Microfungi Associated with Peach Branch Diseases in China.

Peach (Prunus persica L.) is one of the most important and oldest stone fruits grown in China. Even though P. persica is one of the most commonly grown stone fruits in China, little is known about the biodiversity of microfungi associated with peach branch diseases. In the present study, samples were collected from a wide range of peach growing areas in China, and fungal pathogens associated with peach branch diseases were isolated. In total, 85 isolates were obtained and further classified into nine genera and 10 species. Most of the isolates belonged to Botryosphaeriaceae (46), including Botryosphaeria, Diplodia, Neofusicoccum, Phaeobotryon, and Lasiodiplodia species; Ascochyta, Didymella, and Nothophoma species representing Didymellaceae were also identified. Herein, we introduce Ascochyta prunus and Lasiodiplodia pruni as novel species. In addition, we report the first records of Nothophoma pruni, Neofusicoccum occulatum, and Phaeobotryon rhois on peach worldwide, and Didymella glomerata, Nothophoma quercina, and Phaeoacremonium scolyti are the first records from China. This research is the first comprehensive investigation to explore the microfungi associated with peach branch disease in China. Future studies are necessary to understand the pathogenicity and disease epidemiology of these identified species.

Advances in understanding grapevine downy mildew: From pathogen infection to disease management.

Plasmopara viticola is geographically widespread in grapevine-growing regions. Grapevine downy mildew disease, caused by this biotrophic pathogen, leads to considerable yield losses in viticulture annually. Because of the great significance of grapevine production and wine quality, research on this disease has been widely performed since its emergence in the 19th century. Here, we review and discuss recent understanding of this pathogen from multiple aspects, including its infection cycle, disease symptoms, genome decoding, effector biology, and management and control strategies. We highlight the identification and characterization of effector proteins with their biological roles in host-pathogen interaction, with a focus on sustainable control methods against P. viticola, especially the use of biocontrol agents and environmentally friendly compounds.

Coronatine-Induced Maize Defense against Gibberella Stalk Rot by Activating Antioxidants and Phytohormone Signaling.

One of the most destructive diseases, Gibberella stalk rot (GSR), caused by Fusarium graminearum, reduces maize yields significantly. An induced resistance response is a potent and cost-effective plant defense against pathogen attack. The functional counterpart of JAs, coronatine (COR), has attracted a lot of interest recently due to its ability to control plant growth and stimulate secondary metabolism. Although several studies have focused on COR as a plant immune elicitor to improve plant resistance to pathogens, the effectiveness and underlying mechanisms of the suppressive ability against COR to F. graminearum in maize have been limited. We investigated the potential physiological and molecular mechanisms of COR in modulating maize resistance to F. graminearum. COR treatment strongly enhanced disease resistance and promoted stomatal closure with H2O2 accumulation, and 10 µg/mL was confirmed as the best concentration. COR treatment increased defense-related enzyme activity and decreased the malondialdehyde content with enhanced antioxidant enzyme activity. To identify candidate resistance genes and gain insight into the molecular mechanism of GSR resistance associated with COR, we integrated transcriptomic and metabolomic data to systemically explore the defense mechanisms of COR, and multiple hub genes were pinpointed using weighted gene correlation network analysis (WGCNA). We discovered 6 significant modules containing 10 candidate genes: WRKY transcription factor (LOC100279570), calcium-binding protein (LOC100382070), NBR1-like protein (LOC100275089), amino acid permease (LOC100382244), glutathione S-transferase (LOC541830), HXXXD-type acyl-transferase (LOC100191608), prolin-rich extensin-like receptor protein kinase (LOC100501564), AP2-like ethylene-responsive transcription factor (LOC100384380), basic leucine zipper (LOC100275351), and glycosyltransferase (LOC606486), which are highly correlated with the jasmonic acid-ethylene signaling pathway and antioxidants. In addition, a core set of metabolites, including alpha-linolenic acid metabolism and flavonoids biosynthesis linked to the hub genes, were identified. Taken together, our research revealed differentially expressed key genes and metabolites, as well as co-expression networks, associated with COR treatment of maize stems after F. graminearum infection. In addition, COR-treated maize had higher JA (JA-Ile and Me-JA) levels. We postulated that COR plays a positive role in maize resistance to F. graminearum by regulating antioxidant levels and the JA signaling pathway, and the flavonoid biosynthesis pathway is also involved in the resistance response against GSR.

