Search

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.

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.

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 β-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.

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.

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.

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.

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.

Ustilaginoidea virens, causing rice false smut (RFS) is an economically important ascomycetous fungal pathogen distributed in rice-growing regions worldwide. Here, we identified a novel transcription factor UvCGBP1 (Cutinase G-box binding protein) from this fungus, which is unique to ascomycetes. Deletion of UvCGBP1 affected development and virulence of U. virens. A total of 865 downstream target genes of UvCGBP1 was identified using ChIP-seq and the most significant KEGG enriched functional pathway was the MAPK signaling pathway. Approximately 36% of target genes contain the AGGGG (G-box) motif in their promoter. Among the targets, deletion of UvCGBP1 affected transcriptional and translational levels of UvPmk1 and UvSlt2, both of which were important in virulence. ChIP-qPCR, yeast one-hybrid and EMSA confirmed that UvCGBP1 can bind the promoter of UvPmk1 or UvSlt2. Overexpression of UvPmk1 in the ∆UvCGBP1-33 mutant restored partially its virulence and hyphae growth, indicating that UvCGBP1 could function via the MAPK pathway to regulate fungal virulence. Taken together, this study uncovered a novel regulatory mechanism of fungal virulence linking the MAPK pathway mediated by a G-box binding transcription factor, UvCGBP1.

The post-translational modification lysine 2-hydroxyisobutyrylation (Khib ) plays an important role in gene transcription, metabolism, and enzymatic activity. Khib sites have been identified in rice (Oryza sativa). However, the Khib status of proteins in rice flowers during pathogen infection remains unclear. Here, we report a comprehensive identification of Khib -modified proteins in rice flowers, and the changes in these proteins during infection with the fungal pathogen Ustilaginoidea virens. By using a tandem mass tag-based quantitative proteomics approach, we identified 2,891 Khib sites on 964 proteins in rice flowers. Our data demonstrated that 2-hydroxyisobutyrylated proteins are involved in diverse biological processes. Khib levels were substantially reduced upon infection with U. virens. Chromatin immunoprecipitation polymerase chain reaction (PCR) and reverse transcription quantitative PCR analyses revealed that histone Khib is involved in the expression of disease-resistance genes. More importantly, most quantified sites on core histones H3 were downregulated upon U. virens infection. In addition, the histone deacetylases HDA705, HDA716, SRT1, and SRT2 are involved in the removal of Khib marks in rice. HDA705 was further confirmed to negatively regulate rice disease resistance to pathogens U. virens, Magnaporthe oryzae, and Xanthomonas oryzae pv. oryzae (Xoo). Our data suggest that U. virens could modulate Khib in rice flowers during infection.

Monilinia fructicola has been widely reported as the causal agent of brown rot disease on many Rosaceae family fruits worldwide. It has been reported on stone fruits, e.g., peach, plum, cherry, apricot and mume; as well as pome fruits, e.g., apple, pear and hawthorn. Loquat is a member of the Eriobotrya genus in subfamily Maloideae along with apple, pear and hawthorn. So far, loquat has not been reported as the host of any Monilia species. In June 2019, brown rot symptoms were observed on loquat fruits in an orchard in Wuhan, Hubei Province, China. Thirty single spore isolates were obtained and identified as M. fructicola based on morphological characteristics and molecular analysis. This is the first report of loquat brown rot disease caused by Monilia species in the world. Furthermore, upon artificial inoculation, all three Monilia species from peach in China, i.e., M. fructicola, M. mumecola and M. yunnanensis, could cause typical brown rot disease on loquat fruits. At the same time, M. fructicola isolates from loquat showed virulence similar to those isolates from peach when the pathogenicity test was conducted on peach fruits. These results suggested that loquat could be infected by other Monilia species and that isolates from loquat also have potential to damage other Rosaceae family fruits in practice.

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 .

