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.

Bioproducts of Pseudomonas chlororaphis suppress DMI fungicide-induced CsCYP51A and CsCYP51B gene expression in Colletotrichum siamense and generate synergistic effects with metconazole and propiconazole.

Mixtures of fungicides with different modes of action are commonly used as disease and resistance management tools, but little is known of mixtures of natural and synthetic products. In this study, mixtures of metabolites from the rhizobacterium Pseudomonas chlororaphis strain ASF009 formulated as Howler EVO with below label rates (50 µg/ml) of conventional sterol demethylation inhibitor (DMI) fungicides were investigated for control of anthracnose of cherry (Prunus avium) caused by Colletotrichum siamense. Howler mixed with metconazole or propiconazole synergistically reduced disease severity through lesion growth. Realtime PCR showed that difenoconazole, flutriafol, metconazole, and propiconazole induced the expression of DMI target genes CsCYP51A and CsCYP51B in C. siamense. The addition of Howler completely suppressed the DMI fungicide-induced expression of both CYP51 genes. We hypothesize that the downregulation of DMI fungicide-induced expression of the DMI target genes may, at least in part, explain the synergism observed in detached fruit assays.

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.

Pseudomonas chlororaphis metabolites reduce MfCYP51 expression and yield synergistic efficacy in mixture with reduced rates of propiconazole against DMI-resistant Monilinia fructicola isolates.

Brown rot caused by Monilinia fructicola is one of the most important diseases affecting peach production in the southeastern USA. Management often involves the use of demethylation inhibitor (DMI) fungicides, but efficacy can be compromised due to overexpression of the MfCYP51 gene encoding the 14α-demethylase of the ergosterol biosynthesis pathway. This study aimed to investigate the influence of the biorational fungicide Howler EVO containing Pseudomonas chlororaphis ASF009 metabolites, on the expression of MfCYP51 in M. fructicola and associated synergy with a DMI fungicide for control of DMI-resistant strains. Mycelia from two DMI-sensitive and three DMI-resistant M. fructicola isolates were exposed or not to propiconazole (0.3 µg/ml), Howler (78.5 µg/ml), or the combination propiconazole + Howler for 6 h prior to RNA extraction. Real-time PCR indicated that Howler reduced the constitutive expression of MfCYP51 in DMI sensitive and two of three DMI-resistant isolates. Propiconazole-induced expression of the DMI target gene was significantly reduced by Howler and by the mixture of Howler plus propiconazole in all isolates. Detached fruit studies on apple revealed that the combination of Howler plus a reduced label rate of Mentor (50 µg/ml propiconazole) was synergistic against brown rot caused by a DMI-resistant isolate in high and low inoculum spore concentration experiments (synergy values of 40.1 and 4.9, respectively). We hypothesize that the synergistic effects against M. fructicola resistant to DMI fungicides based on MfCYP51 gene overexpression can be attributed to reduced 14α demethylase production due to transcription inhibition, which may necessitate fewer DMI fungicide molecules to arrest fungal growth. The use of Howler /DMI mixtures for brown rot control warrants further investigation because such mixtures could potentially allow for reduced DMI fungicide use rates in the field without compromising yield or increased resistance selection.

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.

Melatonin targets MoIcl1 and works synergistically with fungicide isoprothiolane in rice blast control.

Melatonina natural harmless molecule-displays versatile roles in human health and crop disease control such as for rice blast. Rice blast, caused by the filamentous fungus Magnaporthe oryzae, is one devastating disease of rice. Application of fungicides is one of the major measures in the control of various crop diseases. However, fungicide resistance in the pathogen and relevant environmental pollution are becoming serious problems. By screening for possible synergistic combinations, here, we discovered an eco-friendly combination for rice blast control, melatonin, and the fungicide isoprothiolane. These compounds together exhibited significant synergistic inhibitory effects on vegetative growth, conidial germination, appressorium formation, penetration, and plant infection by M. oryzae. The combination of melatonin and isoprothiolane reduced the effective concentration of isoprothiolane by over 10-fold as well as residual levels of isoprothiolane. Transcriptomics and lipidomics revealed that melatonin and isoprothiolane synergistically interfered with lipid metabolism by regulating many common targets, including the predicted isocitrate lyase-encoding gene MoICL1. Furthermore, using different techniques, we show that melatonin and isoprothiolane interact with MoIcl1. This study demonstrates that melatonin and isoprothiolane function synergistically and can be used to reduce the dosage and residual level of isoprothiolane, potentially contributing to the environment-friendly and sustainable control of crop diseases.

