Alfalfa (Medicago sativa L.) is the most important pasture crop in China in terms of its planting area and status in animal husbandry. Verticillium wilt is a devastating disease worldwide, and China has listed it as an import plant quarantine object. The causal agent was previously known as Verticillium albo-atrum and renamed Verticillium alfalfae in 2011. However, on the list of quarantine diseases for the plants imported to China, the causal organism of alfalfa Verticillium wilt was still designated as V. albo-atrum. This undoubtedly brings much confusion to the quarantine work. In China, the disease was ever found in Zhangye City, Gansu Province, in 2016, and has now been widely distributed in the Hexi Corridor, the most important commercial alfalfa production area in China. It is vital to encourage relevant researchers to pay more attention to this dangerous disease so as to strengthen integrated control measures of the disease. This paper reviews research progress on Verticillium wilt of alfalfa, including the causal pathogen species, distribution, infection cycle and management measures, and suggests future research directions.

Blister blight disease of tea (Camellia sinensis) is caused by basidiomycetous fungus which attacks the tender tea leaves, which is a critical fungal disease for tea production and seriously restricts the development of the tea industry. The pathogen is an obligate parasitic fungus and difficult to culture limiting the research processes, such as various infection means and complicated infection mechanisms. This article summarises the research progresses on disease cycle, disease epidemics, pathogen, host-pathogen interactions, integrated disease management and prospects for future research on blister blight disease.

Chitin is an important component of fungal cell wall. It imparts strength and rigidity to fungal cell wall due to its crystalline nature. Chitin plays an important role in infection-related morphogenesis of phytopathogenic fungi, such as the infection structure and the infection hyphae. During the interaction between plants and plant pathogenic fungi, chitinases secreted by plants can degrade chitin to chitin oligomers. As a pathogen-associated molecular pattern (PAMP), chitin oligomer can be recognized by the membrane-located chitin receptors to stimulate plant immune responses. To successfully infect plants, plant pathogenic fungi have evolved a variety of strategies to inhibit the chitin-triggered plant immunity responses. Specifically, plant pathogenic fungi secrete many proteins such as effector proteins, polysaccharides or chitin deacetylases, chitinases and proteases to inhibit the plant defense responses. These proteins are secreted into the apoplast or inside the cell to destroy the plant immune system, which promotes the nutrient acquisition, colonization, growth, and development of the pathogenic fungi. This review summarizes the molecular mechanism of chitin recognition in plants and the main strategies to inhibit chitin-triggered plant immune responses of plant pathogenic fungi. We also discuss future research trends in this field.

The authors in this study collected Polygonatum cyrtonema leaves with typical symptoms of leaf spot disease, isolated its fungal pathogen, identified the fungal pathogen by adopting morphological characteristics and polygene sequence analysis, probed deep into the biological characteristics of the fungal pathogen, and determined the inhibitory effect of fungicides on its colony growth using the mycelial growth rate method. As clearly illustrated by the results, the conidia of the isolated fungal pathogen are colorless, and most of them are pear or long pear-shaped, with the size of (10-12) μm × (5-7) μm. As evidently proven by the combined analysis of multiple genes, the pathogen has 100% homology with Phyllostacta capitalensis. Combined with morphological characteristics and polygene sequence analysis, the pathogen was identified as Ph. capitalensis, which is the first report of Ph. capitalensis infecting Po. cyrtonema; PDA medium is advantageous for mycelial growth and sporulation. To be specific, the mycelium grows fastest and produces the largest number of spores at 25 ℃. When the pH is 5.0 - 10.0, the mycelium can grow. The optimum pH for spore production is 7.0. The optimum carbon and nitrogen sources for mycelium growth are sucrose and trypsin, respectively, and the optimum carbon source for spore production is glucose. Nonetheless, no spore was produced when yeast, glycine, and beef extract were used as nitrogen sources. Light and dark alternation are suitable for its growth, and light conditions can facilitate the spore production of pathogen. Among the nine tested drugs, triadimefon, EC₅₀=0.292 mg·L^-1, has a strong inhibitory effect on Ph. capitalinsis. The results obtained in this study provide a theoretical basis for the control of Po. cyrtonema in production.

To investigate the causal agent of samara brown spot occurred on Acer truncatum Bunge in Guiyang City, Guizhou Province, we collected diseased samara for pathogen isolation. YB26, a representative fungal strain causing the disease through pathogenicity test, was identified as Alternaria alternata based on combined results of morphological characteristics and multigene (rDNA-ITS, Alt a1 and GAPDH) phylogenetic analysis. Then the biological characteristics of A. alternata strain YB26 were determined by testing the mycelial growth rate under different carbon and nitrogen sources, temperatures, pH values, media and light conditions. The results showed that the suitable conditions for vegetative growth of YB26 were mannitol as carbon source, beef extract as nitrogen source, Sabouraud glucose agar (SDA) as culture medium, temperature of 28 ℃, pH value of 7, and incubation under darkness. Furthermore, the inhibitory effects of 7 fungicides against strain YB26 were tested, among which 25% pyrisoxazole had the best antifungal activity, with the EC₅₀ value of 0.8323 mg·L^-1. This is the first report of samara brown spot on A. truncatum caused by A. alternata. The results provide a basis for the diagnosis and control of the disease on A. truncatum.