Dual RNA-Seq Reveals Temperature-Mediated Gene Reprogramming and Molecular Crosstalk between Grapevine and Lasiodiplodia theobromae.

High temperatures associated with a fluctuating climate profoundly accelerate the occurrence of a myriad of plant diseases around the world. A comprehensive insight into how plants respond to pathogenic microorganisms under high-temperature stress is required for plant disease management, whereas the underlying mechanisms behind temperature-mediated plant immunity and pathogen pathogenicity are still unclear. Here, we evaluated the effect of high temperature on the development of grapevine canker disease and quantified the contribution of temperature variation to the gene transcription reprogramming of grapevine and its pathogenic agent Lasiodiplodia theobromae using a dual RNA-seq approach. The results showed that both grapevine and the pathogen displayed altered transcriptomes under different temperatures, and even the transcription of a plethora of genes from the two organisms responded in different directions and magnitudes. The transcription variability that arose due to temperature oscillation allowed us to identify a total of 26 grapevine gene modules and 17 fungal gene modules that were correlated with more than one gene module of the partner organism, which revealed an extensive web of plant-pathogen gene reprogramming during infection. More importantly, we identified a set of temperature-responsive genes that were transcriptionally orchestrated within the given gene modules. These genes are predicted to be involved in multiple cellular processes including protein folding, stress response regulation, and carbohydrate and peptide metabolisms in grapevine and porphyrin- and pteridine-containing compound metabolisms in L. theobromae, implying that in response to temperature oscillation, a complex web of signaling pathways in two organism cells is activated during infection. This study describes a co-transcription network of grapevine and L. theobromae in the context of considering temperature variation, which provides novel insights into deciphering the molecular mechanisms underlying temperature-modulated disease development.

Molecular Identification and Pathogenicity of Diaporthe eres and D. hongkongensis (Diaporthales, Ascomycota) Associated with Cherry Trunk Diseases in China.

This study aimed to identify fungal species associated with trunk diseases of sweet cherries (Prunus avium) in several commercial cherry orchards in Beijing, Guizhou and Shandong provinces, China. In total, eighteen fungal strains that fitted well into the species concept of Diaporthe were isolated. Based on both morphological and multi-locus phylogenetic analyses of internal transcribed spacer region (ITS), beta-tubulin (tub-2), calmodulin (Cal) and translation elongation factor 1-α (tef1-α) sequencing data, fourteen isolates were identified as Diaporthe eres, while four isolates were classified as D. hongkongensis. Here, we report D. hongkongensis causing sweet cherry branch dieback disease and, further, we confirmed the host association of D. eres with sweet cherries in China. A pathogenicity assay revealed the ability of both D. eres and D. hongkongensis to cause shoot necrosis and stem lesions on Prunus avium cv. 'Brooks' (mean lesion lengths of 1.86 cm and 1.56 cm, respectively). The optimal temperature for the growth of both Diaporthe species was tested. The optimal growth temperature for D. hongkongensis was 30 °C, and the 25-28 °C temperatures were the most favorable for the growth of D. eres strains. This research advances the understanding of fungal trunk diseases in fruit crops, particularly gummosis and branch dieback disease in Chinese cherry orchards, and will aid growers in making decisions about cultural practices and disease management.

First Report of Cylindrocladiella peruviana Associated with Grapevine Black Foot Disease in China.