Site-directed transformants with E407K substitution in Bcmdl1 possesses different fitness from field anilinopyrimidine resistant isolates with E407K mutation in Botrytis cinerea.

Botrytis cinerea is the causal agent of devastating disease gray mold on numerous crops worldwide. To control gray mold, anilinopyrimidine (AP) fungicides have been widely applied since the 1990s. However, the development of resistance in B. cinerea brought a new challenge to this disease control. Due to the unknown mode of action, the mechanism of AP resistance is still ambiguous. In our previous study, mutation E407K in Bcmdl1 was identified to be associated with AP resistance. Since this mutation is the major mechanism of AP resistance in our cases, it is essential to investigate the fitness of E407K strains before designing anti-resistance management strategies. Besides using field-resistant isolates with the E407K mutation, strains with E407K substitution obtained by site-directed mutagenesis were also used to estimate the specific effect of this mutation or substitution on fitness. The fitness of E407K strains were evaluated by determining mycelial growth, sporulation, conidial germination, virulence, acid production, osmotic and oxidative sensitivity, and sclerotial production and viability. Field resistant isolates with E407K mutation produced fewer sclerotia on intermediate medium (IM) but more conidia on PDA when compared with sensitive isolates, whereas site-directed transformants with E407K substitution did not show any fitness costs. The competitive ability of E407K strains was also evaluated on apple fruit using conidial mixtures at three initial ratios of resistant and sensitive isolates at 1:9, 1:1, and 9:1, respectively. Similar with fitness, impaired competitive ability was observed in field resistant isolates but not site-directed transformants at all initial ratios tested. These results indicated that field strains associated with AP resistance suffer a fitness penalty not linked directly to the E407K substitution in Bcmdl1.

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.

A secreted fungal effector suppresses rice immunity through host histone hypoacetylation.

Histone acetylation is a critical epigenetic modification that regulates plant immunity. Fungal pathogens secrete effectors that modulate host immunity and facilitate infection, but whether fungal pathogens have evolved effectors that directly target plant histone acetylation remains unknown. Here, we identified a secreted protein, UvSec117, from the rice false smut fungus, Ustilaginoidea virens, as a key effector that can target the rice histone deacetylase OsHDA701 and negatively regulates rice broad-spectrum resistance against rice pathogens. UvSec117 disrupts host immunity by recruiting OsHDA701 to the nucleus and enhancing OsHDA701-modulated deacetylation, thereby reducing histone H3K9 acetylation levels in rice plants and interfering with defense gene activation. Host-induced gene silencing of UvSec117 promotes rice resistance to U. virens, thus providing an alternative way for developing rice false smut-resistant plants. This is the first direct evidence demonstrating that a fungal effector targets a histone deacetylase to suppress plant immunity. Our data provided insight into a counter-defense mechanism in a plant pathogen that inactivates host defense responses at the epigenetic level.

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.

Risk and molecular mechanisms for boscalid resistance in Penicillium digitatum.

