timely monitoring of resistance to these fungicides in orchards could prevent control failure in practice. RESULTS: In the current study, we developed methods based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a systems to detect MBC and DMI resistance based on the E198A mutation in the ß-tubulin (MfTub2) gene and the presence of the Mona element in the upstream region of the MfCYP51, respectively. For MBC resistance, RPA primers were designed that artificially incorporated PAM sites to facilitate the CRISPR/Cas12a reaction. Subsequently, specific tcrRNAs were designed based on the E198A mutation site. For the detection of the Mona element, we designed RPA primers M-DMI-F2/M-DMI-R1 that in combination with crRNA1 detected 'Mona' and distinguished resistant from sensitive strains. CONCLUSION: Both methods exhibited high sensitivity and specificity, requiring only a simple isothermal device to obtain results within 1 h at 37 °C. The FQ-reporter enabled visualization with a handheld UV or white light flashlight. This method was successfully used with purified DNA from lab cultures and crude DNA from symptomatic fruit tissue, highlighting its potential for on-site detection of resistant strains in orchards. © 2024 Society of Chemical Industry.

Mixing Elemental Sulfur or Chlorothalonil with QoI and DMI Fungicides for Management of Late Leaf Spot of Peanut.

Reduced sensitivity to demethylation inhibitor (DMI) and quinone outside inhibitor (QoI) fungicides in Nothopassalora personata, the cause of late leaf spot of peanut (Arachis hypogaea) complicates management of this disease in the southeastern U.S. Mixtures with protectant fungicides may help preserve the utility of members of both DMI and QoI fungicide groups for leaf spot management. Field experiments were conducted in Tifton, GA from 2019 to 2021 and in Plains, GA during 2019 and 2020. The primary objective was to determine the effects of mixtures of DMI fungicides, tebuconazole and mefentrifluconazole, and QoI fungicides, azoxystrobin and pyraclostrobin, with micronized elemental sulfur on late leaf spot in fields with populations of N. personata with suspected reduced sensitivity to DMI and QoI fungicides. In four of the experiments, the efficacies of elemental sulfur and chlorothalonil as mixing partners were also compared. In most cases, standardized area under the disease progress curve (sAUDPC) and final percent defoliation were less for all DMI and QoI fungicides mixed with sulfur or chlorothalonil than for the respective fungicides alone. In most cases, sAUDPC and final percent defoliation were similar for sulfur and chlorothalonil when mixed with the respective DMI or QoI fungicide. These results indicate that mixtures of DMI or QoI fungicides with either micronized sulfur or chlorothalonil can improve control of late leaf spot compared to the DMI or QoI fungicide alone. These results also indicate that elemental sulfur has potential as an alternative to chlorothalonil in tank mixes where that protectant fungicide is currently being used as a mixing partner to improve leaf spot control.

Resistance of Ascochyta rabiei isolates from chickpeas (Cicer arietinum L.) to fungicides.

Ascochyta blight is a disease that causes significant yield losses in chickpea crops in Turkey under favorable environmental conditions. The fungal pathogen Ascochyta rabiei is the causative agent of this disease. The antifungal activity of previous fungicides against A. rabiei was not effective due to the heterothallic nature of the fungus. The aim of this study was to determine the sensitivity of A. rabiei to fungicides (25.2 g kg-1 boscalid + 12.8 g kg-1 pyraclostrobin; 50 % tebuconazole + 25 % trifloxystrobin; 62.5 g L-1 propiconazole + 37.5 g L-1 azoxystrobin; 80 % thiram; 80 % kükürt (sulphur); 80 % mancozeb; 80 % maneb) under in vitro and field conditions. Pure cultures of A. rabiei were isolated from infected chickpea plants collected in Bogazlayan, Sarikaya, Sorgun, Merkez and Yerköy. A total of 14 A. rabiei isolates and 4 references were evaluated. The field test was conducted at Yozgat Bozok University, Yerköy Agricultural Application and Research Center Station. The trials began on March 14, 2021. The experimental area was divided into plots and the susceptible chickpea variety Sari98 was used for the study. Two artificial inoculations were carried out approximately on the 40th and 80th days after sowing. Twenty-four hours after inoculation, the chickpea plants were sprayed with the fungicides Nativo® WG 75, Bellis®, Dikotan® M45 and Thiovit Jet® using a handheld sprayer. In vitro testing revealed that A. rabiei was resistant to kükürt (sulphur), thiram, maneb, and mancozeb. A field study showed that the percentage of A. rabiei isolates treated with the mancozeb fungicide was between 14 and 21 % of the control. Therefore, effective disease management strategies should include not only the use of fungicides, but also alternative approaches such as the use of resistant varieties. Moreover, the study focused on phenotypic resistance and suggests that future research should investigate the genetic and molecular mechanisms underlying A. rabiei resistance to enable better resistance management.