Pepper (Capsicum annuum L.), belonging to the Solanaceae family, is an economically important vegetable crop in China. In April 2021, severe downy mildew-like symptoms were observed on pepper in Ningxiang County, Changsha City, Hunan Province. The diseased pepper plants exhibited large, interveinal chlorotic lesions on the adaxial surface of the leaves, which coalesced and turned necrotic as disease progressed. Meanwhile, white to gray mycelia could be seen on the abaxial side. Based on morphological characteristics, rDNA-ITS and cox2 sequence analysis, and the result of pathogenicity test, the pathogen was identified as Peronospora capsici. This is the first report of downy mildew caused by P. capsici on pepper in Hunan Province, China, and the sequence information of P. capsici was for the first time deposited in the NCBI nucleotide database.

Lasiodiplodia theobromae is an important plant pathogenic fungus, infecting more than 500 species of woody plants and causing serious damages worldwide. The secreted proteins of pathogenic fungi play important roles in pathogen invasion, expansion, and colonization in plants. Although there are more reports on the involvement of classical secreted proteins in fungal pathogenicity, the roles of non-classical secreted proteins in the pathogenesis of plant pathogenic fungi have rarely been investigated. In this study, 238 candidate non-classical secreted protein-coding genes were obtained by bioinformatic prediction based on the whole genome sequence of L. theobromae. Gene functional prediction and GO enrichment analysis showed that these genes are enriched in the carbon-sulfur lyase activity pathway. The annotation results based on PHI-base showed that 15 non-classical protein-coding genes might be related to the pathogenicity of L. theobromae. Predication of subcellular location using ApoplastP showed that 4 of them were located in plant apoplast and 11 were located in plant proplastids. Reverse transcription quantitative PCR (RT-qPCR) analysis results further verified that the transcriptional expression levels of the above candidate non-classical secreted protein-coding genes were significantly changed after treatment with grapevine branch tissues, indicating that these non-classical secreted proteins may play important roles in grapevine-L. theobromae interaction. The results lay a foundation for further understanding of the pathogenic mechanism of non-classical secreted proteins of L. theobromae.

Lotus is the most important aquatic vegetable in China and has the largest cultivation area. With the expansion of cultivation and the simplification of agricultural practices, various lotus diseases have been becoming more serious, among which lotus rhizome rot disease is the most harmful and the most difficult to control. At present, there are few research reports on the pathogenesis of Fusarium spp. causing lotus rhizome rot disease around the world. From the microscopic and ultra-microscopic levels, this study revealed the changes to the rhizome and its vascular tissues during Fusarium infection. The observation of paraffin-embedded tissue sections showed that with the development of the disease, necrotic cells stained by toluidine blue appeared around the vascular bundles, and the closer to the bundles, the more numerous necrotic cells and the more densely distributed. In order to further reveal the influence of pathogen infection on vascular bundles, brightfield optical and scanning electron microscopy were used to observe the lotus fibers in the diseased lotus rhizomes. The results showed that the smooth and compact lotus fibers became rough and dispersed with the disease tending to be more severe, and a large number of galls-like protrusions and other possible metabolic attachments were attached to the surface. These changes to the vascular bundles are likely to reduce the water and nutrient transportation, and finally the aboveground parts of the plant exhibited the symptoms of water loss and wilting. The results of this study revealed the cellular biological mechanism of the disease symptom development, and provided a basis of histology for the breeding of resistant varieties in the future.

Ligustrum japonicum leaf spot occurs universally in Wenhuilu campus of Yangzhou university, Yangzhou, Jiangsu, China every spring. The aim of this study was to identify the pathogen species, determine the biological characteristics, and screen out effective pesticides for the prevention and control of the disease.The multiple assays were conducted for the pathogen identification including tissue separation and single-spore purification, pathogenicity test both in vitro and vivo to fulfill Koch’s postulates, and morphological and phylogenetic analysis based on a combined ITS, GAPDH, RPB2 and TEF1 sequence dataset, the biological characterization,and the sensitivity test to the five fungicides. We obtained the purified fungal culture which was further identified as Alternaria alternata, a new pathogen causing leaf spot on L. japonicum. The effect of various carbon and nitrogen sources for growing of the representative isolate was investigated and A. alternata isolate was suitable for growing on the optimized medium supplied with sucrose, fructose, and potassium nitrate at 25 ℃, pH 9.0. In addition, 5 fungicides were assessed on the inhibitory effect against mycelial growth of A. alternata isolate in which azoxystrobin displayed the strongest inhibitory activity with EC₅₀ value of 0.080 6 μg·mL^-1, followed byprochloraz, pyraclostrobin and tebuconazole with EC₅₀ values of 2.272 2 μg·mL^-1, 3.934 9 μg·mL^-1 and 6.400 0 μg·mL^-1, respectively, while difenoconazole exhibited the least sensitivity at EC₅₀ value of 15.486 0 μg·mL^-1. These results indicated that these fungicides could be used for the prevention and control of L. japonicum leaf spot disease.