Black foot disease is one of the Grapevine Trunk Diseases (GTDs) that occurs in many grape growing regions and causes vine decline. Black foot disease has been reported in China in 2021 (Ye et al. 2021). In May 2022, serious root rot and nearly half brown necrosis in cross section were observed in several grapevines (Vitis vinifera cv. Cabernet Sauvignon) in Xianyang County, Shaanxi Province, China, with the incidence of 2% in 0.267 hectares. Samples (12-year-old vines) of symptomatic root were collected and taken back to the laboratory. Small fragments from the margin between healthy and diseased tissue were cut into 5 mm × 5 mm pieces. The surface was sterilized using 2% NaOCl for 2 min, followed by 75% ethanol for 30 s and rinsed three times with sterilized water. After the small pieces were dried on sterilized filter paper, they were placed onto potato dextrose agar (PDA) plates with lactic acid at 25°C for 2-3 days. The pure culture was obtained by single spore isolation. In this study, two strains were associated with black foot disease. Colony characteristics were observed in 7-day PDA plates, with cotton wool mycelium and light yellow to brown colony in reverse. Conidia were cylindrical to peanut shaped, 0-1 septa and blunt round at both ends. Size of the conidia was 7.12 to 13.89 × 2.55 to 5.16 µm (average 9.82 × 3.41 µm, n=50). For molecular phylogenetic analysis, genomic DNA of the two strains (JZB3320008 and JZB3320009) was extracted. PCR amplification was performed using four phylogenetic regions (ITS, tub2, tef1 and his3) amplified with primers ITS1/ITS4 (White et al. 1990), T1/Bt2b (O'Donnell and Cigelnik 1997; Glass and Dnoaldson 1995), EF-688F/EF-1251R (Alves et al. 2008) and CYLH3F/CYLH3R (Crous et al. 2004), respectively. Maximum likelihood (ML) inference phylogenetic tree was constructed to confirm the identity of the two strains. ML tree reveals that the two strains clustered with Cylindrocladiella peruviana with 100% bootstrap support value. The sequences of four regions were deposited in GenBank (accession numbers OQ202205-OQ202206 for ITS, OQ225938-OQ225939 for tub2, OQ225936-OQ225937 for tef1 and OQ225934-OQ225935 for his3). Based on the morphological characteristics and molecular phylogenetic analysis, the two strains were identified as C. peruviana. To confirm the pathogenicity, the rooted cuttings (cv. Marselan) in the greenhouse were inoculated by immersing the roots in the conidial suspension (106 conidia/mL; volume, 300 mL) for 30 min, while the control was immersed in sterilized water (volume, 300 mL) for the same time. Nine plants were inoculated with C. peruviana and the same number was used as the control. These grapevines were kept in the greenhouse at 25°C. After 14 days of inoculation, the aboveground of inoculated plants showed symptoms with red leaves, while the control showed no symptoms. After 34 days of inoculation, the inoculated plants showed the reduction of the number of foliage and reduced vigor. They died with brown stem base and vascular discoloration in longitudinal section, while the control showed no symptoms. Cylindrocladiella peruviana was re-isolated by the discoloration regions and the Koch's rule was verified. To our knowledge, the pathogen has been reported in Spain and California (Agustí-Brisach et al. 2012; Koike et al. 2016). This is the first report of Cylindrocladiella peruviana causing grapevine black foot disease in China. We will better diagnose and prevent the disease in the future.

Belowground microbiota analysis indicates that Fusarium spp. exacerbate grapevine trunk disease.

BACKGROUND: Grapevine trunk diseases (GTDs) are disease complexes that are major threats to viticulture in most grapevine growing regions. The microbiomes colonizing plant belowground components form complex associations with plants, play important roles in promoting plant productivity and health in natural environments, and may be related to GTD development. To investigate associations between belowground fungal communities and GTD symptomatic or asymptomatic grapevines, fungal communities associated with three soil-plant compartments (bulk soils, rhizospheres, and roots) were characterized by ITS high-throughput amplicon sequencing across two years. RESULTS: The fungal community diversity and composition differs according to the soil-plant compartment type (PERMANOVA, p < 0.001, 12.04% of variation explained) and sampling year (PERMANOVA, p < 0.001, 8.83%), whereas GTD symptomatology exhibited a weaker, but still significant association (PERMANOVA, p < 0.001, 1.29%). The effects of the latter were particularly prominent in root and rhizosphere community comparisons. Many GTD-associated pathogens were detected, but their relative abundances were not correlated (or were negatively correlated) to symptomatology. Fusarium spp., were enriched in symptomatic roots and rhizospheres compared to asymptomatic counterparts, suggesting that their abundances were positively correlated with symptomatic vines. Inoculation tests revealed that Fusarium isolates, similar to Dactylonectria macrodidyma, a pathogen associated with black foot disease, caused dark brown necrotic spots on stems in addition to root rot, which blackened lateral roots. Disease indices were higher with co-inoculation than single inoculation with a Fusarium isolate or D. macrodidyma, suggesting that Fusarium spp. can exacerbate disease severity when inoculated with other known GTD-associated pathogens. CONCLUSIONS: The belowground fungal microbiota of grapevines varied from soil-plant compartments, the years and whether showed GTD symptoms. The GTDs symptoms were related to the enrichment of Fusarium spp. rather than the relative abundances of GTD pathogens. These results demonstrate the effects of fungal microbiota of roots and rhizospheres on GTDs, while providing new insights into opportunistic pathogenesis of GTDs and potential control practices.

