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.

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.

MoAti1 mediates mitophagy by facilitating recruitment of MoAtg8 to promote invasive growth in Magnaporthe oryzae.

Mitophagy is a selective autophagy for the degradation of damaged or excessive mitochondria to maintain intracellular homeostasis. In Magnaporthe oryzae, a filamentous ascomycetous fungus that causes rice blast, the most devastating disease of rice, mitophagy occurs in the invasive hyphae to promote infection. To date, only a few proteins are known to participate in mitophagy and the mechanisms of mitophagy are largely unknown in pathogenic fungi. Here, by a yeast two-hybrid screen with the core autophagy-related protein MoAtg8 as a bait, we obtained a MoAtg8 interactor MoAti1 (MoAtg8-interacting protein 1). Fluorescent observations and protease digestion analyses revealed that MoAti1 is primarily localized to the peripheral mitochondrial outer membrane and is responsible for recruiting MoAtg8 to mitochondria under mitophagy induction conditions. MoAti1 is specifically required for mitophagy, but not for macroautophagy and pexophagy. Infection assays suggested that MoAti1 is required for mitophagy in invasive hyphae during pathogenesis. Notably, no homologues of MoAti1 were found in rice and human protein databases, indicating that MoAti1 may be used as a potential target to control rice blast. By the host-induced gene silencing (HIGS) strategy, transgenic rice plants targeted to silencing MoATI1 showed enhanced resistance against M. oryzae with unchanged agronomic traits. Our results suggest that MoATI1 is required for mitophagy and pathogenicity in M. oryzae and can be used as a target for reducing rice blast.

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.

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.

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.

Identifying pathogenicity-related genes in the pathogen Colletotrichum magnum causing watermelon anthracnose disease via T-DNA insertion mutagenesis.

Fruit rot caused by Colletotrichum magnum is a crucial watermelon disease threatening the production and quality. To understand the pathogenic mechanism of C. magnum, we optimized the Agrobacterium tumefaciens-mediated transformation system (ATMT) for genetic transformation of C. magnum. The transformation efficiency of ATMT was an average of around 245 transformants per 100 million conidia. Southern blot analysis indicated that approximately 75% of the mutants contained a single copy of T-DNA. Pathogenicity test revealed that three mutants completely lost pathogenicity. The T-DNA integration sites (TISs) of three mutants were Identified. In mutant Cm699, the TISs were found in the intron region of the gene, which encoded a protein containing AP-2 complex subunit σ, and simultaneous gene deletions were observed. Two deleted genes encoded the transcription initiation protein SPT3 and a hypothetical protein, respectively. In mutant Cm854, the TISs were found in the 5'-flanking regions of a gene that was similar to the MYO5 encoding Myosin I of Pyricularia oryzae (78%). In mutant Cm1078, the T-DNA was integrated into the exon regions of two adjacent genes. One was 5'-3' exoribonuclease 1 encoding gene while the other encoded a WD-repeat protein retinoblastoma binding protein 4, the homolog of the MSl1 of Saccharomyces cerevisiae.

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.

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.

Mitochondrial Inner Membrane ABC Transporter Bcmdl1 Is Involved in Conidial Germination, Virulence, and Resistance to Anilinopyrimidine Fungicides in Botrytis cinerea.

