The velvet family proteins mediate low resistance to isoprothiolane in Magnaporthe oryzae.

Isoprothiolane (IPT) resistance has emerged in Magnaporthe oryzae, due to the long-term usage of IPT to control rice blast in China, yet the mechanisms of the resistance remain largely unknown. Through IPT adaptation on PDA medium, we obtained a variety of IPT-resistant mutants. Based on their EC50 values to IPT, the resistant mutants were mainly divided into three distinct categories, i.e., low resistance (LR, 6.5 ≤ EC50 < 13.0 µg/mL), moderate resistance 1 (MR-1, 13.0 ≤ EC50 < 25.0 µg/mL), and moderate resistance 2 (MR-2, 25.0 ≤ EC50 < 35.0 µg/mL). Molecular analysis of MoIRR (Magnaporthe oryzae isoprothiolane resistance related) gene demonstrated that it was associated only with the moderate resistance in MR-2 mutants, indicating that other mechanisms were associated with resistance in LR and MR-1 mutants. In this study, we mainly focused on the characterization of low resistance to IPT in M. oryzae. Mycelial growth and conidial germination were significantly reduced, indicating fitness penalties in LR mutants. Based on the differences of whole genome sequences between parental isolate and LR mutants, we identified a conserved MoVelB gene, encoding the velvet family transcription factor, and genetic transformation of wild type isolate verified that MoVelB gene was associated with the low resistance. Based on molecular analysis, we further demonstrated that the velvet family proteins VelB and VeA were indispensable for IPT toxicity and the deformation of the VelB-VeA-LaeA complex played a vital role for the low IPT-resistance in M. oryzae, most likely through the down-regulation of the secondary metabolism-related genes or CYP450 genes to reduce the toxicity of IPT.

Transcriptional Activator UvXlnR Is Required for Conidiation and Pathogenicity of Rice False Smut Fungus Ustilaginoidea virens.

Transcription factors play critical roles in diverse biological processes in fungi. XlnR, identified as a transcriptional activator that regulates the expression of the extracellular xylanase genes in fungi, has not been extensively studied for its function in fungal development and pathogenicity in rice false smut fungus Ustilaginoidea virens. In this study, we characterized UvXlnR in U. virens and established that the full-length, N-terminal, and C-terminal forms have the ability to activate transcription. The study further demonstrated that UvXlnR plays crucial roles in various aspects of U. virens biology. Deletion of UvXlnR affected growth, conidiation, and stress response. UvXlnR mutants also exhibited reduced pathogenicity, which could be partially attributed to the reduced expression of xylanolytic genes and extracellular xylanase activity of U. virens during the infection process. Our results indicate that UvXlnR is involved in regulating growth, conidiation, stress response, and pathogenicity.

Phytophthora sojae effector Avr1d functions as an E2 competitor and inhibits ubiquitination activity of GmPUB13 to facilitate infection.

Oomycete pathogens such as Phytophthora secrete a repertoire of effectors into host cells to manipulate host immunity and benefit infection. In this study, we found that an RxLR effector, Avr1d, promoted Phytophthora sojae infection in soybean hairy roots. Using a yeast two-hybrid screen, we identified the soybean E3 ubiquitin ligase GmPUB13 as a host target for Avr1d. By coimmunoprecipitation (Co-IP), gel infiltration, and isothermal titration calorimetry (ITC) assays, we confirmed that Avr1d interacts with GmPUB13 both in vivo and in vitro. Furthermore, we found that Avr1d inhibits the E3 ligase activity of GmPUB13. The crystal structure Avr1d in complex with GmPUB13 was solved and revealed that Avr1d occupies the binding site for E2 ubiquitin conjugating enzyme on GmPUB13. In line with this, Avr1d competed with E2 ubiquitin conjugating enzymes for GmPUB13 binding in vitro, thereby decreasing the E3 ligase activity of GmPUB13. Meanwhile, we found that inactivation of the ubiquitin ligase activity of GmPUB13 stabilized GmPUB13 by blocking GmPUB13 degradation. Silencing of GmPUB13 in soybean hairy roots decreased P. sojae infection, suggesting that GmPUB13 acts as a susceptibility factor. Altogether, this study highlights a virulence mechanism of Phytophthora effectors, by which Avr1d competes with E2 for GmPUB13 binding to repress the GmPUB13 E3 ligase activity and thereby stabilizing the susceptibility factor GmPUB13 to facilitate Phytophthora infection. This study unravels the structural basis for modulation of host targets by Phytophthora effectors and will be instrumental for boosting plant resistance breeding.