The woody plant-degrading pathogen Lasiodiplodia theobromae effector LtCre1 targets the grapevine sugar-signaling protein VvRHIP1 to suppress host immunity.

Lasiodiplodia theobromae is a causal agent of Botryosphaeria dieback, which seriously threatens grapevine production worldwide. Plant pathogens secrete diverse effectors to suppress host immune responses and promote the progression of infection, but the mechanisms underlying the manipulation of host immunity by L. theobromae effectors are poorly understood. In this study, we characterized LtCre1, which encodes a L. theobromae effector that suppresses BAX-triggered cell death in Nicotiana benthamiana. RNAi-silencing and overexpression of LtCre1 in L. theobromae showed impaired and increased virulence, respectively, and ectopic expression in N. benthamiana increased susceptibility. These results suggest that LtCre1 is as an essential virulence factor for L. theobromae. Protein-protein interaction studies revealed that LtCre1 interacts with grapevine RGS1-HXK1-interacting protein 1 (VvRHIP1). Ectopic overexpression of VvRHIP1 in N. benthamiana reduced infection, suggesting that VvRHIP1 enhances plant immunity against L. theobromae. LtCre1 was found to disrupt the formation of the VvRHIP1-VvRGS1 complex and to participate in regulating the plant sugar-signaling pathway. Thus, our results suggest that L. theobromae LtCre1 targets the grapevine VvRHIP1 protein to manipulate the sugar-signaling pathway by disrupting the association of the VvRHIP1-VvRGS1 complex.

LtGAPR1 Is a Novel Secreted Effector from Lasiodiplodia theobromae That Interacts with NbPsQ2 to Negatively Regulate Infection.

The effector proteins secreted by a pathogen not only promote the virulence and infection of the pathogen but also trigger plant defense response. Lasiodiplodia theobromae secretes many effectors that modulate and hijack grape processes to colonize host cells, but the underlying mechanisms remain unclear. Herein, we report LtGAPR1, which has been proven to be a secreted protein. In our study, LtGAPR1 played a negative role in virulence. By co-immunoprecipitation, 23 kDa oxygen-evolving enhancer 2 (NbPsbQ2) was identified as a host target of LtGAPR1. The overexpression of NbPsbQ2 in Nicotiana benthamiana reduced susceptibility to L. theobromae, and the silencing of NbPsbQ2 enhanced L. theobromae infection. LtGAPR1 and NbPsbQ2 were confirmed to interact with each other. Transiently, expressed LtGAPR1 activated reactive oxygen species (ROS) production in N. benthamiana leaves. However, in NbPsbQ2-silenced leaves, ROS production was impaired. Overall, our report revealed that LtGAPR1 promotes ROS accumulation by interacting with NbPsbQ2, thereby triggering plant defenses that negatively regulate infection.

Magnaporthe oryzae endoplasmic reticulum membrane complex regulates the biogenesis of membrane proteins for pathogenicity.

In eukaryotes, the majority of newly synthesized integral membrane proteins are inserted into the endoplasmic reticulum (ER) membrane before transferred to their functional sites. The conserved ER membrane complex (EMC) takes part in the insertion process for tail-anchored membrane proteins. However, the function of EMC in phytopathogenic fungi has not been characterized. Here, we report the identification and functional characterization of two EMC subunits MoEmc5 and MoEmc2 in Magnaporthe oryzae. The knockout mutants ΔMoemc5 and ΔMoemc2 exhibit substantial defect in autophagy, pathogenicity, cell wall integrity, and magnesium ion sensitivity. We demonstrate that the autophagy process was severely impaired in the ΔMoemc5 and ΔMoemc2 mutants because of the low-protein steady-state level of Atg9, the sole membrane-associated autophagy protein. Furthermore, the protein level of membrane proteins Chs4, Fks1, and MoMnr2 is also significantly reduced in the ΔMoemc5 and ΔMoemc2 strains, leading to their supersensitivity to Calcofluor white, Congo red, and magnesium. In addition, MoEmc5, but not MoEmc2, acts as a magnesium transporter independent of its EMC function. Magnaporthe oryzae EMC regulates the biogenesis of membrane proteins for autophagy and virulence; therefore, EMC subunits could be potential targets for fungicide design in the future.

Transcriptomic Analysis of Resistant and Wild-Type Botrytis cinerea Isolates Revealed Fludioxonil-Resistance Mechanisms.