Rhizoctonia solani AG-1 IB is an important soil-borne fungal pathogen that causes sore shin of tobacco seedlings. In this study, we obtained a new 45.9 Mbp genome sequence of R. solani AG-1 IB strain LK4 isolated from diseased tobacco seedlings using Illumina HiSeq PE 150 and Oxford Nanopore PromethION platforms. The genome consists of 112 contigs with a N50 value of 0.89 Mbp and a GC content of 52.13%. Results of genome synteny analysis showed that translocation and translocation + inversion are more common in strain LK4 as compared with those in the genome sequences of other four publicly-released R. solani AG-1 IB strains, suggesting that strain LK4 is different from the four strains in genome structure. A total of 7,978 protein-coding genes, 231 no-coding RNAs, and 5,191 repeat sequences were predicted. The NR, GO, KEGG, Swiss-Prot, COG, PHI, Signal P, CAZy, DFVF, TCDB, and P450 databases were used to annotate and categorize protein-coding genes, and 7,375 protein-coding genes were annotated at least once. This new genome assembly and annotation resource will help elucidate the pathogenic mechanism of R. solani AG-1 IB and improve the management of tobacco sore shin disease.

In recent years, branch blight has occurred in a large area in Zanthoxylum bungeanum cultivation regions of Shaanxi province, and seriously affects the growth, yield and quality of Z. bungeanum. To identify the pathogen causing branch blight of Z. bungeanum, tissue separation method and Koch’s postulate were applied to obtain the pathogen of Z. bungeanum branch blight. It was then identified as Diaporthe eres by histologic sections, morphological characteristics and the sequences of ITS, β-tubulin and TEF1-α. The in vitro toxicity tests of fungicides against D. eres showed all the tested fifteen fungicides exhibited inhibitory effects on the colony growth of D. eres with different degrees. Among the biogenic fungicides, 3% Polyoxin WP and 0.3% Matrine EC showed the strongest inhibitory effects with EC₅₀ values of 0.046 and 0.608 mg·L^-1, respectively. Among the chemical fungicides, the compound fungicides showed relatively stronger inhibitory effects, and 30% Pyraclostrobin·Tebuconazole SC, 30% Tebuconazole·Carbendazim SC and 12% Myclobutanil·Triadimefon exhibited the most obvious inhibitory effects with EC₅₀ values of 0.002, 0.115 mg·L^-1 and 0.125 mg·L^-1, respectively. The present study can provide scientific guidance for the effective control of Z. bungeanum branch blight.

In recent years, dieback disease caused by Rugonectria rugulosa is an important new disease that has seriously threatened the healthy production of Falcataria moluccana. It is critical to establish rapid and accurate molecular detection techniques for disease prevention and management. In this study, the specific primer pairs ITS-36F/ITS-324R for Rugonectria rugulosa was designed based on ITS sequences. The outer and inner primer pairs, ITS1/ITS4 and ITS-36F/ITS-324R, were used for the nested primers. With the optimized reaction conditions, the 300 bp single targeted band was amplified, and the detection limit was 1 fg·μL^-1. Rugonectria rugulosa could be successfully detected by the nested-PCR from the diseased samples. The established nested-PCR assay was rapid, specific and efficient, which could be used for monitoring and early diagnosing for the potential F. moluccana dieback disease.

The diseased plants of Achyranthes bidentata showing typical symptoms of Fusarium wilt were collected from Jiaozuo, Henan Province, China. Tissue separation method was used to obtain potential pathogenic fungal isolates, and the pathogenicity of these isolates was determined by root-dipping inoculation method. Fusarium proliferatum was identified as the causal agent of the disease based on morphological characteristics, ITS sequence and polygenic analysis (EF1-α, Tub, RPB2 and PRO1/2). F. proliferatum isolates showed maximum radial growth at 28 ℃ and pH 7.0 on oatmeal agar medium under dark conditions. The pathogen could utilize multiple carbon and nitrogen sources, with the best carbon and nitrogen sources of sucrose and peptone, respectively. To our knowledge, this is the first report of F. proliferatum causing Fusarium wilt on A. bidentata. The results provide a scientific basis for diagnosis and control of Fusarium wilt of A. bidentata. Toxicity test of the six fungicides on F. proliferatum showed that these fungicides had certain inhibitory effects on the pathogen, and tebuconazole and fludioxonil exhibited relative higher inhibitory effects, with EC₅₀ values of 3.03 and 2.36 mg·L^-1, respectively.

Fusarium pseudograminearum is one of the dominant pathogens causing wheat crown rot in China. To identify its chemotypes quickly and simply, the 4 reported methods for Fusarium chemotype identification were tested for their applicability to this pathogen. The results showed that these methods could not effectively differentiate the three chemotypes of F. pseudograminearum. In this case, we explored a new method to identify the chemotypes of F. pseudograminearum by designing specific primers. Using this new developed method, the chemotypes of 479 strains of F. pseudograminearum collected from Hebei, Shandong, Henan and Jiangsu provinces, China were identified. Besides rare NIV producers, theses isolates are mainly DON producers. Most isolates from Hebei, Shandong and Henan provinces belong to 15-ADON in chemotype, but the isolates from Jiangsu Province primarily belong to the chemotype of 3-ADON. The geographic distribution of the 3-ADON and 15-ADON isolates of F. pseudograminearum is similar to that of F. graminearum species complex.