Pyrimethanil is an anilinopyrimidine (AP) fungicide that is highly effective in controlling green mold caused by Penicillium digitatum but has not yet been registered in China to control postharvest diseases of citrus. In this study, baseline sensitivity of P. digitatum to pyrimethanil was established based on the effective concentrations for 50% inhibition (EC50) values of 127 isolates collected from five major citrus-growing regions of China. The distribution of these EC50 values was unimodal but with a long right tail. The mean ± SD EC50 value was 0.137 ± 0.046 μg/ml, and the minimum and maximum were 0.073 and 0.436 μg/ml, respectively. Pyrimethanil in potato dextrose agar (PDA) at 0.20 μg/ml decreased methionine production in the mycelia by 21.6% and reduced the activity of cell wall-degrading enzymes cellulase and pectinase by 9.1 and 32.8%, respectively. Twelve pyrimethanil-resistant mutants were obtained by consecutive subculturing of 12 arbitrarily selected sensitive isolates on pyrimethanil-amended PDA for four generations, and the resistance factors ranged from 69 to 3,421. There was no cross-resistance between pyrimethanil and prochloraz (r = 0.377, P = 0.123). Compared with their parental isolates, pyrimethanil-resistant mutants had reduced pathogenicity to citrus fruit but higher tolerance to hydrogen peroxide. No differences were detected in tolerance to NaCl, CaCl2, Congo red, or sodium dodecyl sulfate. The exogenous addition of methionine into PDA partially alleviated pyrimethanil toxicity to the sensitive isolates but had no significant effect on toxicity to the resistant mutants. Sequencing of cystathionine γ-synthase encoding genes CGS1 and CGS2, the potential target genes for pyrimethanil, showed that there was no nucleotide mutation in the coding region of CGS of the pyrimethanil-resistant mutants. However, the relative expression of CGS1 and CGS2 of the pyrimethanil-resistant mutants was reduced by 42.5 and 57.4%, respectively. These results have important implications for applications of pyrimethanil to control P. digitatum and for understanding the modes of action and resistance mechanisms of pyrimethanil.

Rice false smut (RFS) caused by Ustilaginoidea virens is one of the most important diseases in the majority of rice-growing areas worldwide. Rice false smut causes not only yield loss, but also threatens human or animal health by producing cyclopeptide mycotoxins. Cultivar resistance is the most economical, effective and environmentally friendly approach to control RFS. However, development of RFS-resistant rice cultivar still faces big challenges. In the field, disease severity of RFS is largely affected by rice growth period and variable weather conditions. To date, quite a few cultivars with stable resistance to RFS have been identified and could be used as resistant resource for disease-resistance breeding (Sun et al., 2020).

Goldthread (

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.

Magnaporthe oryzae causes rice blast disease, which seriously threatens the safety of food production. Understanding the mechanism of appressorium formation, which is one of the key steps for successful infection by M. oryzae, is helpful to formulate effective control strategies of rice blast. In this study, we identified MoWhi2, the homolog of Saccharomyces cerevisiae Whi2 (Whisky2), as an important regulator that controls appressorium formation in M. oryzae. When MoWHI2 was disrupted, multiple appressoria were formed by one conidium and pathogenicity was significantly reduced. A putative phosphatase, MoPsr1, was identified to interact with MoWhi2 using a yeast two‐hybridization screening assay. The knockout mutant ΔMopsr1 displayed similar phenotypes to the ΔMowhi2 strain. Both the ΔMowhi2 and ΔMopsr1 mutants could form appressoria on a hydrophilic surface with cAMP levels increasing in comparison with the wild type (WT). The conidia of ΔMowhi2 and ΔMopsr1 formed a single appressorium per conidium, similar to WT, when the target of rapamycin (TOR) inhibitor rapamycin was present. In addition, compared with WT, the expression levels of MoTOR and the MoTor signalling activation marker gene MoRS3 were increased, suggesting that inappropriate activation of the MoTor signalling pathway is one of the important reasons for the defects in appressorium formation in the ΔMowhi2 and ΔMopsr1 strains. Our results provide insights into MoWhi2 and MoPsr1‐mediated appressorium development and pathogenicity by regulating cAMP levels and the activation of MoTor signalling in M. oryzae., MoWhi2 interacting with MoPsr1 contributes to appressorium development and pathogenicity by regulating intracellular cAMP levels and the activation of MoTor signalling in Magnaporthe oryzae.