Botrytis cinerea, the causal agent of gray mold, is one of the most destructive pathogens of cherry tomatoes, causing fruit decay and economic loss. Fludioxonil is an effective fungicide widely used for crop protection and is effective against tomato gray mold. The emergence of fungicide-resistant strains has made the control of B. cinerea more difficult. While the genome of B. cinerea is available, there are few reports regarding the large-scale functional annotation of the genome using expressed genes derived from transcriptomes, and the mechanism(s) underlying such fludioxonil resistance remain unclear. The present study prepared RNA-sequencing (RNA-seq) libraries for three B. cinerea strains (two highly resistant (LR and FR) versus one highly sensitive (S) to fludioxonil), with and without fludioxonil treatment, to identify fludioxonil responsive genes that associated to fungicide resistance. Functional enrichment analysis identified nine resistance related DEGs in the fludioxonil-induced LR and FR transcriptome that were simultaneously up-regulated, and seven resistance related DEGs down-regulated. These included adenosine triphosphate (ATP)-binding cassette (ABC) transporter-encoding genes, major facilitator superfamily (MFS) transporter-encoding genes, and the high-osmolarity glycerol (HOG) pathway homologues or related genes. The expression patterns of twelve out of the sixteen fludioxonil-responsive genes, obtained from the RNA-sequence data sets, were validated using quantitative real-time PCR (qRT-PCR). Based on RNA-sequence analysis, it was found that hybrid histidine kinase, fungal HHKs, such as BOS1, BcHHK2, and BcHHK17, probably involved in the fludioxonil resistance of B. cinerea, in addition, a number of ABC and MFS transporter genes that were not reported before, such as BcATRO, BMR1, BMR3, BcNMT1, BcAMF1, BcTOP1, BcVBA2, and BcYHK8, were differentially expressed in the fludioxonil-resistant strains, indicating that overexpression of these efflux transporters located in the plasma membranes may associate with the fludioxonil resistance mechanism of B. cinerea. All together, these lines of evidence allowed us to draw a general portrait of the anti-fludioxonil mechanisms for B. cinerea, and the assembled and annotated transcriptome data provide valuable genomic resources for further study of the molecular mechanisms of B. cinerea resistance to fludioxonil.

Identification and Characterization of Colletotrichum Species Associated with Cherry Leaf Spot Disease in China.

Leaf spot is a common and serious disease of sweet cherry worldwide and has become a major concern in China. From 2018 to 2020, disease investigations were carried out in Beijing City, Sichuan, Shandong, and Liaoning Provinces in China, and 105 Colletotrichum isolates were obtained from diseased samples. Isolates were identified by morphological characterization coupled with multigene phylogenetic analyses based on six loci (internal transcribed spacer region, glyceraldehyde 3-phosphate dehydrogenase, calmodulin, actin, chitin synthase, and ß-tubulin). A total of 13 Colletotrichum species were identified, namely Colletotrichum aenigma, C. gloeosporioides, C. fructicola, C. siamense, C. temperatum, C. conoides, C. hebeiense, C. sojae, C. plurivorum, C. karsti, C. truncatum, C. incanum, and C. dematium. Among these, C. aenigma (25.7%) was the most prominent species isolated from diseased leaves, followed by C. gloeosporioides (19.0%) and C. fructicola (12.4%). Pathogenicity was tested on detached leaves of cv. 'Tieton' and 'Summit' and young seedlings of cv. 'Brooks' under greenhouse conditions. All 13 species were pathogenic to cherry leaves, and C. aenigma, C. conoides, and C. dematium showed high levels of virulence. Seedlings inoculated with the isolates developed similar symptoms to those seen in the orchards. This study provides the first reports for 11 of the 13 Colletotrichum species on sweet cherry in the world, excluding C. aenigma and C. fructicola. This is the first comprehensive study of Colletotrichum species associated with cherry leaf spot in China, and the results will provide basic knowledge to develop sustainable control measures for cherry leaf spot.

First Report of Bacterial Wilt Caused by Enterobacter mori of Strawberry in Beijing, China.