Botrytis cinerea causes gray mold on thousands of plants, leading to huge losses in production. Anilinopyrimidine (AP) fungicides have been applied to control B. cinerea since the 1990s. Although resistance to AP fungicides was detected soon after their application, the mechanism of AP resistance remains to be elucidated. In this study, a sexual cross between resistant and sensitive isolates was performed, and the genomes of parental isolates and progenies were sequenced to identify resistance-related single nucleotide polymorphisms (SNPs). After screening and verification, mutation E407K in the Bcmdl1 gene was identified and confirmed to confer resistance to AP fungicides in B. cinerea. Bcmdl1 was predicted to encode a mitochondrial protein that belonged to a half-type ATP-binding cassette (ABC) transporter. Although Bcmdl1 was a transporter, it did not mediate resistance to multiple fungicides but mediated resistance specifically to AP fungicides. On the other hand, reductions in conidial germination and virulence were observed in Bcmdl1 knockout transformants compared to the parental isolate and complemented transformants, illustrating the biological functions of Bcmdl1. Subcellular localization analysis indicated that Bcmdl1 was localized in mitochondria. Interestingly, the production of ATP was reduced after cyprodinil treatment in Bcmdl1 knockout transformants, suggesting that Bcmdl1 was involved in ATP synthesis. Since Mdl1 could interact with ATP synthase in yeast, we hypothesize that Bcmdl1 forms a complex with ATP synthase, which AP fungicides might target, thereby interfering with the metabolism of energy. IMPORTANCE Gray mold, caused by B. cinerea, causes huge losses in the production of many fruits and vegetables. AP fungicides have been largely adopted to control this disease since the 1990s, and the development of resistance to AP fungicides initiates new problems for disease control. Due to the unknown mode of action, information on the mechanism of AP resistance is also limited. Recently, mutations in mitochondrial genes were reported to be related to AP resistance. However, the mitochondrial process of these genes remains to be elucidated. In this study, we identified several AP resistance-related mutations by quantitative trait locus sequencing (QTL-seq) and confirmed that mutation E407K in Bcmdl1 conferred AP resistance. We further characterized the expression patterns, biological functions, subcellular localization, and mitochondrial processes of the Bcmdl1 gene. This study deepens our understanding of the mechanism of resistance to and mode of action of AP fungicides.

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.

UvATG6 Interacts with BAX Inhibitor 1 Proteins and Plays Critical Roles in Growth, Conidiation, and Virulence in Ustilaginoidea virens.

Autophagy and apoptosis are evolutionarily conserved catabolic processes involved in regulating development and cellular homeostasis. Bax inhibitor 1 (BI-1) and autophagy protein 6 (ATG6) perform essential functions in these roles, such as cellular differentiation and virulence in various filamentous fungi. However, the functions of ATG6 and BI-1 proteins in development and virulence in the rice false smut fungus Ustilaginoidea virens are still poorly understood. In this study, UvATG6 was characterized in U. virens. The deletion of UvATG6 almost abolished autophagy in U. virens and reduced growth, conidial production and germination, and virulence. Stress tolerance assays showed that UvATG6 mutants were sensitive to hyperosmotic, salt, and cell wall integrity stresses but were insensitive to oxidative stress. Furthermore, we found that UvATG6 interacted with UvBI-1 or UvBI-1b and suppressed Bax-induced cell death. We previously found that UvBI-1 could suppress Bax-induced cell death and was a negative regulator of mycelial growth and conidiation. Unlike UvBI-1, UvBI-1b could not suppress cell death. UvBI-1b-deleted mutants exhibited decreased growth and conidiation, while the UvBI-1 and UvBI-1b double deletion reduced the phenotype, indicating that UvBI-1 and UvBI-1b antagonistically regulate mycelial growth and conidiation. In addition, the UvBI-1b and double mutants exhibited decreased virulence. Our results provide evidence of the cross talk of autophagy and apoptosis in U. virens and give clues for studying other phytopathogenic fungi. IMPORTANCE Ustilaginoidea virens causes destructive panicle disease in rice, significantly threatening agricultural production. UvATG6 is required for autophagy and contributes to growth, conidiation, and virulence in U. virens. Additionally, it interacts with the Bax inhibitor 1 proteins UvBI-1 and UvBI-1b. UvBI-1 suppresses cell death induced by Bax, unlike UvBI-1b. UvBI-1 negatively regulates growth and conidiation, while UvBI-1b is required for these phenotypes. These results indicate that UvBI-1 and UvBI-1b may antagonistically regulate growth and conidiation. In addition, both of them contribute to virulence. Additionally, our results suggest cross talk between autophagy and apoptosis, contributing to the development, adaptability, and virulence of U. virens.

One-Pot Assay for Rapid Detection of Benzimidazole Resistance in Venturia carpophila by Combining RPA and CRISPR/Cas12a.

