Postharvest diseases are very severe and harm the development of kiwifruit production. 773 kiwifruits were collected from Shaanxi, Sichuan, Hubei, Shandong and Jiangxi provinces, and 739 fungal isolates were obtained. Combined the ITS sequences and the results of the pathogenicity test, 722 isolates were classified into 11 genera, namely Botryosphaeria, Diaporthe, Neofusicoccum, Alternaria, Botrytis, Fusarium, Cladosporium, Sclerotinia, Rhizopus, Lasiodiplodia, and Pestalotiopsis. Among them, Botryosphaeria dothidea and Diaporthe spp. are the dominant groups, accounting for 45.29% and 45.01%, respectively. Pathogen diversity analysis showed that pathogens were similar among the same areas.

Sugarcane leaf scald is a quarantine disease that has been reported in many sugarcane planting areas in China. It is a serious threat to sugarcane production in China. This study aimed to isolate, identify, and preserve the pathogen isolates that caused leaf scald disease in different sugarcane varieties in Guangxi. To evaluate their pathogenicity, 40 isolates of Xanthomonas albilineans were selected for genetic diversity analysis and pathogenicity assay. According to their sequence differences and phylogenetic tree analysis of housekeeping genes of ATP binding cassette transporter, atpD, gyrB, and virB gene of type IV secretion system, 40 isolates were divided into two types. Observation of the symptoms on the inoculated plants and disease index investigation revealed that the pathogenicity among 40 strains was significantly different; of which, 5 strains displayed strong pathogenicity, 4 strains had medium while 31 strains exhibited weak pathogenicity.

Paris yunnanensis is a well-known medicinal plant distributed in Southwestern China especially in Yunnan. During 2016, P. yunnanensis plants exhibiting symptoms of mottling, shrinking, necrosis and mosaic were observed and collected from Mangshi county, Dehong autonomous prefecture of Yunnan Province. Using the virus universal primers of genera Potyvirus, Tobamovirus, Luteovirus, Begomovirus and the specific primers of paris polyphylla virus X (PPVX), viral pathogens were detected in 13 symptomatic P. yunnanensis leaf samples. The result showed that the expected size amplicon was detected from the diseased plants with the Potyvirus primers of CIFor/CIRev but not with other virus primers. Cloning and sequencing analysis of the amplicons revealed that they shared 99% nucleotide (nt) sequence identity with chilli veinal mottle virus (ChiVMV) YN-tobacco isolate in the ci gene core region, suggesting that ChiVMV infection on P. yunnanensis. Ninety-three viral diseased P. yunnanensis leaf samples collected during 2016 to 2018 from counties of Longling (Baoshan City), Mangshi (Dehong autonomous prefecture), Lushui (Nujiang autonomous prefecture), Gucheng (Lijiang City), Shangri-La (Diqing autonomous prefecture) in Yunnan Province, and Yanbian county (Panzhihua City) in Sichuan Province were then tested by RT-PCR using the specific primers of ChiVMV. The results showed that except for the 8 samples from Lushui county, 25 out of 85 samples from the other five counties were infected with ChiVMV. The amplicons from ChiVMV isolates of Lijiang and Diqing were then selected for cloning and sequencing. Sequencing analysis revealed that the two isolates, designated as YLJ-CL1 (MW408193) and YDQ-CL1 (MW404246) respectively, shared 100% nt sequence identity with each other, and had 82.4%-99.1% nt sequence identity to other ChiVMV isolates in the 1 269 nt regions including the partial NIb and complete cp genes. Phylogenetic tree based on the 1 269 nt sequences of the selected ChiVMV isolates showed that the isolates from P. yunnanensis were closely related to the isolate from Yunnan tobacco. This is the first report of ChiVMV infecting the plant of genus Paris as well as the plant in family Melanthiaceae.

Corynespora cassiicola is a worldwide phytopathogenic fungus. The strawberry leaf spot caused by this pathogen poses a certain potential threat to the healthy development of the strawberry industry. Scytalone dehydratase (SCD) is a key enzyme in the melanin biosynthesis pathway of fungal dihydroxynaphthalene (DHN) melanin, which plays an important role in the pathogenesis of plant pathogens. In this study, the gene-knockout mutants of CcSCD1 were obtained by homologous recombination and verified by RT-PCR. Compared with the wild type, the knockout mutants of CcSCD1 exhibited no pigmentation, sparse hyphae, significantly decreased spore production, colorless conidia and attenuated pathogenicity. These results suggest that CcSCD1 is involved in melanin biosynthesis, vegetative growth, conidiation and pathogenicity of C. cassiicola.

Fusarium pseudograminearum is a soil-borne fungal pathogen causing Fusarium crown rot, which has been becoming one of major pathogens that threaten wheat production in China. The APSES family is found exclusively in fungi and has been reported to control several cellular processes in these organisms. In this study, we identified a conserved StuA ortholog FpStuA, which was highly expressed in conidia and infection stages. Three FpStuA deletion mutants were obtained by polyethylene glycol (PEG)-mediated protoplast stable transformation. Compared with the wild-type strain, Δfpstua mutants grew relatively slowly and had few aerial hyphae.Δfpstua mutants displayed about 70% reduction in conidiation compared to the WT. Moreover, conidia of Δfpstua mutants were smaller and fewer separation. Pathogenicity assays showed that Δfpstua mutants were dramatically decreased in virulence on barley leaves, wheat coleoptiles and wheat roots. Furthermore, deoxynivalenol production was significantly reduced in Δfpstua mutants. Taken together, these results indicated that FpStuA was important for growth, conidiation and virulence in F. pseudograminearum.