The succinate dehydrogenase inhibitor (SDHI) fungicide boscalid is an excellent broad-spectrum fungicide but has not been registered in China to control Penicillium digitatum, the causal agent of green mold of citrus. The present study evaluated the risk and molecular mechanisms for boscalid resistance in P. digitatum. Resistance induction with four arbitrarily selected sensitive isolates of P. digitatum by ultraviolet (UV) irradiation on conidia plated on boscalid-amended potato dextrose agar (PDA) and consecutive growing on boscalid-amended PDA produced five highly resistant isolates with EC50 values greater than 1000 µg/mL and two resistant isolates with EC50 lower than 200 µg/mL. Boscalid resistance of the five mutants with EC50 values above 1000 µg/mL was stable after successive transfers on PDA for 16 generations. However, for the other two mutants with EC50 lower than 200 µg/mL, the EC50 values decreased significantly after successive transfers. There was significant cross-resistance between boscalid and carboxin (r = 0.925, P < 0.001), but no significant cross-resistance was detected between boscalid and fludioxonil (r = 0.533，P = 0.095) or between boscalid and prochloraz (r = -0.543，P = 0.088). The seven resistant mutants varied greatly in the mycelia growth, sporulation, pathogenicity, and sensitivities to exogenous stresses including NaCl, salicylhydroxamic acid (SHAM), and H2O2. Alignment of the deduced amino acid sequence showed that there was no point mutation in the target enzyme succinate dehydrogenase (Sdh) subunits SdhA, SdhC, or SdhD in each of the seven resistant mutants, and the mutation of a conserved histidine residue to tyrosine (H243Y) in the subunit SdhB (i.e., iron­sulfur protein) occurred in only three highly resistant isolates. Molecular docking indicated that mutation H243Y could not prevent the binding of boscalid into the quinone-binding site of SDH in the presence of the heme moiety. However, for SDH without the heme moiety, boscalid could bind into a deeper site with a much higher affinity, and the mutation H243Y spatially blocked the docking of boscalid into the deeper site. This may be the molecular mechanism for boscalid resistance caused by SdhB-H243Y mutation.

Occurrence and Detection of Carbendazim Resistance in Botryosphaeria dothidea from Apple Orchards in China.

Botryosphaeria dothidea causes white rot, which is among the most devastating diseases affecting apple crops globally. In this study, we assessed B. dothidea resistance to carbendazim by collecting samples from warts on the infected branches of apple trees or from fruits exhibiting evidence of white rot. All samples were collected from different orchards in nine provinces of China in 2018 and 2019. In total, 440 B. dothidea isolates were evaluated, of which 19 isolates from three provinces were found to exhibit carbendazim resistance. We additionally explored the fitness and resistance stability of these isolates, revealing that they were no less fit than carbendazim-sensitive isolates in terms of pathogenicity, sporulation, and mycelial growth and that the observed carbendazim resistance was stable. Sequencing of the ß-tubulin gene in carbendazim-resistant isolates showed the presence of a substitution at codon 198 (GAG to GCG) that results in an alanine substitution in place of glutamic acid (E198A) in all 19 resistant isolates. A loop-mediated isothermal amplification (LAMP) method was then developed to rapidly and specifically identify this E198A mutation. This LAMP method offers value as a tool for rapidly detecting carbendazim-resistant isolates bearing this E198A mutation and can thus be used for the widespread monitoring of apple crops to detect and control the development of such resistance.

Identification of Monilia Species in Tibet and Characterization of M. yunnanensis in China.

Samples of peach and plum fruits with brown rot symptoms were collected from Tibet in 2019 and 2020, and the causal agent was identified as Monilia yunnanensis, which represents the first characterization of Monilia spp. on peach and plum in Tibet. Morphological investigation showed that some conidia from naturally diseased fruits were larger than those observed in previously isolated M. yunnanensis. Some conidia of M. yunnanensis isolates from Tibet produced more than two, even up to six germ tubes from different parts of each conidium, instead of one or two germ tubes developing from the pointy sides of each conidium. The alignment of ribosomal internal transcribed spacer region sequences revealed that some isolates from Tibet displayed a mutation at the 374th position from adenine (A) to cytosine (C). Although abovementioned differences were observed between isolates from Tibet and other regions, phylogenetic analysis indicated that all of the M. yunnanensis isolates from different stone fruits and different regions in China were clustered together without obvious genetic differentiation. These results revealed that hosts and geographic environments did not play a major role in the evolution of M. yunnanensis.

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.

First Report of Anthracnose Caused by Colletotrichum jinshuiense on Goldthread in China.