The Identification, Characterization, and Functional Analysis of the Sugar Transporter Gene Family of the Rice False Smut Pathogen, Villosiclava virens.

False smut, caused by Villosiclava virens, is becoming increasingly serious in modern rice production systems, leading to yield losses and quality declines. Successful infection requires efficient acquisition of sucrose, abundant in rice panicles, as well as other sugars. Sugar transporters (STPs) may play an important role in this process. STPs belong to a major facilitator superfamily, which consists of large multigenic families necessary to partition sugars between fungal pathogens and their hosts. This study identified and characterized the STP family of V. viren, and further analyzed their gene functions to uncover their roles in interactions with rice. Through genome-wide and systematic bioinformatics analyses, 35 STPs were identified from V.virens and named from VvSTP1 to VvSTP35. Transmembrane domains, gene structures, and conserved motifs of VvSTPs have been identified and characterized through the bioinformatic analysis. In addition, a phylogenetic analysis revealed relationship between VvSTPs and STPs from the other three reference fungi. According to a qRT-PCR and RNA-sequencing analysis, VvSTP expression responded differently to different sole carbon sources and H2O2 treatments, and changed during the pathogenic process, suggesting that these proteins are involved in interactions with rice and potentially functional in pathogenesis. In total, 12 representative VvSTPs were knocked out through genetic recombination in order to analyze their roles in pathogenicity of V. virens. The knock-out mutants of VvSTPs showed little difference in mycelia growth and conidiation, indicating a single gene in this family cannot influence vegetative growth of V. virens. It is clear, however, that these mutants result in a change in infection efficiency in a different way, indicating that VvSTPs play an important role in the pathogenicity of virens. This study is expected to contribute to a better understanding of how host-derived sugars contribute to V. virens pathogenicity.

A secreted fungal subtilase interferes with rice immunity via degradation of SUPPRESSOR OF G2 ALLELE OF skp1.

Serine protease subtilase, found widely in both eukaryotes and prokaryotes, participates in various biological processes. However, how fungal subtilase regulates plant immunity is a major concern. Here, we identified a secreted fungal subtilase, UvPr1a, from the rice false smut (RFS) fungus Ustilaginoidea virens. We characterized UvPr1a as a virulence effector localized to the plant cytoplasm that inhibits plant cell death induced by Bax. Heterologous expression of UvPr1a in rice (Oryza sativa) enhanced plant susceptibility to rice pathogens. UvPr1a interacted with the important rice protein SUPPRESSOR OF G2 ALLELE OF skp1 (OsSGT1), a positive regulator of innate immunity against multiple rice pathogens, degrading OsSGT1 in a protease activity-dependent manner. Furthermore, host-induced gene silencing of UvPr1a compromised disease resistance of rice plants. Our work reveals a previously uncharacterized fungal virulence strategy in which a fungal pathogen secretes a subtilase to interfere with rice immunity through degradation of OsSGT1, thereby promoting infection. These genetic resources provide tools for introducing RFS resistance and further our understanding of plant-pathogen interactions.

First Report of Brown Rot Caused by Monilia yunnanensis on Sweet Cherry in China.