Fungicide resistance in Botrytis cinerea and identification of Botrytis species associated with blueberry in Michigan.

Botrytis blossom blight and fruit rot, caused by Botrytis cinerea, is a significant threat to blueberries, potentially resulting in substantial economic losses if not effectively managed. Despite the recommendation of various cultural and chemical practices to control this pathogen, there are widespread reports of fungicide resistance, leading to decreased efficacy. This study aimed to characterize the resistance profile of B. cinerea isolated from blighted blossoms and fruit in 2019, 2020 and 2022 (n = 131, 40, and 37 for the respective years). Eight fungicides (fludioxonil, thiabendazole, pyraclostrobin, boscalid, fluopyram, fenhexamid, iprodione, and cyprodinil) were tested using conidial germination at specific discriminatory doses. Additionally, 86 isolates were phylogenetically characterized using the internal transcribed spacer regions (ITS) and the protein coding genes: glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and RNA polymerase II second largest subunit (RPB2). This revealed higher fungicide resistance frequencies in 2020 and 2022 compared to 2019. Over all 3 years, over 80% of the isolates were sensitive to fludioxonil, fluopyram, and fenhexamid. Pyraclostrobin and boscalid showed the lowest sensitivity frequencies (<50%). While multi-fungicide resistance was observed in all the years, none of the isolates demonstrated simultaneous resistance to all tested fungicides. Botrytis cinerea was the most prevalent species among the isolates (74) with intraspecific diversity detected by the genes. Two isolates were found to be closely related to B. fabiopsis, B. galanthina, and B. caroliniana and 10 isolates appeared to be an undescribed species. This study reports the discovery of a potentially new species sympatric with B. cinerea on blueberries in Michigan.

CbCyp51-Mediated Demethylation Inhibitor Resistance Is Modulated by Codon Bias.

Cercospora leaf spot, caused by the fungus Cercospora beticola, is the most destructive foliar disease of sugarbeet worldwide. Resistance to the sterol demethylation inhibitor (DMI) fungicide tetraconazole has been previously correlated with synonymous and nonsynonymous mutations in CbCyp51. Here, we extend these analyses to the DMI fungicides prothioconazole, difenoconazole, and mefentrifluconazole in addition to tetraconazole to confirm whether the synonymous and nonsynonymous mutations at amino acid positions 144 and 170 are associated with resistance to these fungicides. Nearly half of the 593 isolates of C. beticola collected in the Red River Valley of North Dakota and Minnesota in 2021 were resistant to all four DMIs. Another 20% were resistant to tetraconazole and prothioconazole but sensitive to difenoconazole and mefentrifluconazole. A total of 13% of isolates were sensitive to all DMIs tested. We found five CbCyp51 haplotypes and associated them with phenotypes to the four DMIs. The most predominant haplotype (E170_A/L144F_C) correlated with resistance to all four DMIs with up to 97.6% accuracy. The second most common haplotype (E170_A/L144) consisted of isolates associated with resistance phenotypes to tetraconazole and prothioconazole while also exhibiting sensitive phenotypes to difenoconazole and mefentrifluconazole with up to 98.4% accuracy. Quantitative PCR did not identify differences in CbCyp51 expression between haplotypes. This study offers an understanding of the importance of codon usage in fungicide resistance and provides crop management acuity for fungicide application decision-making.

Fungicide resistance in Colletotrichum fructicola and Colletotrichum siamense causing peach anthracnose in China.