The potato early blight caused by Alternaria solani is an important disease in potato production, which seriously affects the yield and quality of potatoes. To reveal the interaction mechanism between A. solani and its host plant potato, we screened the effector candidates produced by A. solani during its interaction with potato plant using RNA transcriptome sequencing technology and biological software (signalp-4.1, tmhmm-2.0c, phobius101 and ProtComp v3) combined with characteristics analysis of effector proteins. As a result, 137 differentially expressed candidate proteins were obtained, and four (Gene03396, Gene06670, Gene02858 and Gene06508)of them were selected for further analysis. Based on the result of RNA-seq analysis, Gene03396, Gene06670 and Gene02858 had an up-regulated expression pattern at three time points (3, 4 and 5 dpi) compared with that at 0 dpi, whereas Gene06508 had a downregulated expression pattern. The four proteins do not have a common motif according to the MEME analysis result. Transient overexpression of the target effector candidate genes in tobacco showed that Gene03396 and Gene02858 directly induced tobacco cell necrosis, and Gene06670 inhibited programmed cell death (PCD) induced by BCL2-associated X, indicating that effector candidates function by stimulating plant immunity or inhibiting host defense response. The subcellular localization results revealed that Gene03396 was located on the cell membrane, while Gene06670 and Gene02858 may be located in the intercellular space. These results provide a basis for further identifying the effector proteins of A. solani and revealing the underlying pathogenic mechanism of the pathogen.

In this study, 10 bacterial strains with inhibitory activities against Colletotrichum gloesporioides from mango were isolated from the epidermis of healthy mangoes. Six strains were identified as Bacillus subtilis, three strains were identified as B. amyloliquefaciens, and one was identified as B. velezensis based on morphological observation, physiological, biochemical, and molecular analysis. The results of dual culture method and double Petri dish assay showed that bacterial suspensions and volatile compounds (VOCs) of 10 strains had inhibitory effects against C. gloesporioides, among which the strain N-16-2 showed the best inhibitory effect (>79%). The effect of VOCs produced by 10 strains on the control of post-harvest mango anthracnose was measured in glass desiccators, and the results showed that control effects of all ten strains were stronger than 60% against mango anthracnose, with strain YM-11-C showing the best control effect of 70%. Moreover, solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) were used to identify the components of the bacterial VOCs. The results showed that the VOCs of ten bacteria mainly contained six types of compounds, including phenols, esters, ketones, olefins, alkanes, quinones, and etc. Antifungal activities of six synthetic VOCs (Diethyl Phthalate, Hexanedioic acid,bis(2-ethylhexyl) ester, Butylated Hydroxytoluene, Squalene, 2-Dodecanone, 2,5- Cyclohexadiene-1,4-dione,2,6-bis(1,1-dimethylethyl)-) were tested, and the results showed that Diethyl Phthalate, 2-Dodecanone, and 2,5-Cyclohexadiene-1,4-dione,2,6-bis(1,1-dimethylethyl)- had significant inhibitory activities, with the quinone 2,5-Cyclohexadiene-1,4-dione,2,6-bis(1,1-dimethylethyl)- showing the best inhibitory effect against mango C. gloesporioides from mango. This is the first report of inhibitory effects and control effects of VOCs produced by B. subtilis, B. amyloliquefaciens, and B. velezensis on C. gloesporioides and mango anthracnose. It is also the first report of inhibitory effects and control effects of compound 2,5-Cyclohexadiene-1,4-dione,2,6-bis(1,1-dimethylethyl)- on C. gloesporioides and mango anthracnose.

Rice blast caused by the hemibiotrophic fungi Magnaporthe oryzae is one of the most serious diseases of cultivated rice worldwide. Effector proteins play essential roles during M. oryzae-rice interaction. In our pre-vious study, a high-quality secretome database of M. oryzae has been constructed using shotgun-based proteomics approach. In this study, we characterize an uncharacterized protein with DUF967 domain (named as MoUPE4), which is predicted as a candidate effector. MoUPE4 lacks N-terminal signal peptide and is classified as a non-classically secreted protein. The expression level of MoUPE4 gene is highly induced at the early stage of M. oryzae infection. Furthermore, MoUPE4 could suppress BAX-mediated cell death and H₂O₂ accumulation in tobacco by using the Agrobacterium-mediated transient expression system. The overexpression of MoUPE4 gene in M. oryzae does not affect fungal growth, sporulation, conidia germination, appressorium formation, and sensitivity to stress factors. However, MoUPE4 overexpression significantly increases M. oryzae virulence. These results collectively suggest that MoUPE4 is a new effector that plays an important role in M. oryzae pathogenicity.

Fritillaria thunbergii Miq. is one of the traditional Chinese medicines in Zhejiang Province. In recent years, a new mildew spot disease has occurred in the main producing area of F. thunbergii of Pan′an County. The large amount of mold layer appeared on the surface of diseased stems and leaves seriously affecting the growth and development of F. thunbergii. In this study, the pathogen was isolated and the two purified cultures YZ-1 and YZ-3 were obtained, which caused the typical symptoms similar to that in the field by the pathogenicity test assay. Combined with the biological morphology which was obviously similar to Cladosporium spp. and the analysis of sequence set of ITS, ACT and EF-α gene, isolates YZ-1 and YZ-3 was identified as Cladosporium pseudocladosporioides, the pathogen of mildew spot disease of Fritillaria thunbergii. The optimum temperature and pH for mycelial growth of the pathogen were at 25 ℃ and value 7-8, respectively. Maltose was the optimum carbon source, and beef extract and tryptone were the optimum nitrogen sources. In this paper, the toxicity of three different plant-derived antifungal agents to the pathogen was also determined in laboratory. It was found that 0.5% of berberine aqueous solution exhibited the best inhibitory effect on the pathogen growth on PDA plates. This study provides a scientific basis for the identification and prevention of Cladosporium mildew of Fritillaria thunbergii.