Chinese cherry industry has developed rapidly over the past few years, with the planting acreage continuously expanding, from Shandong province to Liaoning, Shaanxi, Hebei, Sichuan etc. Monilia spp. are the most important causal agents of brown rot of cherry, to date, M. fructicola, M. mumecola, and M. fructigena were reported to cause brown rot of cherry in China (Chen et al. 2013; Yin et al. 2014; Liu et al. 2012). In May 2023, fruit of sweet cherry cultivar 'Hongdeng' (Prunus avium L.) with symptoms resembling brown rot were collected from Tongchuan City, Shaanxi Province. Conidia on diseased tissues were spread on a water agar (WA, 1.5% agar and distilled water) medium and isolated with a glass needle under a professional single spore separation microscope (Wuhan Heipu Science and Technology Ltd., Wuhan, China). If no conidia were present, fruit pieces (5 × 5 mm) at the intersection of healthy and diseased tissues were surface sterilized with a sodium hypochlorite solution (1%) for 30 s and washed three times in sterilized water, followed by 75% ethanol for 30 s, then washed three times in sterilized water. After the tissue pieces were dried, they were placed on potato dextrose agar (PDA; 200 g of potato, 20 g of dextrose, and agar at 20 g/L) and incubated at 22 °C for about twenty days to produce spores and then single spore isolation was carried out. Thirty single-spore isolates were obtained and all were morphologically similar. The isolates produced white-gray colonies with even margins and concentric rings of sporogenous mycelium after 3 days incubation, and abundant black-colored stromata on the PDA medium after 15 days of incubation at 22°C. Conidia were one-celled, hyaline, ellipsoid to lemon shape (14.12 × 10.37 µm), with 1-2 germs which is similar to M. yunnanensis on peach. The genomic DNA of the isolates was extracted as described previously (Chi et al. 2009). The pathogen identity was confirmed by multiplex PCR which resulted in a 237bp amplicon, which is diagnostic of M. yunnanensis (Hu et al. 2011). Further sequencing of the internal transcribed spacer (ITS) region 1 and 2 and 5.8S gene (accession number: OR192774) indicated 100% identity with that of M. yunnanensis isolates (accession numbers: MW355895, ON024742). The average daily growth of mycelium on PDA at 22°C was 11.44 mm. Koch's postulates were fulfilled by inoculating 20 mature sweet cherry fruits of cv. 'Van' with mycelial plugs in a drilled hole. After 3 days of incubation at 22℃ in an airtight plastic tray with wet paper, the inoculated fruit developed typical brown rot symptoms. The developing spores on inoculated fruit were confirmed to be M. yunnanensis based on ITS sequence. All control fruit inoculated with a PDA plug remained healthy. M. yunnanensis was first reported as the causal agent of brown rot of peach in China (Hu et al. 2011). Later studies demonstrated that it is also pathogen on other fruits, e.g. hawthorn (Zhao et al. 2013), plum (Yin et al. 2015), apricot (Yin et al. 2017), apple, and pear in China (Zhu et al. 2016). To our knowledge, this is the first report of cherry brown fruit rot caused by M. yunnanensis, indicating the high risk of this species to cherry production, and effective strategies must be taken to prevent the possible control failure in practice.

Gap-Free Nuclear and Mitochondrial Genomes of Ustilaginoidea virens JS60-2, a Fungal Pathogen Causing Rice False Smut.

Rice false smut (RFS), caused by Ustilaginoidea virens, has become a major disease in recent years, and mycotoxins produced by U. virens often threaten food safety. To study fungal pathogenesis and identify potential targets for developing new fungicides, gap-free nuclear and complete mitochondrial genomes of U. virens JS60-2 were sequenced and assembled. Using the second and third generation sequencing data, we assembled a 38.02-Mb genome that consists of seven contigs with the contig N50 being 6.32-Mb. In total, 8,486 protein-coding genes were annotated in the genome, including 21 secondary metabolism gene clusters. We also assembled the complete mitochondrial genome, which is 102,498 bp, with 28% GC content. The JS60-2 genomes assembled in this study will facilitate research on U. virens and contribute to RFS control. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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

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

Identification, Genetic Diversity, and Chemical Control of Xanthomonas arboricola pv. pruni in China.