Citrus melanose is an important disease that seriously damages citrus production. It is caused by Diaporthe species, which are known as important pathogens, endophytes or saprobes with broad host ranges. Over 170 species of Diaporthe were reported around the world and the complexity of species structures makes it hard to be identified. Phylogenetic analysis based on translation elongation factor 1-ɑ (TEF1) gene, s-tubulin (TUB) gene, calmodulin (CaM) gene, histone (HIS) gene and ITS locus was a reliable method to identify Diaporthe species In this study, the species structure of Diaporthe species from different tissues within individual trees was investigated. Altogether, 494 single spore isolates were obtained, and 92 of them were selected to sequence the five loci (ITS, TEF1, HIS, TUB, CaM) for phylogenetic analysis. Based on the combined multi-locus dataset, phylogenetic trees constructed by using two methods (MP and ML) showed that D. citri was the dominant species no matter on fruits, leaves or twigs. Diaporthe species showed the greatest diversity on fruits and the least diversity on twigs. D. eres was only isolated from twigs. The results of this study may enhance our understanding for the species diversity of Diaporthe species on different tissues from same trees, such information would be useful for the control of Diaporthe diseases in the future.

Long non-coding RNAs (lncRNAs) play an important role in biological processes but regulation and function of lncRNAs remain largely unelucidated, especially in fungi. Ustilaginoidea virens is an economically important fungus causing a devastating disease of rice. By combining microscopic and RNA-seq analyses, we comprehensively characterized lncRNAs of this fungus in infection and developmental processes and defined four serial typical stages. RNA-seq analyses revealed 1724 lncRNAs in U. virens, including 1084 long intergenic non-coding RNAs (lincRNAs), 51 intronic RNAs (incRNAs), 566 natural antisense transcripts (lncNATs) and 23 sense transcripts. Gene Ontology enrichment of differentially expressed lincRNAs and lncNATs demonstrated that these were mainly involved in transport-related regulation. Functional studies of transport-related lncRNAs revealed that UvlncNAT-MFS, a cytoplasm localized lncNAT of a putative MFS transporter gene, UvMFS, could form an RNA duplex with UvMFS and was required for regulation of growth, conidiation and various stress responses. Our results were the first to elucidate the lncRNA profiles during infection and development of this important phytopathogen U. virens. The functional discovery of the novel lncRNA, UvlncNAT-MFS, revealed the potential of lncRNAs in regulation of life processes in fungi.

Lysine 2-hydroxyisobutyrylation (Khib ) is a newly identified post-translational modification (PTM) that plays important roles in transcription and cell proliferation in eukaryotes. However, its function remains unknown in phytopathogenic fungi. Here, we performed a comprehensive assessment of Khib in the rice false smut fungus Ustilaginoidea virens, using Tandem Mass Tag (TMT)-based quantitative proteomics approach. A total of 3 426 Khib sites were identified in 977 proteins, suggesting that Khib is a common and complex PTM in U. virens. Our data demonstrated that the 2-hydroxyisobutyrylated proteins are involved in diverse biological processes. Network analysis of the modified proteins revealed a highly interconnected protein network that included many well-studied virulence factors. We confirmed that the Zn-binding reduced potassium dependency3-type histone deacetylase (UvRpd3) is a major enzyme that removes 2-hydroxyisobutyrylation and acetylation in U. virens. Notably, mutations of Khib sites in the mitogen-activated protein kinase (MAPK) UvSlt2 significantly reduced fungal virulence and decreased the enzymatic activity of UvSlt2. Molecular dynamics simulations demonstrated that 2-hydroxyisobutyrylation in UvSlt2 increased the hydrophobic solvent-accessible surface area and thereby affected binding between the UvSlt2 enzyme and its substrates. Our findings thus establish Khib as a major post-translational modification in U. virens and point to an important role for Khib in the virulence of this phytopathogenic fungus.