Goldthread (Coptis chinensis Franch) is one of the most widely used Chinese traditional medicine plants with remarkable medicinal properties (Mizrahi et al. 2014). In July 2019, a new anthracnose-like leaf spot disease was observed in Banqiao Town, Enshi, Hubei, China. The incidence rate ranged from 10% to 20%. Infected leaves firstly showed oil-like dots, further gradually expanded to irregular whorls with a pale center and dark-brown edge. Petiole infection led to leaves dropping when severe occurrence. Black acervuli were developed on the infected leaves with abundant setae, especially near veins. To identify the causal agent, 4-mm2 tissues were derived from the disease-health junction and surface-disinfected with 0.1% mercury dichloride for 1 min and 75% ethanol for 30 s respectively. They were placed on a PDA plate and incubated at 25°C after being rinsed with sterile water three times. Isolates were purified by single spore isolation. Colonies on PDA were white to pale-gray with dense aerial mycelia, and the underside was yellowish to olive. Colonies grow 77.5 to 81.5 mm in 1 week. No conidia were observed during vegetable growth, but conidiomatal acervuli were found on infected leaves. Setae were 1-3 septate, dark-brown, 78.0 to 134.5 µm (mean = 108 ± 23.4) long, 4.1 to 9.1 µm (mean = 6.1 ± 1.1) diameter, cylindrical to conical, apices acute. Conidiophores hyaline to pale brown, septate. Conidia were hyaline, unicellular, aseptate, curved, cylindrical, often guttulate, measuring 20.1 to 28.0 × 3.5 to 5.4 µm (mean = 25.4 ± 1.7 × 4.5 ± 0.5 µm), L/W ratio = 5.6. Hyphae septate branched, hyaline to pale brown, 1.6 to 4.5 in diameter. Hyphopodial appressoria pale to medium brown, smooth-walled, globose or obovoid, 6.3 to 9.9 × 4.1 to 7.6 µm (mean = 8.3 ± 0.9 × 7.6 ± 0.7 µm), L/W ratio = 1.1. Morphological features were similar to the description of C. jinshuiense (Fu et al. 2019). To identify its phylogenetic position, maximum-likelihood (ML) analyses of two isolates (Esh8 and Esh 11) were implemented with a concatenation of multiple sequences of the internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), beta-tubulin (TUB2), and chitin-synthase 1 (CHS-1) using MEGA 7. The sequences were amplified using primers ITS1/ITS4, GDF1/GDR1, ACT-512F/ACT-783R, T1/Bt2b, CHS-79F/CHS-354R (Weir et al. 2012) and deposited in GenBank with accession numbers MW440484 - MW440485 (ITS), MW676256 - MW676257 (GAPDH), MW676252 - MW676253 (ACT), MW676254 - MW676255 (TUB2) and MW676258 - MW676259 (CHS-1). Results indicated they were clustered with C. jinshuiense in the C. dematium species complex. Isolates were inoculated onto injured healthy leaves (20 leaves) with mycelial plugs, ten leaves being inoculated with blank plugs were used as control. Disease symptoms were consistent with those observed in the field after five days post-inoculation with a 100% incidence rate, while no symptom was observed on the control leaves. And same isolates were isolated from six inoculated leaves with 100% re-isolation frequency. These results fulfilled Koch's postulates. In a previous study, C. boninense was identified as the causal agent of goldthread anthracnose in Chongqing, China (Ding et al. 2020). To our knowledge, this study is the first report of anthracnose on goldthread caused by C. jinshuiense in China.

MoWhi2 Mediates Mitophagy to Regulate Conidiation and Pathogenesis in Magnaporthe oryzae.

Mitophagy refers to the specific process of degrading mitochondria, which is an important physiological process to maintain the balance of mitochondrial quantity and quality in cells. At present, the mechanisms of mitophagy in pathogenic fungi remain unclear. Magnaporthe oryzae (Syn. Pyricularia oryzae), the causal agent of rice blast disease, is responsible for the most serious disease of rice. In M. oryzae, mitophagy occurs in the foot cells and invasive hyphae to promote conidiation and infection. In this study, fluorescent observations and immunoblot analyses showed that general stress response protein MoWhi2 is required for mitophagy in M. oryzae. In addition, the activation of the autophagy, pexophagy and cytoplasm-to-vacuole targeting (CVT) pathway upon nitrogen starvation was determined using the GFP-MoATG8, GFP-SRL and MoAPE1-GFP strains and the ΔMowhi2 mutant in these backgrounds. The results indicated that MoWhi2 is specifically required for mitophagy in M. oryzae. Further studies showed that mitophagy in the foot cells and invasive hyphae of the ΔMowhi2 was interrupted, leading to reduced conidiation and virulence in the ΔMowhi2 mutant. Taken together, we found that MoWhi2 contributes to conidiation and invasive growth by regulating mitophagy in M. oryzae.