Peach bacterial spot caused by Xanthomonas arboricola pv. pruni has become widespread in most peach-producing areas of China and has caused devastating losses to the peach industry. However, little is known about the population biology and epidemiology of X. arboricola pv. pruni in China, thus no effective management strategy is available. Altogether, 321 symptomatic samples of peach bacterial spot from 12 provinces in China were collected from which 612 bacterial isolates were obtained. Based on 16S rDNA sequence comparison in GenBank, the obtained isolates were identified as Pantoea spp. (514) and Xanthomonas spp. (98). The pathogenicity test demonstrated that the causal agent of the peach bacterial spot was the Xanthomonas spp. instead of the Pantoea spp. Based on morphological observation, physiological and biochemical characterization, and molecular identification, the Xanthomonas spp. were further identified to be X. arboricola pv. pruni. Then, 41 X. arboricola pv. pruni isolates representing different populations were selected and analyzed with repetitive element sequence based-PCR and intersimple sequence repeat markers to understand the genetic diversity and population structure along with four X. arboricola pv. pruni isolates from plum and three isolates of X. arboricola pv. juglandis as comparison. A total of 98 polymorphic alleles were identified, with a mean value of percentage of polymorphic loci of 14. Genetic diversity and phylogenetic analysis revealed the profound heterogeneity between X. arboricola pv. juglandis and X. arboricola pv. pruni, moderate genetic differentiation within X. arboricola pv. pruni, and obvious host specificity but weak geographical differentiation in X. arboricola population. Finally, the efficiency of bactericides on X. arboricola pv. pruni was evaluated in vitro and in vivo. The parallel repeated field trials in two orchards demonstrated that 80% Mancozeb (1:800) and 47% Kocide (1:800, 1:1,500, and 1:2,000) had excellent control efficacies for X. arboricola pv. pruni, especially as the control efficacy of Kocide could even reach 90%. This study conducted a systematic investigation for the occurrence, population variance, and chemical control of X. arboricola pv. pruni. It improved the understanding of the pathogen populations of peach bacterial spot in China and provided solid theoretical and practical guidance for X. arboricola pv. pruni control.

Sensitivity of Colletotrichum nymphaeae to Six Fungicides and Characterization of Fludioxonil-Resistant Isolates in China.

Colletotrichum nymphaeae is the dominant species causing anthracnose disease of peach in China. In this study, 140 isolates of C. nymphaeae were assessed for their sensitivity to six fungicides. It was found that C. nymphaeae was highly resistant to carbendazim, procymidone, and boscalid but sensitive to pyraclostrobin and prochloraz. For fludioxonil, the fungus exhibited differential sensitivities (i.e., approximately 14% of isolates were resistant to fludioxonil and the resistance was stable). Fludioxonil-resistant isolates had a mean EC50 value of 2.2380 µg/ml, whereas the mean EC50 value was 0.0194 µg/ml in fludioxonil-sensitive isolates. The mean EC50 values of C. nymphaeae for pyraclostrobin and prochloraz were 0.0083 µg/ml and 0.002 µg/ml, respectively. No cross-resistance was observed between fungicides from different groups. Mycelial growth rate, control efficacy, and osmotic stress responses were significantly different (P < 0.05) between fludioxonil-sensitive (FluS) and -resistant (FluR) isolates, but no significant difference was observed (P > 0.05) in virulence and sporulation between FluS and FluR isolates. No mutation was detected in coding regions of the CnOs-1, Cal, Hk1, Hog1, TPI, and Mrr1 genes. Interestingly, with fludioxonil treatment, the expression of ABC transporter gene atrB was significantly overexpressed in some resistant isolates. However, overexpression of the atrB gene was not detected in one moderately and one highly resistant isolate, indicating that other unknown mechanisms may be involved. Current findings uncovered several effective chemicals and provided the foundation for designing management strategies to practically control peach anthracnose with the most effective demethylation inhibitor fungicides and quinone outside inhibitor fungicides.

Cytological Observation of the Infectious Process of Venturia carpophila on Peach Leaves.