High resistance to benzimidazole fungicides in Venturia carpophila is caused by the point mutation E198K of the ß-tubulin (TUB2) gene. Traditional methods for detection of fungicide resistance are time-consuming, which are routinely based on tedious operation, reliance on expensive equipment, and specially trained people. Therefore, it is important to establish efficient methods for field detection of benzimidazole resistance in V. carpophila to make suitable management strategies and ensure food safety. Based on recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a, a rapid one-pot assay ORCas12a-BRVc (one-pot RPA-CRISPR/Cas12 platform) was established for the detection of benzimidazole resistance in V. carpophila. The ORCas12a-BRVc assay enabled one-pot detection by adding components at the bottom and wall of the tube separately, solving the problems of aerosol contamination and decreased sensitivity caused by competing DNA substrates between Cas12a cleavage and RPA amplification. The ORCas12a-BRVc assay could accomplish the detection with a minimum of 7.82 × 103 fg µL-1 V. carpophila genomic DNA in 45 min at 37 °C. Meanwhile, this assay showed excellent specificity due to the specific recognition ability of the Cas12a-crRNA complex. Further, we combined a method that could rapidly extract DNA from V. carpophila within 2 min with the ORCas12a-BRVc to achieve more rapid and simple detection of V. carpophila with benzimidazole resistance in fields. The ORCas12a-BRVc assay has the advantages of simplicity, rapidity, high sensitivity, high specificity, and ease of operation without the need for precision instruments and the need to isolate and culture pathogens. This assay is the first application of the one-pot platform based on the combination of RPA and CRISPR/Cas12a in fungicide resistance detection and can be used for monitoring of resistant populations in fields, providing guidance on making suitable management strategies for peach scab.

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.

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.

Colletotrichum Species Associated with Anthracnose Disease of Watermelon (Citrullus lanatus) in China.

Colletotrichum species are important plant pathogens, causing anthracnose in virtually every crop grown throughout the world. However, little is known about the species that infect watermelon. A total of 526 strains were isolated from diseased watermelon samples of eight major watermelon growing provinces in China. Phylogenetic analyses using seven loci (ITS, gadph, chs-1, his3, act, tub2, and gs) coupled with morphology of 146 representative isolates showed that they belonged to 12 known species of Colletotrichum, including C. aenigma, C. chlorophyti, C. fructicola, C. jiangxiense, C. karstii, C. magnum, C. nymphaeae, C. nigrum, C. orbiculare, C. plurivorum, C. sojae, and C. truncatum and three new species, here described as C. citrulli, C. kaifengense, and C. qilinense. Colletotrichum orbiculare was the dominant species. Pathogenicity tests revealed that all isolates of the species described above were pathogenic, with C. magnum and C. kaifengense being the most aggressive to leaves and fruits, respectively. This is the first report of C. aenigma, C. chlorophyti, C. fructicola, C. jiangxiense, C. nymphaeae, C. nigrum, C. plurivorum, and C. sojae on watermelon. These findings shed light on the Colletotrichum spp. involved in watermelon anthracnose and provide useful information for implementing effective control of watermelon anthracnose in China.

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.

Diaporthe citri: A Fungal Pathogen Causing Melanose Disease.

Citrus melanose is a fungal disease caused by Diaporthe citri F.A. Wolf. It is found in various citrus-growing locations across the world. The host range of D. citri is limited to plants of the Citrus genus. The most economically important hosts are Citrus reticulata (mandarin), C. sinensis (sweet orange), C. grandis or C. maxima (pumelo), and C. paradisi (grapefruit). In the life cycle of D. citri throughout the citrus growing season, pycnidia can be seen in abundance on dead branches, especially after rain, with conidia appearing as slimy masses discharged from the dead twigs. Raindrops can transmit conidia to leaves, twigs, and fruits, resulting in disease dispersion throughout small distances. Persistent rains and warm climatic conditions generally favor disease onset and development. The melanose disease causes a decline in fruit quality, which lowers the value of fruits during marketing and exportation. High rainfall areas should avoid planting susceptible varieties. In this article, information about the disease symptoms, history, geographic distribution, epidemiology, impact, and integrated management practices, as well as the pathogen morphology and identification, was reviewed and discussed.

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.