Peach is one of the popular and economically important fruit crops in China. Peach cultivation is hampered due to attacks of anthracnose disease, causing significant economic losses. Colletotrichum fructicola and Colletotrichum siamense belong to the Colletotrichum gloeosporioides species complex and are considered major pathogens of peach anthracnose. Application of different groups of fungicides is a routine approach for controlling this disease. However, fungicide resistance is a significant drawback in managing peach anthracnose nowadays. In this study, 39 isolates of C. fructicola and 41 isolates of C. siamense were collected from different locations in various provinces in China. The sensitivity of C. fructicola and C. siamense to some commonly used fungicides, i.e., carbendazim, iprodione, fluopyram, and propiconazole, was determined. All the isolates of C. fructicola collected from Guangdong province showed high resistance to carbendazim, whereas isolates collected from Guizhou province were sensitive. In C. siamense, isolates collected from Hebei province showed moderate resistance, while those from Shandong province were sensitive to carbendazim. On the other hand, all the isolates of C. fructicola and C. siamense showed high resistance to the dicarboximide (DCF) fungicide iprodione and succinate dehydrogenase inhibitor (SDHI) fungicide fluopyram. However, they are all sensitive to the demethylation inhibitor (DMI) fungicide propiconazole. Positive cross-resistance was observed between carbendazim and benomyl as they are members of the same methyl benzimidazole carbamate (MBC) group. While no correlation of sensitivity was observed between different groups of fungicides. No significant differences were found in each fitness parameter between carbendazim-resistant and sensitive isolates in both species. Molecular characterization of the ß-tubulin 2 (TUB2) gene revealed that in C. fructicola, the E198A point mutation was the determinant for the high resistance to carbendazim, while the F200Y point mutation was linked with the moderate resistance to carbendazim in C. siamense. Based on the results of this study, DMI fungicides, e.g., propiconazole or prochloraz could be used to control peach anthracnose, especially at locations where the pathogens have already developed the resistance to carbendazim and other fungicides.

Validation of Fungicide Spray Strategies and Selection for Fenhexamid Resistance in Botrytis cinerea on Greenhouse-Grown Grapevines.

In this study, in planta assays were conducted to assess the effects of fungicide spray tactics, such as the reduction of the labeled fungicide dose and mixture with a multisite fungicide, on fungicide resistance selection and disease control using Vitis vinifera 'Cabernet Sauvignon' grown in a greenhouse for 2 years. The entire clusters were inoculated with Botrytis cinerea isolates at varying frequencies of fenhexamid resistance, followed by fungicide sprays and disease and fenhexamid resistance investigations at critical phenological stages. Our findings indicate that the lower dose of the at-risk fungicide, fenhexamid, effectively managed fenhexamid resistance and disease as well as the higher, labeled dose. In addition, a mixture with the multisite fungicide captan generally resulted a net-positive effect on both resistance management and disease control.

Assessing the predictability of fungicide resistance evolution through in vitro selection.

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

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

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

Discovery and biological profile of pyridachlometyl.

Pyridachlometyl is a novel tubulin dynamics modulator fungicide developed by Sumitomo as a new agent designed to tackle fungicide resistance. Pyridachlometyl is being developed as a first-in-class molecule with an anti-tubulin mode of action, the chemical structure of which is characterized by a unique tetrasubstituted pyridazine ring. The first commercial product 'Fuseki flowable' received initial registration in 2023 in Japan. The concepts of the discovery project, optimization of chemical structures, and biological profiles are reviewed herein. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Machine Learning-Based Identification of Mating Type and Metalaxyl Response in Phytophthora infestans Using SSR Markers.

Phytophthora infestans is the causal agent of late blight in potato. The occurrence of P. infestans with both A1 and A2 mating types in the field may result in sexual reproduction and the generation of recombinant strains. Such strains with new combinations of traits can be highly aggressive, resistant to fungicides, and can make the disease difficult to control in the field. Metalaxyl-resistant isolates are now more prevalent in potato fields. Understanding the genetic structure and rapid identification of mating types and metalaxyl response of P. infestans in the field is a prerequisite for effective late blight disease monitoring and management. Molecular and phenotypic assays involving molecular and phenotypic markers such as mating types and metalaxyl response are typically conducted separately in the studies of the genotypic and phenotypic diversity of P. infestans. As a result, there is a pressing need to reduce the experimental workload and more efficiently assess the aggressiveness of different strains. We think that employing genetic markers to not only estimate genotypic diversity but also to identify the mating type and fungicide response using machine learning techniques can guide and speed up the decision-making process in late blight disease management, especially when the mating type and metalaxyl resistance data are not available. This technique can also be applied to determine these phenotypic traits for dead isolates. In this study, over 600 P. infestans isolates from different populations-Estonia, Pskov region, and Poland-were classified for mating types and metalaxyl response using machine learning techniques based on simple sequence repeat (SSR) markers. For both traits, random forest and the support vector machine demonstrated good accuracy of over 70%, compared to the decision tree and artificial neural network models whose accuracy was lower. There were also associations (p < 0.05) between the traits and some of the alleles detected, but machine learning prediction techniques based on multilocus SSR genotypes offered better prediction accuracy.

Apple Bitter Rot and Glomerella Leaf Spot: A Comprehensive Review of Causal Species and Their Biology, Fungicide Sensitivities, and Management Strategies.