Peach scab caused by Venturia carpophila is one of the most destructive fungal diseases of peach worldwide, and it seriously affects peach production. Until now,the infectious process and pathogenesis of V. carpophila on peach have remained unclear. Here we present the infection behavior of V. carpophila at the ultrastructural and cytological levels in peach leaves with combined microscopic investigations (i.e., light microscopy, confocal laser scanning microscopy, scanning electron microscopy, and transmission electron microscopy). V. carpophila germinated at the tip of conidia and produced short germ tubes on peach leaf surfaces at 2 days post inoculation (dpi). At 3 dpi, swollen tips of germ tubes differentiated into appressoria. At 5 dpi, penetration pegs produced by appressoria broke through the cuticle layer and then differentiated into thick subcuticular hyphae in the pectin layer of the epidermal cell walls. At 10 dpi, the subcuticular hyphae extensively colonized in the pectin layer. The primary hyphae ramified into secondary hyphae and proliferated along with the incubation. At 15 dpi, the subcuticular hyphae divided laterally to form stromata between the cuticle layer and the cellulose layer of the epidermal cells. At 30 dpi, conidiophores developed from the subcuticular stromata. Finally, abundant conidiophores and new conidia appeared on leaf surfaces at 40 dpi. These results provide useful information for further a understanding of V. carpophila pathogenesis.

Whole-Genome Sequence of Diaporthe citri Isolate NFHF-8-4, the Causal Agent of Citrus Melanose.

Diaporthe species are the causal agents of melanose, stem-end rot, and gummosis diseases of citrus. D. citri is the predominant species on different citrus varieties. These diseases exceedingly reduce quality and marketability of fresh fruits. Melanose on fruits especially causes massive economic losses. The infection mechanisms of D. citri are still unclear and the genome sequence of D. citri has not been released. In order to systemically explore the interaction between citrus and D. citri, we sequenced the whole-genome of D. citri NFHF-8-4, which was isolated from a sample with melanose in Jiangxi Province. The NFHF-8-4 genome sequence will provide valuable information for studying the development process, infection process, and resistance to fungicides mechanisms in D. citri.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome Sequence of Venturia carpophila, the Causal Agent of Peach Scab.

Venturia carpophila, the causal agent of scab disease on peach, is a host-specific fungus that is widely distributed around the world, including China. In our previous study, samples were collected from 14 provinces in China, and 750 isolates were obtained by single-spore separation. Here, we reported the first highly contiguous whole-genome sequence (35.87 Mb) of the V. carpophila isolate ZJHZ1-1-1, which included 33 contigs with N50 value of 2.01 Mb and maximum contig length of 3.39 Mb. The high-quality genome sequence and annotation resource will be useful to study the fungal biology, pathogen-host interaction, fungicide resistance, characterization of important genes, population genetic diversity, and development of molecular markers for genotyping and species identification.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

First Report of Atypical Scab Caused by Venturia asperata on Apple in China.

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.

Effect of Chemical Seed Treatment on Rice False Smut Control in Field.

Rice false smut, caused by the pathogen Ustilaginoidea virens, is a severe emerging disease in China. It affects not only the quality of rice but also yields of rice production. To make clear the effect of chemical seed treatment on the rice false smut control in fields, during 2014 to 2017, four fungicides with different modes of action were used to treat rice seeds contaminated by false smut balls. In rice-growing seasons, samples of rice tissues were taken for detection of U. virens by using a specific nested PCR method at different rice-growing stages. In addition, the occurrence of rice false smut was investigated at maturation stage. Results showed that U. virens in plant tissues decreased significantly at the seedling stage upon chemical seed treatment. Four chemical treatments decreased the detection rate significantly (P < 0.01) compared with the water treatment, but no significant difference was observed among four chemical treatments. However, the detection rate did not decease significantly at the tillering and booting stages. Similarly, the final occurrence of rice false smut did not show significant difference between each chemical and water treatment. These results suggested that chemical seed treatment had only limited efficacy in preventing occurrence of rice false smut; application of fungicides at the booting stage or integrated use of fungicides and agricultural practices might give a better control for this disease.