In recent years, a new important disease on Zanthoxylum bungeanum was found in Guizhou Province, and named as the brown spot of Z. bungeanum. To identify the pathogen, the typical disease samples were collected from producing areas in Duanshan Town, Huishui County, Guizhou Province for isolation. The pathogen was obtained by tissue isolation and pathogenicity test. Based on the morphological observation and sequence analysis of internal transcribed spacer (ITS), RNA polymerase II (RPB2) and large ribosomal subunit (LSU), Alternaria tenuissima was identified as the pathogen causing Z. bungeanum brown spot. The experiments for the antifungal activity screening in vitro showed that all the ten fungicides selected in this study showed certain inhibitory effects on the colony growth of A. tenuissima, where 25% Pyraclostrobin SC, 30% Oxime·tebuconazole SC, and 60% Difenoconazole WG exhibited relatively higher inhibitory effects with the EC₅₀ values of 0.044 9 , 0.129 5 and 0.676 9 μg·mL^-1, respectively. The above results would provide a helpful guidance for effectively controlling Z. bungeanum brown spot.

Three Broussonetia papyifera samples suspected to be infected by Begomovirus, with yellow mosaic leaves, were collected from Lianping county, Heyuan city, Guangdong province. Total DNA was extracted from suspected samples individually, and was used as template for PCR detection with degenerate Begomovirus pri-mers AV₄₉₄/CoPR. The PCR detection result showed that three suspected samples were infected by Begomovirus. The full genome sequence of virus isolated from Broussonetia papyifera in Guangdong (GS-2021) was obtained by RCA amplification, followed by enzyme digestion, cloning and sequencing. GS-2021 was a bipartite virus, including DNA-A and DNA-B components. The full sequence of DNA-A (GS-2021-A) was 2 777 nt in size, and encoded seven ORFs. The full sequence of DNA-B (GS-2021-B) was 2 742 nt in size, and encoded two ORFs. GS-2021 shared the higher similarity with all isolates of clerodendrum golden mosaic China virus (ClGMCNV). GS-2021-A shared a 93.0%-93.9% identity with DNA-A of all isolates of ClGMCNV, and the highest identity (93.9%) is with the Fujian Fz7 isolate (GenBank accession number: FJ011668). GS-2021-B shared an 86.3%-89.6% identity with DNA-B of all isolates of ClGMCNV, and the highest identify (89.6%) is with the Fujian Fz7 isolate (GenBank accession number: FJ011669). GS-2021 was closely related to five isolates of ClGMCNV from Fujian, Zhejiang, Jiangsu and the United States, which belonged to the same clade. In addition, GS-2021 clustered with Fz7 isolate from Fujian in a small clade, and had the closest relationship with it. Recombination analysis showed that there was no obvious gene recombination event in GS-2021. Based on the latest demarcation threshold for Begomovirus, GS-2021 was a new strain of ClGMCNV. In this study, Begomovirus was detected on Broussonetia papyifera for the first time. The full viral genome sequence of this virus was obtained and identified as a new strain of ClGMCNV. This result shows that Broussonetia papyifera is a newly discovered natural host for Begomovirus.

In the horticultural base of Zhangzhou, Fujian, a new leaf spot disease was observed in Heliconia rostrata Ruiz & Pav. The pathogen causing this disease was identified by pathogenicity assays, morphological characteristics and gene sequence analysis on ITS, TEF and GPD. The results showed that the leaf spot of H. rostrata was caused by Bipolaris setariae. This is the first report of a leaf spot disease caused by B. setariae on H. rostrata globally, which will help to a timely detection and efficient management of Bipolaris leaf spot.

The leaves of Camellia japonica with typical symptoms of gray spot disease were collected to isolate and identify the pathogen. Through the morphological observation of the pathogen and phylogenetic analysis of LSU,ITS, TUB2 and TEF gene sequences, it was determined that the pathogen was a fungus of Pestalotiopsis sp..

Apple chlorotic leaf spot virus (ACLSV) is a viral pathogen infecting fruits of the family Rosaceae. Its pathogenesis, however, is still unknown. In this work we employed the movement protein (MP) of ACLSV, which is a gene silencing suppressor, as a bait protein to screen for interacting proteins in Nicotiana occidentalis 37B using a yeast two-hybrid technique. We constructed a cDNA library with a capacity of 9.6×10⁶ CFU and an average insert size over 1 kb, and finally screened out 15 different proteins which are involved in the process of photosynthesis, ATP metabolism, peroxides metabolism, amino acids metabolism and stress resistance, providing a basis for decoding the pathogenic mechanism of ACLSV.