Strawberry (Fragaria × ananassa) is an economically important crop in China, and a crucial part of urban agriculture in Beijing. In November 2020, wilt symptoms were observed in strawberry seedlings in several greenhouses in the Pinggu District of Beijing city (40.14° N; 117.12° E). The average disease incidence was 20%. Water-soaked lesions appeared along the veins of diseased strawberry leaves and bacterial ooze was also present on severely affected leaves. Bisected crowns had a reddish-brown discoloration in the xylem which later turned black. Three diseased strawberry seedlings were collected for pathogen identification. Isolations were conducted from stem, crown, leaf, and roots of diseased strawberry plants. Samples were surface sterilized by immersion in 70% ethanol for 30 s and rinsed three times with sterile distilled water, before being placed on potato dextrose agar (PDA) medium and incubated at 28℃. Several bacterial colonies grew on the medium after 24 h. Colonies were then purified on Lysogeny broth (LB) agar plates using the streak plate method. Twenty-nine isolates were obtained from 36 diseased tissue samples, which were from stem(10), crown(12), leaf(2) and roots(5) separately. All isolates appeared white, round, opaque and smooth on LB plates. To identify the isolates, genomic DNA was extracted from nine purified bacterial colonies (CM1 to CM9). The fragments of atpD, gyrB, infB and rpoB gene were amplified and sequenced with primers atpD 01-F/ atpD 02-R, gyrB 01-F/ gyrB 02-R, and infB 01-F/ infB 02-R (Brady et al. 2008) and RpoB-F/ RpoB-R (Mollet et al. 1997), respectively. All atpD, gyrB, infB and rpoB sequences belonging to the isolates were identical. The sequences of atpD, gyrB, infB and rpoB gene of isolates CM1 and CM3 were deposited in GenBank under accession numbers ON055247, ON055248, ON055249, ON055250, ON055251, ON055252, OL771192 and OL771193. BLAST searches were conducted with the sequences of atpD, gyrB, infB and rpoB. The atpD, gyrB, infB and rpoB sequences of the obtained isolate showed 99.53%, 99.06%, 99.19% and 99.80% identity with the corresponding sequences of Enterobacter mori strains, respectively. Phylogenetic analysis was performed using the maximum likelihood (ML) method with the CIPRES Science Gateway platform (http://www.phylo.org/) based on the combined atpD, gyrB, infB and rpoB sequences (Brady et al. 2013; Palmer et al. 2018). In the phylogenetic tree, the isolates were clustered together with E. mori strain LMG 25706. To confirm the pathogenicity, 200 µL of bacterial suspensions (108 CFU/mL) of the two isolates were injected into the crown of six healthy Fragaria × ananassa cv. Bennihope strawberry seedlings respectively with 1 mL sterilized syringe, and the control seedlings were injected with sterile water. The seedlings were kept in a moist chamber (28°C, 16-h light and 8-h dark period) for 2 days. Then all the seedlings were transferred to the greenhouse with conditions similar to those where the diseased plants were collected. Forty days after inoculation, old leaves started to wilt and leaf midvein necrosis, along with xylem discoloration, was observed in inoculated plants. No symptoms were observed in the control group. Pathogenicity tests were conducted three times with similar results. The bacteria were re-isolated from the symptomatic diseased strawberry plants and confirmed as E. mori by morphological and sequence analyses as above, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of strawberry bacterial wilt caused by E. mori. Due to the significant crop loss from this disease, more research is needed in epidemiology and disease management.

First Report of Fusarium commune Associated with a Root Rot of Grapevine in China.