Sensitivity of Colletotrichum fructicola and Colletotrichum siamense of Peach in China to Multiple Classes of Fungicides and Characterization of Pyraclostrobin-Resistant Isolates.

Anthracnose, mainly caused by Colletotrichum gloeosporioides species complex including Colletotrichum fructicola and Colletotrichum siamense, is a devastating disease of peach. Chemical control has been widely used for years, but management failures have increased with the commonly used fungicides. Therefore, screening of sensitivity of Colletotrichum spp. to fungicides with different modes of action is needed to make proper management strategies for peach anthracnose. In this study, the sensitivity of 80 isolates of C. fructicola and C. siamense was screened for pyraclostrobin, procymidone, prochloraz, and fludioxonil based on mycelial growth inhibition at discriminatory doses. Results showed that C. fructicola and C. siamense isolates were highly resistant to procymidone and fludioxonil with 100% resistance frequencies to both fungicides, but sensitive to prochloraz, i.e., no resistant isolates were found. For pyraclostrobin, 74% of C. fructicola isolates showed high resistance, 26% showed low resistance, and all of the C. siamense isolates showed low resistance. No positive cross-resistance was observed between pyraclostrobin and azoxystrobin even when they are members of the same quinone outside inhibitor (QoI) fungicide group or between pyraclostrobin and non-QoIs. Resistant isolates to QoI fungicides were evaluated for the fitness penalty. Results showed that no significant differences except for the mycelial growth rates that were detected between high- and low-resistance isolates of C. fructicola. Molecular characterization of the Cyt b gene revealed that the G143A point mutation was the determinant of the high resistance in C. fructicola. This study demonstrated the resistance status of C. fructicola and C. siamense to different fungicides and briefly discussed implications of that resistance. Demethylation inhibitor fungicides were found to be the best option among the different chemicals studied here, to control peach anthracnose in China.

Sensitivity of Venturia carpophila from China to Five Fungicides and Characterization of Carbendazim-Resistant Isolates.

Peach scab is a fungal disease caused by Venturia carpophila that can significantly reduce peach yield and quality. Fungicide application is the main control measure for peach scab worldwide. To better understand the fungicide-resistance status and devise suitable management strategies, the sensitivity of 135 single-spore V. carpophila isolates to the commonly used fungicides carbendazim, iprodione, propiconazole, azoxystrobin, and boscalid were determined using a microtiter plate test method. Results showed that the mean effective concentrations to cause inhibitions by 50% (EC50) of tested isolates to iprodione, propiconazole, azoxystrobin, and boscalid were 16.287, 0.165, 0.570, and 0.136 µg/ml, respectively. The EC50 values of V. carpophila isolates to four fungicides displayed unimodal frequency distributions, indicating no resistance occurred to these fungicides. On the contrary, bimodal frequency distribution was observed for carbendazim, indicating that V. carpophila developed resistance to carbendazim. Resistance was widely detected from all 14 provinces studied. Molecular analysis showed that the point mutation E198K of the TUB2 gene determined high resistance, whereas E198G conferred moderate resistance. Moderate and high resistances were stable, and the resistant isolates did not show significant fitness penalties. On the contrary, some resistant isolates showed better competitiveness under certain stresses. This is the first report to detect the sensitivity of V. carpophila to fungicides, which enables future monitoring of fungicide resistance and provides basic information to allow the design of suitable peach scab management strategies.

The Bax inhibitor UvBI-1, a negative regulator of mycelial growth and conidiation, mediates stress response and is critical for pathogenicity of the rice false smut fungus Ustilaginoidea virens.