To analyze the genetic diversity and molecular evolution characteristics of chili veinal mottle virus (ChiVMV) in Guizhou tobacco, 52 tobacco samples suspected to be infected with ChiVMV in this study were collected from Xingyi City, Southwest of Guizhou Province in 2021-2022. The tobacco samples were detected by RT-PCR using universal primers for detection of Potyvirus, and the derived fragments were cloned and sequenced by ChiVMV specific primers. The genetic diversity and molecular evolution of 16 ChiVMV Guizhou isolates from tobacco were analyzed basing on the sequences of coat protein (CP) gene and related sequences reported in GenBank. The results showed that the nucleotide sequence identity between CP genes of the obtained ChiVMV Guizhou isolates and those of the other 36 isolates in GenBank was 84.79%-99.65%. In the phylogenetic tree, 52 ChiVMV isolates from different origins were phylogenetically divided into 3 different clusters. The clustering results manifested obvious geographical distribution characteristics. The results of genetic diversity and evolution analysis demonstrate that all of the isolates populations showed high genetic diversity due to the influence of geographical distribution. Significant genetic differentiation and low frequency of gene exchange happened among populations. The results of our study have potential implications for the breeding of ChiVMV resistant varieties and the control of ChiVMV.

To clarify the occurrence and epidemics of Monosporascus spot root rot and its effective control, biological characteristics of the fungal pathogen M. cannonballus were studied under different media, carbon and nitrogen sources, temperatures and pH values, and their susceptibility to fungicides were determined by using the mycelial growth rate method. The results showed that the suitable medium for mycelial growth of MC8 was potato sucrose medium, oat meal agar and potato dextrose agar, and the perithecia could be produced on potato sucrose medium, oat meal agar and corn meal medium. Carbon and nitrogen sources did not significantly promote the mycelial growth of MC8, but lactose could significantly promote the production of perithecia, which was first reported worldwide. The optimum range of pH value for mycelium growth was 5~7. The optimum temperature for mycelium growth and perithecia production of M. cannonballus was 30 ℃. The pathogenicity test showed that MC8 could infect watermelon, melon, pumpkin and gourd. The fungicide sensitivity test showed that prochloraz and azoxystrobin had the best inhibitory effect on the mycelial growth of M. cannonballus strain MC8 on PDA.

Camellia anthracnose is one of the main diseases on C. oleifera, which is mainly managed by chemical control. However, the use of fungicide had caused many problems, such as environmental pollution and outbreaks of fungicide-resistance strains. In this study, the effects of different concentrations of sodium silicate, different concentrations of sodium silicate correspond to pH, and different sodium silicate concentration pH=6.7 were investigated on the germination of spores, colony diameter, and hyphal growth of C. oleifera anthracnose, pathogenesis and changes of defensive enzyme activity under artificial inoculation conditions after root irrigation and foliar spraying of sodium silicate at different concentrations. The results showed that three groups of treatments could inhibit the growth of C. oleifera anthracnose pathogen, among which the different concentrations of sodium silicate treatment group had the best inhibition effect on C. oleifera anthracnose pathogen, and the spore germination inhibition rate of 20 mmol·L^-1 sodium silicate treatment was 84.02% and the colony diameter inhibition rate was 77.61%. In the root irrigation treatment, the diameter of the disease spots on C. oleifera leaves treated with 15 mmol·L^-1 sodium silicate was the smallest. The lesion diameter was the smallest when the sodium silicate concentration was 1.5 mmol·L^-1 during spraying. After inoculation of different concentrations of sodium silicate and different treatment methods, the defensive enzyme activities of C. oleifera changed.

Fusarium sacchari is a predominant pathogen causing pokkah boeng disease (PBD) on sugarcane in Guangxi Province, China. Thus, it is of great importance to elucidate the pathogenic mechanism of this pathogen for the development of effective control strategies for PBD prevention. Proton-coupled oligopeptide transporters that transport dipeptide and tripeptide play vital roles in the growth and development of prokaryotic and eukaryotic organisms. In this study, we investigated the expression level of FsPTR2E, a gene encoding a proton-coupled oligopeptide transporter in F. sacchari, during conidiation and infection of its host, and characterized the biological phenotypes of FsPTR2E knockout mutants. The results showed that: 1) FsPTR2E was significantly up-regulated on macroconidium-induction medium (SNA + carnation leaf medium) after incubation for 24-72 h, as compared with that incubated on PDA medium; 2) the expression level of FsPTR2E was significantly decreased at 72 h post-inoculation onto sugarcane plants compared with that at the mycelial stage; 3) deletion of FsPTR2E did not significantly affect mycelial growth, conidial germination and tolerance to stress, but resulted in a significant decrease in macrocondial and microcondial production; 4) The ΔFsPTR2E mutant was enhanced in virulence on sugarcane plants compared with the wild-type strain; 5) ΔFsPTR2E grew as well as the wild-type strain on media with dipeptides as the only nitrogen source. These results demonstrate that FsPTR2E is involved in sporulation and pathogenicity in F. sacchari but may not be involved in dipeptide transport.