The demethylation inhibitor (DMI) fungicide prochloraz has been widely used in China to control citrus green mold, which is caused by Penicillium digitatum. The 50% effective concentration (EC50) values of prochloraz for 129 isolates of P. digitatum collected in 2017 from citrus groves of four provinces of China ranged from 0.0032 to 0.4582 mg/liter. Analysis of the distribution of natural logarithms of EC50 values indicated that 111 isolates with EC50 values lower than 0.05 mg/liter could be considered sensitive to prochloraz. Relative baseline sensitivity was established based on the 111 sensitive isolates, and the mean EC50 value was 0.0090 ± 0.0054 mg/liter (SD). Prochloraz at 60, 100, and 140 mg/liter provided preventive efficacies of 67.8, 93.0, and 96.4%, respectively. Prochloraz at 0.005 and 0.01 mg/liter disrupted cell membrane integrity of conidia but reduced cell membrane permeability of mycelia. Prochloraz at 0.01 mg/liter reduced ergosterol content in mycelia by 41.8%. Two prochloraz-resistant isolates with EC50 values of 3.97 and 5.68 mg/liter were attained by consecutive subculturing on prochloraz-amended PDA. Studies on the expression levels of three potential target genes, CYP51A, CYP51B, and CYP51C, demonstrated that whether in the absence or presence of prochloraz, only CYP51B in the resistant isolates was overexpressed at least 10-fold higher than that of the sensitive ones. Sequencing of the three genes showed that only CYP51B in the resistant isolates had a 199-bp insertion in the promoter region. In addition, only CYP51B displayed point mutations of G405S, G389C, and Y390S in the coding regions in the resistant isolates. These results were important for understanding the resistance mechanisms of P. digitatum to prochloraz.

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.

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 .

Ustilaginoidea virens is an economically important fungus causing a devastating grain disease, rice false smut. An insertional mutagenesis screen was used to explore biological mechanisms underlying infection process of U. virens. T184, a new mutant was identified, with abnormal conidial morphology and deficient virulence. Analysis of the T-DNA inserted gene UvPal1 in the mutant confirmed it as a putative homologue of a cellular morphogenetic protein in yeast, Pal1, whose function has not been well characterized. Deletion of UvPal1 affected hyphal growth, cell morphology, stress adaptation and virulence. UvPal1 could interact with the endocytic proteins, UvEde1 and UvSla2, but was not required for receptor-mediated endocytosis. A yeast two-hybrid (Y2H) analysis was further carried out to screen the UvPal1-interacting proteins, resulting in the identification of 16 putative interacting proteins. Interestingly, UvPal1 interacted with a septin protein, UvCdc11 in vivo and in vitro, and also affected subcellular localization of UvCdc11 protein. Deletion of the four core septins impaired the growth, morphogenesis, stress response and virulence. Collectively, effects on cell morphology, oxidative stress response and virulence are similar to those of UvPal1, suggesting that UvPal1 physically interacts with UvCdc11 to mediate the septin complex to maintain the cellular morphology and virulence of U. virens.

Background Peach scab, caused by Venturia carpophila, can significantly reduce both the yield and quality of peach fruit. It is difficult to distinguish peach scab from other peach diseases such as black spot and bacterial spot. An efficient assay is needed to identify V. carpophila in order to develop scientific management strategies. Results A set of loop-mediated isothermal amplification (LAMP) primers was designed based on the internal transcribed spacer (rDNA-ITS) sequence to detect V. carpophila. Compared with the conventional polymerase chain reaction (PCR) method, the LAMP method not only exhibited higher sensitivity and specificity in the detection of V. carpophila, but also required simpler equipment and less operational time. The minimum detectable concentration of V. carpophila genomic DNA with the LAMP method was 56.6 fg μl-1 , which was 100 times lower than with the conventional PCR method. When eight fungal species including V. carpophila (23 isolates from 14 provinces) and one bacterial species were used with LAMP detection, only V. carpophila showed a color change, from brown to yellowish green, and ladder-like bands in electrophoresis, indicating successful amplification. Moreover, when crude DNAs of peach fruit samples were used in LAMP detection, amplification was observed only from diseased fruits, and not from healthy fruits and the negative control. Conclusion The LAMP assay shows simplicity, rapidity, high sensitivity and specificity, and will be useful in distinguishing scab caused by Venturia carpophila from other diseases with similar symptoms. © 2020 Society of Chemical Industry.

Ustilaginoidea virens is an economically important biotrophic fungal pathogen that causes rice false smut worldwide. However, the regulatory mechanisms of smut ball formation under U. virens infection remain unclear. Here, we identified an important transcription factor, UvCom1, from this pathogen, which regulates the formation of smut balls on rice spikelets. UvCom1 contains two conserved internal repeat 1 (RPT) domains and is found only in fungi, with specific conservation in species of Basidiomycetes and Ascomycetes. UvCom1 protein N- or C-terminal fusion vectors both showed transactivation activity in yeast. Deletion of UvCom1 significantly affected the vegetative growth and conidiation of U. virens. UvCom1 negatively regulated the responses to oxidative, osmotic, and cell wall stresses. Remarkably, UvCom1 was found to be essential for the formation of rice smut balls, and UvCom1 deletion mutants lost the ability to stably utilize nutrients from the rice host. UvCom1 was also highly expressed at the mycelial expansion stage. Transcriptomic analysis and quantitative real-time PCR revealed that UvCom1 could affect the expression of genes significantly enriched in transmembrane transport. This study demonstrates that UvCom1 is a key transcription factor governing smut ball formation of this biotrophic fungus.