Bax inhibitor-1 (BI-1), an evolutionarily conserved protein, is a suppressor of cell death induced by the proapoptotic protein Bax and is involved in the response to biotic and abiotic stress in animals, plants and yeast. Rice false smut caused by Ustilaginoidea virens is one of the destructive rice diseases worldwide. Although BI-1 proteins are widely distributed across filamentous fungi, few of them are functionally characterized. In this study, we identified a BI-1 protein in U. virens, UvBI-1, which contains a predicted Bax inhibitor-1-like family domain and could suppress the cell death induced by Bax. By co-transformation of the CRISPR/Cas9 construct along with donor DNA fragment containing the hygromycin resistance gene, we successfully generated Uvbi-1 deletion mutants. The UvBI-1 deletion showed an increase in mycelia vegetative growth and conidiation, suggesting this gene acts as a negative regulator of the growth and conidiation. In addition, the Uvbi-1 mutants exhibited higher sensitivity to osmotic and salt stress, hydrogen peroxide stress, and cell wall or membrane stress than the wild-type strain. Furthermore, UvBI-1 deletion was found to cause increased production of secondary metabolites and loss of pathogenicity of U. virens. Taken together, our results demonstrate that UvBI-1 plays a negative role in mycelial growth and conidiation, and is critical for stress tolerance, cell wall integrity, secondary metabolites production and pathogenicity of U. virens. Therefore, this study provides new evidence on the conserved function of BI-1 among fungal organisms and other species.

Natural allelic variations provide insights into host adaptation of Phytophthora avirulence effector PsAvr3c.

Filamentous pathogens, such as fungi and oomycetes, secrete avirulence (AVR) effectors that trigger plant immune responses and provide striking examples of host adaptations. Avr effector genes display different types of allelic variations, including deletions, epigenetic silencing and sequence polymorphisms, to avoid detection. However, how effector sequence polymorphisms enable pathogens to dodge host immune surveillance remains largely unknown. PsAvr3c is a Phytophthora AVR gene that is recognized by soybean carrying Rps3c. PsAvr3c natural alleles display a rich diversity of single nucleotide polymorphisms in field isolates. We combined both site-directed mutagenesis and population sequence surveys to identify a serine substitution of glycine at position 174 in PsAvr3c that resulted in evasion of Rps3c-mediated soybean immunity. The S174G substitution did not affect the nuclear localization of PsAvr3c in planta, which is required to activate Rps3c, but it significantly impaired the binding affinity of PsAvr3c with a previously identified spliceosome-associated protein GmSKRPs. Silencing GmSKRPs specifically impaired PsAvr3c-triggered cell death in Rps3c soybean. This study uncovered a plant Phytophthora pathogen that adapted to a resistant plant through a key amino acid mutation and subsequently reduced the binding affinity with a plant immune regulator to evade host resistance.

Genome-wide identification and analysis of the basic leucine zipper (bZIP) transcription factor gene family in Ustilaginoidea virens.

The basic leucine zipper (bZIP) transcription factor (TF) family is one of the largest and most diverse TF families widely distributed across the eukaryotes. The bZIP TF family plays an important role in growth, development, and response to abiotic or biotic stresses, which have been well characterized in plants, but not in plant pathogenic fungi. In this study, we performed genome-wide and systematic bioinformatics analysis of bZIP genes in the fungus Ustilaginoidea virens, the causal agent of rice false smut disease. We identified 28 bZIP family members in the U. virens genome by searching for the bZIP domain in predicted genes. The gene structures, motifs, and phylogenetic relationships were analyzed for bZIP genes in U. virens (UvbZIP). Together with bZIP proteins from two other fungi, the bZIP genes can be divided into eight groups according to their phylogenetic relationships. Based on RNA-Seq data, the expression profiles of UvbZIP genes at different infection stages were evaluated. Results showed that 17 UvbZIP genes were up-regulated during the infection period. Furthermore, 11 infection-related UvbZIP genes were investigated under H2O2 stress and the expression level of eight genes were changed, which confirmed their role in stress tolerance and pathogenicity. In summary, our genome-wide systematic characterization and expression analysis of UvbZIP genes provided insight into the molecular function of these genes in U. virens and provides a reference for other pathogens.