Geminiviruses led to a serious threat to crop production. To explore the research hotspots and frontiers of geminiviruses, 3 235 related literatures in the core collection database of Web of Science from 1978 to 2022 were selected as the research objects. The software CiteSpace was used to analyze the research hotspots, cooperation networks and cocitation networks of these literatures. The results showed that geminivirus research has entered a prosperous stage in the 21st century, and more and more researchers have devoted themselves to this field. Keyword burst detection analysis showed that “Begomovirus”, “transmission”, and “resistance” are the research hotspots in recent years. Three articles authored by Chinese scholars have high sigma values, and their research has been at the forefront. Cooperative module network analysis showed that the United States of America started early in the field of geminivirus research, whose numbers of published papers and betweenness centrality are in the leading position. The People′s Republic of China ranks the third in the world in terms of the number of published papers, but the intermediary centrality is higher than that of the United States of America which ranks first in the world, and India which ranks second in the world, indicating that geminivirus research in China is showing a positive trend of development. In China, the scholar with the largest number of articles in the field of geminiviruses is Zhou Xueping, whose position ranks second in the world, and is a representative person in the exploration stage of geminiviruses in our country. Co-citation network analysis showed that “geminivirus-BR1 gene expression”, “mosaic virus movement protein”, “retinoblastoma protein”, “whitefly-transmitted geminiviruses”, and “red blotch virus” are the research frontiers in the field of geminiviruses at different periods of development. With the research on geminiviruses going into depth, it is speculated that future research will go on keeping the focus on the mechanisms of virus transmission, breeding of virus-resistant crops, interactions of plant-virus-vector insects, and so on.

For precise and efficient control of downy mildew on grapes, we investigated the effects of the carboxylic acid amide Oomycide dimethomoph at different concentrations on the fungal diversity at healthy and di-seased grape phyllosphere, which might provide a reference for the development of microbial pesticides. Spraying assay of dimethomoph at low, medium and high concentrations on the ‘Red Earth’ grape variety was conducted during the growth and development with the same amount of sterile water as control (CK). Based on microbial diversity and bioinformatics analysis technology, the diversity of fungi in healthy and diseased grape leaves was analyzed to evaluate the effects of ovicidal agents at different concentrations. A total of 1 588 operational taxonomic units (OTUs) were obtained for the grape samples, covering 552 genera and 894 species of fungi in 10 phyla, 37 orders, 104 orders and 261 families. Treatments with moderate concentrations of dimethomoph (DM, DMP) significantly increased phyllosphere fungal diversity in healthy and morbid groups. The results of non-metric multidimensional scale analysis showed that dimethomoph had no significant effect on grape phyllosphere fungi in healthy state, but had significant effect in diseased state (P<0.05). Ascomycota and Basidiomycota are the dominant phyllosphere phyla, and Cladosporium, Alternaria, Cercospora are the dominant genera. The number of significantly changed genera was the largest by treatment with moderate concentration of dimethomoph. The phyllosphere fungi of grapes are mainly composed of saprophytic, pathogenic, and mixed trophic patterns. The pathotrophic fungi mainly consist of plant pathogens, and when grapes are infected by downy mildew and show diseased symptom, the relative abundance of other plant pathogens decreases.

To rapidly and accurately identify and differentiate four soil-borne pathogens of tobacco, including Ralstonia solanacearum, Pectobacterium carotovorum, Phytophthora parasitica, and Fusarium oxysporum, a multiplex PCR assay was established by designing and screening of specific primers for each pathogen. The primer concentration and annealing temperature of the reaction system were optimized. The specificity and sensitivity of the optimized system were tested, and its practicability in detecting pathogens in tobacco tissue, soil samples and seed samples was verified. The results showed that the optimal primer concentrations were 0.9 μL for RsRipNTF3/RsRipNTR3, 0.6 μL for PccTF4/PccTR4, 1.2 μL for PpRhTF1/PpRhTR1, and 0.8 μL for FoP450TF1/FoP450TR1, and the optimal annealing temperature was 61.1℃. In the quadruple PCR detection system, the specific target fragments of R. solanacearum, Pectobacterium carotovorum, Phytophthora parasitica and F. oxysporum with sizes of 511, 361, 669 and 840 bp could be amplified, respectively, and the detection sensitivity of pathogen DNA can reach 50 pg·μL^-1. The applicability test showed that the assay could accurately detect the pathogens in infected plant tissues and soil samples. Using the optimized reaction system, 80 commercial tobacco varieties were detected for carrying pathogen, it was found that four samples were positive for R. solanacearum or F. oxysporum, while no Pectobacterium carotovorum or Phytophthora parasitica was detected. In conclusion,the multiplex PCR detection method established in this study can sensitively, accurately and high-throughput detect four tobacco soil-borne pathogens, providing a reliable technical method for accurate diagnosis and early prevention and control of tobacco soil-borne diseases.

In order to obtain high-quality and sufficient protoplasts of Botrytis cinerea that can be used for genetic transformation, effects of multiple parameters including mycelial age, combinations of lytic enzymes, types of osmotic stabilizers, enzymatic hydrolysis temperature, and time of enzyme digestion on protoplasts preparation were studied. The optimal lytic enzyme was determined to be the combination of driselase, snailase, and lysing enzyme at an active ingredient of 1%, 0.1% and 1%, respectively, and the mycelial age, composition and concentration of osmotic stabilizer, enzyme digestion temperature, and enzyme digestion time were as follows: mycelia of JA-6 was cultivated on PDA at 25 °C for 36 h, osmotic stabilizer contained 0.6 mol·L^-1 KCl and 50 mmol·L^-1 CaCl₂, and enzyme digestion time was 3 h at 120 r·min^-1 at 28 °C. Enzymatic hydrolysis of 5 g·mL^-1 of B. cinerea mycelium can yield 1.06×10⁷ protoplasts·mL^-1 under above optimal protoplast preparation conditions. No significant differences of the colony morphology, growth rate, conidial production and pathogenicity were observed between the regenerated strain and the wild-type strain. The GFP (green fluorescent protein) gene was subsequently transformed into B. cinerea JA-6 by PEG mediated transformation. The fluorescence signal of the transformants can be stably inherited. The established protoplast preparation method in the present research would meet the requirements of genetic transformation of B. cinerea for further study.