Rice false smut caused by Ustilaginoidea virens is a serious disease of rice (Oryza sativa), severely reducing plant mass and yields worldwide. We performed genome-wide analysis of the CCHC-type zinc-finger transcription factor family in this pathogen. We identified and functionally characterized seven UvCCHC genes in U. virens. The deletion of various UvCCHC genes affected the stress responses, vegetative growth, conidiation, and virulence of U. virens. ∆UvCCHC5 mutants infected rice spikelets normally but could not form smut balls. Sugar utilization experiments showed that the ∆UvCCHC5 mutants were defective in the utilization of glucose, sucrose, lactose, stachyose, and trehalose. Deletion of UvCCHC5 did not affect the expression of rice genes associated with grain filling, as revealed by RT-qPCR. We propose that the ∆UvCCHC5 mutants are impaired in transmembrane transport, and the resulting nutrient deficiencies prevent them from using nutrients from rice to form smut balls. RNA-seq data analysis indicated that UvCCHC4 affects the expression of genes involved in mitochondrial biogenesis, ribosomes, transporters, and ribosome biogenesis. These findings improve our understanding of the molecular mechanism underlying smut ball formation in rice by U. virens.

Monilia mumecola is one of the causal agents of peach brown rot in China. In this study, M. mumecola isolates from different locations and hosts were used to analyze the genetic diversity and to assay the sensitivity to four generally used fungicides: carbendazim, tebuconazole, azoxystrobin, and boscalid. Results showed that isolates from different locations tended to be separated. Interestingly, isolates from different hosts (e.g., peach and apricot) at the same locations generally clustered together, indicating that the M. mumecola isolates may infect different hosts in the same areas. The fungicide sensitivity assay of 93 M. mumecola isolates showed that the average effective concentration for 50% mycelial growth inhibition values for carbendazim, tebuconazole, azoxystrobin, and boscalid were 0.103, 0.034, 0.325, and 0.419 µg/ml, respectively. The sensitivity distributions of the tested isolates to the four fungicides showed continuous unimodal curves, indicating no qualitative shift of resistance. No significant difference of sensitivity to tested fungicides was observed among isolates from either different locations or different hosts.

It is a common practice to add salicylhydroxamic acid (SHAM) into artificial medium in the in vitro sensitivity assay of fungal phytopathogens to the quinone outside inhibitor (QoI) fungicides. The rationale for adding SHAM is to inhibit fungal alternative oxidase, which is presumed to be inhibited by secondary metabolites of plants. Therefore, the ideal characteristics of SHAM should be almost nontoxic to phytopathogens and have no significant effect on control efficacy of fungicides. However, this study showed that the average effective concentration for 50% inhibition (EC50) of mycelial growth values of SHAM were 97.5 and 401.4 μg/ml for Sclerotinia sclerotiorum and Botrytis cinerea, respectively. EC50 values of the three QoI fungicides azoxystrobin, kresoxim-methyl, and trifloxystrobin in the presence of SHAM at 20 and 80 μg/ml for S. sclerotiorum and B. cinerea, respectively, declined by 52.7 to 78.1% compared with those without SHAM. For the dicarboximide fungicide dimethachlone, the average EC50 values in the presence of SHAM declined by 18.2% (P = 0.008) for S. sclerotiorum and 35.9% (P = 0.012) for B. cinerea. Pot experiments showed that SHAM increased control efficacy of the three QoI fungicides against the two pathogens by 43 to 83%. For dimethachlone, SHAM increased control efficacy by 134% for S. sclerotiorum and 86% for B. cinerea. Biochemical studies showed that SHAM significantly inhibited peroxidase activity (P = 0.024) of B. cinerea and esterase activity (P = 0.015) of S. sclerotiorum. The strong inhibitions of SHAM per se on mycelial growth of B. cinerea and S. sclerotiorum and significant influences on the sensitivity of the two pathogens to both the QoI fungicides and dimethachlone as well as inhibitions on peroxidase and esterase indicate that SHAM should not be added in the in vitro assay of sensitivity to the QoI fungicides.