During last decade, species belonging to Fusarium, Rosellinia, Armillaria and Dactylonectria were confirmed as phytopathogens causing grapevine root diseases (Highet and Nair 1995; Teixeira et al. 1995; Calamit et al. 2021; Ye et al. 2021). From 2020 to 2021, grapevine decline was observed in several vineyards in Beijing region, China. Leaves turned yellow with brown necrotic patches and roots were poorly developed, which was suggesting that a root disease was affecting the vines. The disease incidence was up to 10-15% of the vineyard for sample collection. Symptomatic root samples (cv. 'Red Globe') were collected and tissue fragments were excised at the margin of the symptomatic tissue in order to isolate the potential pathogen. The surface was sterilized using 1.5% sodium hypochlorite for 3 min, followed by 70% ethanol for 30 sec, and rinsed three times with sterile distilled water (Ye et al. 2020). Tissues were dried and placed onto potato dextrose agar (PDA) plates, followed by incubation at 25°C under dark conditions for 3 d. Hyphal tips of fungi growing from the samples were transferred onto new PDA plates and incubated until they produced conidia. Next, single spores were transferred onto new PDA plates and incubated at 25°C for 7 d. Eight isolates numbered with JZB3110172 to JZB3110179 were obtained and their culture characters were identical, and the re-isolation percentage was 100%. Colonies were white to orange, with abundant fluffy aerial mycelium. Macroconidia were fusiform with a slightly curved apical cell and a foot-shaped basal cell, and measured 16.2-43.2 µm × 2.7-4.9 µm (n=50); microconidia were cylindrical, straight to slightly curved, 5.1-13 × 2.1-3.9 µm (n=50). Morphological characters of the isolates resembled to Fusarium commune (Skovgaard et al. 2003). For phylogenetic analysis, genomic DNA of the eight isolates was extracted with a DNA extraction kit (DNeasy plant Mini Kit). PCR amplifications of two phylogenetic markers (EF-1α and RPB2) were performed using the primers EF-1/EF-2 (Geiser et al. 2004) and RPB2-5F2/RPB2-7cR (Liu et al. 1995), respectively. The sequences were deposited in GenBank ON457645 to ON457660. Comparison of base pairs on Maximum likelihood (ML) phylogenetic analysis was conducted using the RAxML-HPC2 tool on XSEDE on the CIPRES Science Gateway platform (http://www.phylo.org/). The sequences of EF-1α and RPB2 of the eight isolates showed 99 to 100% similarity to the reference isolates of F. commune. In the phylogenetic tree, the isolates from this study clustered with the representative strains of F. commune (NRRL 52764, NRRL 28387 and NRRL 52744). Based on morphological characters and the phylogenetic results, all of isolates were identified as F. commune. Koch's postulates were conducted on healthy, 3-month-old grapevine 'Marselan'. Plant roots were trimmed with sterile scissors and then soaked in a spore suspension (1.0 × 106 spores mL-1) or sterile water (as the control) for 30 min. The inoculated grapevines were transplanted into pots and kept in the greenhouse at 25°C. After 14 days, all the inoculated plants developed necrosis and turned yellow. No symptoms were observed on the control. Koch's postulates were fulfilled by re-isolating the fungus from necrotic root tissues. The isolates obtained from the artificially infected tissue were identified again as F. commune based on morphological and molecular analyses. Overall, this is the first report of F. commune associated with a grapevine root rot globally, which lays a foundation for further study and developing disease control methods.

Fusarium Species Associated with Cherry Leaf Spot in China.

Sweet cherry is an important fruit crop in China with a high economic value. From 2019 to 2020, a leaf spot disease was reported, with purplish-brown circular lesions in three cultivating regions in China. Twenty-four Fusarium isolates were obtained from diseased samples and were identified based on morphological characteristics and multi-locus phylogenetic analyses. Seven species, including F. luffae (7 isolates), F. lateritium (6 isolates), F. compactum (5 isolates), F. nygamai (2 isolates), F. citri (2 isolates), F. ipomoeae (1 isolate) and F. curvatum (1 isolate) were identified. The pathogenicity test showed that analyzed strains of all species could produce lesions on detached cherry leaves. Therefore, Fusarium was proved to be a pathogen of cherry leaf spots in China. This is the first report of F. luffae, F. compactum, F. nygamai, F. citri, F. ipomoeae and F. curvatum on sweet cherry in China.

Lasiodiplodia theobromae protein LtScp1 contributes to fungal virulence and protects fungal mycelia against hydrolysis by grapevine chitinase.

The LysM proteins have been reported to be important for the virulence and host immunity suppression in herbaceous plant pathogens, whereas far less information is documented in the woody plant pathogen Lasiodiplodia theobromae. To investigate the functional mechanism of LysM protein in L. theobromae, one gene LtScp1 was cloned and characterized detailedly in the current study. Transcription profiling revealed that LtScp1 was highly expressed at the infectious stages. Compared to wild type, overexpression and silencing of LtScp1 in L. theobromae led to significantly increased and decreased lesion areas, respectively. Moreover, LtScp1 was determined to be a secreted protein via a yeast signal peptide trapping system. Interestingly, LtScp1 was confirmed to be modified by the N-glycosylation, which is necessary for the homodimerization of LtScp1 molecules. Furthermore, it was found that LtScp1 interacted with the grapevine chitinase VvChi4 and interfered the ability of VvChi4 to bind chitin. Collectively, these results suggest that LtScp1 functions as a virulence factor to protect the fungus from degradation during the infection.

An NmrA-Like Protein, Lws1, Is Important for Pathogenesis in the Woody Plant Pathogen Lasiodiplodia theobromae.