Bitter rot and Glomerella leaf spot (GLS) are two distinct diseases of apple fruit and foliage caused by members of the ascomycete fungal genus Colletotrichum. While GLS is restricted to subtropical and in some areas to temperate climates, bitter rot is responsible for significant yield loss worldwide, particularly during the post-harvest period. Initially thought to be caused by just two species of Colletotrichum, C. acutatum and C. gloeosporioides, advances in molecular biology and sequencing techniques enabled the identification of 25 different species capable of causing bitter rot and/or GLS of apple belongs to the C. gloeosporioides species complex (CGSC), C. acutatum species complex (CASC) and C. boninense species complex (CBSC). Three species (C. gloeosporioides, C. fructicola, and C. chrysophilum) of CGSC cause both bitter rot and GLS, 18 species (6 of CGSC and 12 of CASC) only cause bitter rot, and four species (C. aenigma and C. asianum of CGSC, C. limetticola of CASC and C. karstii of CBSC) only cause GLS. These species were found to differ in their geographical distribution, environmental and host tissue preference, pathogenicity, and fungicide sensitivities. In this review, we summarize the distribution, life cycle, and pathogenicity mechanisms of all currently known Colletotrichum species responsible for bitter rot and GLS of apple. Furthermore, we describe known apple defense mechanisms and management strategies for the control of these economically significant pathogens and identify gaps in our present understanding for future research.

Transcriptome Analysis Reveals Potential Regulators of DMI Fungicide Resistance in the Citrus Postharvest Pathogen Penicillium digitatum.

Green mold, caused by Penicillium digitatum, is the major cause of citrus postharvest decay. Currently, the application of sterol demethylation inhibitor (DMI) fungicide is one of the main control measures to prevent green mold. However, the fungicide-resistance problem in the pathogen P. digitatum is growing. The regulatory mechanism of DMI fungicide resistance in P. digitatum is poorly understood. Here, we first performed transcriptomic analysis of the P. digitatum strain Pdw03 treated with imazalil (IMZ) for 2 and 12 h. A total of 1338 genes were up-regulated and 1635 were down-regulated under IMZ treatment for 2 h compared to control while 1700 were up-regulated and 1661 down-regulated under IMZ treatment for 12 h. The expression of about half of the genes in the ergosterol biosynthesis pathway was affected during IMZ stress. Further analysis identified that 84 of 320 transcription factors (TFs) were differentially expressed at both conditions, making them potential regulators in DMI resistance. To confirm their roles, three differentially expressed TFs were selected to generate disruption mutants using the CRISPR/Cas9 technology. The results showed that two of them had no response to IMZ stress while ∆PdflbC was more sensitive compared with the wild type. However, disruption of PdflbC did not affect the ergosterol content. The defect in IMZ sensitivity of ∆PdflbC was restored by genetic complementation of the mutant with a functional copy of PdflbC. Taken together, our results offer a rich source of information to identify novel regulators in DMI resistance.

Development and Implementation of a Novel CAPS Assay Reveals High Prevalence of a Boscalid Resistance Marker and Its Co-Occurrence with an Azole Resistance Marker in Erysiphe necator.

Succinate dehydrogenase inhibitors (SDHIs) are frequently used against powdery mildew (PM) fungi, such as Erysiphe necator, the causal agent of grapevine PM. Fungicide resistance, however, hinders effective control. DNA-based monitoring facilitates the recognition of resistance. We aimed (i) to adapt an effective method to detect a widespread genetic marker of resistance to boscalid, a commonly used SDHI, and (ii) to study the co-occurrence of the marker with a marker of resistance to demethylase inhibitor (DMI) fungicides. Sequencing of the sdhB gene identified a nonsynonymous substitution, denoted as sdhB-A794G, leading to an amino acid change (H242R) in the sdhB protein. In vitro fungicide resistance tests showed that E. necator isolates carrying sdhB-A794G were resistant to boscalid. We adopted a cleaved amplified polymorphic sequence-based method and screened more than 500 field samples collected from five Hungarian wine regions in two consecutive years. The sdhB-A794G marker was detected in all wine regions and in both years, altogether in 61.7% of samples, including 20.5% in which both sdhB-A794G and the wild-type were present. The frequency of sdhB-A794G was higher in SDHI-treated vineyards than in vineyards without any SDHI application. A significant difference in the presence of the marker was detected among wine regions; its prevalence ranged from none to 100%. We identified significant co-occurrence of sdhB-A794G with the CYP51-A495T (Y136F) mutation of the CYP51 gene, a known marker of resistance to DMIs. The monitoring of fungicide resistance is fundamental for the successful control of E. necator. Our rapid, cost-effective diagnostic method will support decision-making and fungicide resistance monitoring and management.