Benzimidazole fungicides (MBCs) have been widely used in agriculture since 1970s, and resistance to this class of fungicides in Botrytis cinerea is reported worldwide. Resistance to MBCs in B. cinerea is related to mutations in the target β-tubulin gene (TUB2). Compared with the mutation at codon 200, the substitutions from glutamic acid to alanine (E198A), valine (E198V), or lysine (E198K) at codon 198 are currently the predominant mutations in MBC resistant populations of B. cinerea. In this study, a loop-mediated isothermal amplification (LAMP) method was established for rapid detection of benzimidazole resistance in B. cinerea. On the basis of the three mutations at TUB2 codon 198, three sets of LAMP primers were designed, and each of these primer sets was able to specifically amplify the DNA containing its corresponding mutation, while no amplification was detected with other mutated or the wild type DNA. After optimization, the sensitivity and specificity tests illustrated that this LAMP assay had good sensitivity and specificity to detect specific resistant genotypes in B. cinerea. Result also showed that the LAMP assay had good reproducibility. Above all, boiled mycelia could be used as templates, which simplified the process and increased the efficiency of the assay. Considering its rapidity, simplicity, and high efficiency, the LAMP assay developed in this study is a promising tool for the diagnosis of benzimidazole resistance in B. cinerea, and will contribute to the monitoring of resistance development to MBCs in the future.

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 EC

Background Generating chromosome-scale haplotype resolved assembly is important for functional studies. However, current de novo assemblers are either haploid assemblers that discard allelic information, or diploid assemblers that can only tackle genomes of low complexity. Results Here, Using robust programs, we build a diploid genome assembly pipeline called gcaPDA (gamete cells assisted Phased Diploid Assembler), which exploits haploid gamete cells to assist in resolving haplotypes. We demonstrate the effectiveness of gcaPDA based on simulated HiFi reads of maize genome which is highly heterozygous and repetitive, and real data from rice. Conclusions With applicability of coping with complex genomes and fewer restrictions on application than most of diploid assemblers, gcaPDA is likely to find broad applications in studies of eukaryotic genomes.

Rice false smut is a fungal disease distributed worldwide and caused by Ustilaginoidea virens. In this study, we identified a putative ester cyclase (named as UvEC1) as being significantly upregulated during U. virens infection. UvEC1 contained a SnoaL-like polyketide cyclase domain, but the functions of ketone cyclases such as SnoaL in plant fungal pathogens remain unclear. Deletion of UvEC1 caused defects in vegetative growth and conidiation. UvEC1 was also required for response to hyperosmotic and oxidative stresses and for maintenance of cell wall integrity. Importantly, ΔUvEC1 mutants exhibited reduced virulence. We performed a tandem mass tag (TMT)-based quantitative proteomic analysis to identify differentially accumulating proteins (DAPs) between the ΔUvEC1-1 mutant and the wild-type isolate HWD-2. Proteomics data revealed that UvEC1 has a variety of effects on metabolism, protein localization, catalytic activity, binding, toxin biosynthesis and the spliceosome. Taken together, our findings suggest that UvEC1 is critical for the development and virulence of U. virens.

Background Peach diseases can cause severe yield reduction and decreased quality for peach production. Rapid and accurate detection and identification of peach diseases is of great importance. Deep learning has been applied to detect peach diseases using imaging data. However, peach disease image data is difficult to collect and samples are imbalance. The popular deep networks perform poor for this issue. Results This paper proposed an improved Xception network named as L2MXception which ensembles regularization term of L2-norm and mean. With the peach disease image dataset collected, results on seven mainstream deep learning models were compared in details and an improved loss function was integrated with regularization term L2-norm and mean (L2M Loss). Experiments showed that the Xception model with L2M Loss outperformed the current best method for peach disease prediction. Compared to the original Xception model, the validation accuracy of L2MXception was up to 93.85%, increased by 28.48%. Conclusions The proposed L2MXception network may have great potential in early identification of peach diseases.