Tomato mottle mosaic virus (ToMMV), a member of the genus Tobamovirus, showed stronger pathogenicity than tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV) in most solanaceae crops. To figure out the cytopathological changes of tobacco plants that were infected by ToMMV, we constructed a full-length infectious cDNA clone of ToMMV, and carried out transmission electron microscopy observation. The results showed that the rod-shaped virions of ToMMV were 220-310 nm in length and 16-18 nm in diameter. ToMMV virions were also observed in the seeds of ToMMV-infected Nicotiana tabacum var. Xanthi nc. ToMMV virions were dispersed or angularly-layered in cytoplasm or vacuoles. Multivesicular bodies, small vesicles, myeloid bodies, mitochondrial cristae were observed to be reduced or disappeared in both N. tabacum var. Xanthi nc and N. rustica infected with ToMMV. In addition, broken chloroplast membranes and large amount of small peripheral vesicles protruding into the disintegrated chloroplasts were observed in the infected N. tabacum var. Xanthi nc showing mosaic and distortion symptoms, while large amount of small vesicles were observed in the cells of infected N. tabacum var. Xanthi nc showing leaf narrowing. However, chloroplasts changes were less frequent in N. rustica with systemic necrosis symptoms. In this study, the construction of ToMMV infectious clone and the observation of cytopathological changes in tobacco plants infected with ToMMV, provided important bases for the further study of the pathogenesis of ToMMV and its interaction with the host.

Jackfruit (Artocarpus heteophyllus) is an important tropical fruit in Hainan. In July 2019 and June 2021, a new fruit stalk wilt on jackfruit with an average incidence of about 16% was observed in plantations in Ledong (18°52′11″N, 108°41′57″E) and Wanning (18°69′43″N,110°17′19″E). Five fungus isolates were obtained from diseased samples of two different locations, were identified as Fusarium solani based on morphological characteristics. Isolates of this study and F. solani strains available in GenBank were clustered as a distinct clade with 98% and 100% bootstrap support in phylogenetic analysis, based on sequence similarity of ITS and LSU, respectively. Upon inoculation, the isolated F. solani caused fruit stalk wilt on healthy jackfruit, and F. solani isolates were consistently reisolated from the diseased fruit stalk,thus fulfilling Koch’s postulates.To our knowledge, this is the first report of fruit stalk wilt caused by F. solani on A. heteophyllus in China.

During a survey in 2022, cucumber displayed yellowing and chlorotic symptoms on leaves in Qingzhou and Shouguang, Shandong Province. In order to investigate the virus species, eight samples were collec-ted, and total RNA was extracted from each sample. Three high quality RNA samples were mixed together for the next-generation sequencing (NGS). The NGS clean data were assembled into large contigs and then compared with the GenBank Virus RefSeq database using BLASTn software. The BLAST results were verified with reverse transcription-polymerase chain reaction (RT-PCR), confirming that the cucumber samples were infected by five species of virus, including watermelon silver mottle virus, cucumber mosaic virus, cucurbit aphid-borne yellows virus, zucchini yellow mosaic virus and watermelon mosaic virus. After cloning the full-length of S RNA (GenBank No. OQ184866), we found that the N gene of WSMoV was 828 nt in length,encoding 275 aa, sharing 92%-98% sequence identity with other WSMoV isolates. Further phylogenetic analysis showed that the N gene formed an independent cluster and was mostly related to the cucumber isolate. This is the first report that watermelon silver mottle virus infects cucumber in Shangdong Province.

To clarify the taxonomic status of the phytoplasma causing willow witches’-broom in Gansu Pro-vince, this study used nested PCR technique to clone the 16S rDNA gene using total DNA collected from different regions and species of willow witches’-broom samples as templates, and nine 1 250 bp target fragments were obtained using P1/P7 and R16F2n/R16R2; the sequences of the above genes were compared by homology, The results showed that the 16S rDNA gene of willow witches’-broom phytoplasma strain had the highest homology with Willow proliferation phytoplasma (Willow proliferation) in Inner Mongolia, both above 99.1%; the phylogenetic tree was constructed and found that the 16S rDNA gene of willow witches’-broom clustered with members of 16SrVI-A subgroup. The phylogenetic tree was constructed, and the 16S rDNA gene of willow witches’-broom clustered with members of the 16SrVI-A subgroup; and the virtual RFLP analysis reported that the highest sequence similarity with the one obtained in this study was the reference pattern of the 16SrVI-A subgroup, with a similarity coefficient around 0.98, so the willow witches’-broom phytoplasma in Gansu Province could be classified into the 16SrVI-A subgroup. Meanwhile, the phytoplasma resources were preserved by grafting and identified as vector insects based on amplification of the 16S rDNA gene in the willow shoulder reticulata.