The NmrA-like proteins have been reported to be important nitrogen metabolism regulators and virulence factors in herbaceous plant pathogens. However, their role in the woody plant pathogen Lasiodiplodia theobromae is less clear. In the current study, we identified a putative NmrA-like protein, Lws1, in L. theobromae and investigated its pathogenic role via gene silencing and overexpression experiments. We also evaluated the effects of external carbon and nitrogen sources on Lws1 gene expression via qRT-PCR assays. Moreover, we analyzed the molecular interaction between Lws1 and its target protein via the yeast two-hybrid system. The results show that Lws1 contained a canonical glycine-rich motif shared by the short-chain dehydrogenase/reductase (SDR) superfamily proteins and functioned as a negative regulator during disease development. Transcription profiling revealed that the transcription of Lws1 was affected by external nitrogen and carbon sources. Interaction analyses demonstrated that Lws1 interacted with a putative GATA family transcription factor, LtAreA. In conclusion, these results suggest that Lws1 serves as a critical regulator in nutrition metabolism and disease development during infection.

A Putative Effector LtCSEP1 from Lasiodiplodia theobromae Inhibits BAX-Triggered Cell Death and Suppresses Immunity Responses in Nicotiana benthamiana.

Lasiodiplodia theobromae is a causal agent of grapevine trunk disease, and it poses a significant threat to the grape industry worldwide. Fungal effectors play an essential role in the interaction between plants and pathogens. However, few studies have been conducted to understand the functions of individual effectors in L. theobromae. In this study, we identified and characterized a candidate secreted effector protein, LtCSEP1, in L. theobromae. Gene expression analysis suggested that transcription of LtCSEP1 in L. theobromae was induced at the early infection stages in the grapevine. Yeast secretion assay revealed that LtCSEP1 contains a functional signal peptide. Transient expression of LtCSEP1 in Nicotiana benthamiana suppresses BAX-trigged cell death and significantly inhibits the flg22-induced PTI-associated gene expression. Furthermore, the ectopic expression of LtCSEP1 in N. benthamiana enhanced disease susceptibility to L. theobromae by downregulating the defense-related genes. These results demonstrated that LtCSEP1 is a potential effector of L. theobromae, which contributes to suppressing the plant's defenses.

Campylocarpon fasciculare (Nectriaceae, Sordariomycetes); Novel Emergence of Black-Foot Causing Pathogen on Young Grapevines in China.

The black-foot disease of grapevine is one of the most destructive diseases in viticulture and it is caused by a complex of soil-borne fungi. This study aimed to identify the species associated with black-foot disease in young grapevines in vineyards of China. Fungal isolates were identified as Campylocarpon fasciculare, based on both morphological and multi-locus phylogenetic analysis of ITS, tef1-α and ß-tubulin sequence data. For the first time in China, we report Campylocarpon fasciculare associated with symptomatic young grapevines. Koch's postulates were performed on Vitis vinifera cv. Summer Black (SB) in a greenhouse and to confirm the pathogenicity on grapevines. This work improves the knowledge of black-foot disease in Chinese vineyards and will be helpful to growers in their decisions regarding vinicultural practices, planting and disease management.

Systemic Identification and Functional Characterization of Common in Fungal Extracellular Membrane Proteins in Lasiodiplodia theobromae.

Plant pathogenic fungi deploy secreted proteins into apoplastic space or intracellular lumen to promote successful infections during plant-pathogen interactions. In the present study, fourteen CFEM domain-containing proteins were systemically identified in Lasiodiplodia theobromae and eight of them were functionally characterized. All eight proteins were confirmed to be secreted into extracellular space by a yeast signal peptide trapping system. The transcriptional levels of most CFEM genes, except for LtCFEM2 and LtCFEM6, were significantly elevated during infection. In addition, almost all LtCFEM genes, apart from LtCFEM2, LtCFEM3, and LtCFEM6, were transcriptionally up-regulated at 35°C in contrast to that at 25°C and 30°C. As two elicitors, LtCFEM1 induced local yellowish phenotype and LtCFEM4 triggered cell death in Nicotiana benthamiana leaves. Furthermore, these proteins displayed distinct subcellular localizations when expressed transiently in N. benthamiana. Moreover, two genes, LtCFEM7 and LtCFEM8, were found to be spliced alternatively by RT-PCR and sequencing. Therefore, our data suggest that LtCFEM proteins play important roles in multiple aspects, including pathogenicity and plant immune response, which will enhance our understanding of the sophisticated pathogenic mechanisms of plant opportunistic pathogen L. theobromae.