Type Ⅲ secretion system (T3SS), which deliver a group of type Ⅲ effectors (T3Es) into host cells, is required for the pathogenicity of Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit bacterial canker disease. However, it is not clear how the T3Es be used during pathogen infection. Herein, we investigated the pathogenic role of HopAZ1, one of the 14 T3Es shared by all five Psa biovars. The results showed that HopAZ1 was widely distributed in diverse P. syringae pathovars and probably was acquired before the differentiation of Psa biovars, and may suffered purify selection due to its potential important roles in pathogenesis. The hopAZ1 gene, co-regulated with T3SS, is activated during the early course of Psa infection. However, in-frame deletion of hopAZ1 resulted in increased pathogenicity on host canes. A kiwifruit yeast two-hybrid (Y2H) cDNA library was constructed and used to screen the host targets of HopAZ1, uncovering two potential targets, kiwifruit Cp1 (cysteine peptidase 1, Acc23383) and PR5 (pathogenesis-related 5, Acc28852) proteins. Taken together, we found that HopAZ1 is clearly involved in host-pathogen interaction, and may enhance the defense response of kiwifruit via interacting with Cp1 or PR5 during Psa infection. These results provide important basis for insight of HopAZ1 function and discovery of defense-related genes in kiwifruit.

The objective of this work was to analyze the distribution of Streptomyces scabies on potato plants and its population dynamics in the field. The plant samples from different growing environments, and the soil samples at different plant growth stages were collected. Two molecular techniques, regular PCR and quantitative PCR (qPCR), were used for the qualitative and quantitative analysis of the pathogens. The scab-pathogen-specific amplified band (184 bp) was detected in all diseased tissue samples including roots, stolons, tubers, stems and leaves from diseased field, greenhouse potted plants and minituber seedbeds. However, there were no such bands in the corresponding tissues of healthy plants. The qPCR detection results showed that the spore equivalents per gram of different tissue samples (diseased field/pot plants/minituber seedbed) were: leaves (7.63/8.06/20.49)×10³, stems (2.67/4.75/73.35)×10⁴, stolons (56.84/3.78/79.88)×10⁵, tubers (1.67/2.60/43.47)×10⁷, roots (2.51/51.79/8.31)×10⁵. During the different growth periods of potato in the diseased field, the spore equivalents of pathogens per gram of free soils and rhizosphere soils were (2.12-9.18)×10⁴ and (2.31-14.03)×10⁴ respectively, and the tuber swelling stage was higher than others. Further tracking by quantitative detection was conducted in order to reveal the relative amount of pathogens in different parts of diseased plants. The testing results showed that the range of spore equivalents per gram were leaves (4.62-23.76)×10³, stems (6.12-77.08)×10³, stolons (2.75-4.57)×10⁴, roots (1.41-2.36)×10⁵, tubers 3.43×10⁴-2.04×10⁷ at four stages of seedling, tuber forming, tuber swelling and harvest respectively. This study preliminarily investigated the distribution of Streptomyces scabies on potato plants and its population dynamics in the field. It laid a foundation for further study on the infection mechanism and occurrence regularity of the common scab disease.

Zucchini green mottle mosaic virus (ZGMMV), belonging to the genus Tobamovirus, was newly reported from China in 2018. In this study, we purified ZGMMV from field-grown bottle gourd sample, and inoculated bottle gourd (Lagenaria siceraria), watermelon (Citrullus lanatus), muskmelon (Cucumis melo), cucumber (C. sativus), zucchini (Cucurbita pepo), pumpkin (Cucurbita moschata), luffa (Luffa cylindrica), Nicotiana benthamiana, N. glutinosa and N. occidentalis at the seedling stage. Through the symptom observation, serological and molecular detection, the host symptoms and range were investigated. The results showed that ZGMMV could infect all tested plants. The symptoms were mosaic, mottle and chlorotic spots on the leaves of all inoculated plants. In addition, necrotic spots on stem and flesh decay were observed in watermelon, and chlorotic spots appeared on the fruit surface of muskmelon. We studied the transmission modes through pruning, drip irrigation and seeds. Results showed that ZGMMV can be transmitted by mechanical pruning, drip irrigation water and seeds. It was confirmed that one percent of seed transmission occurred in bottle gourd, watermelon and muskmelon. These results will help us to design effective prevention and control strategy for the virus.

Fusarium pseudograminearum (Fp) is one of the main pathogens of wheat Fusarium crown rot (FCR) that has been spreading and aggravating in Huanghuai winter region. In this study, from the wheat grain filling period in mid-May to the harvest of maize in Kaifeng and Jiaozuo, Henan Province, we invested and collected the sick wheat plants and 13 stubbles with perithecia in the field. The single ascospore was isolated from ascus of perithecia. Based on morphological and molecular characteristics, a total of 327 single spore cultures were identified to be F. pseudograminearumin which 164 and 163 strains belonged to MAT-1 and MAT-2, respectively. To improve sexual stage formation in the laboratory, the mating experiment was carried out using the ascospore cultures with opposite mating type to obtain the optimum conditions started from that reported abroad. The result of mating experiment showed that using Sachs synthetic medium with rice stem node, alternate black and cold white light at 22 ℃ for about 60 days, the KF86×KF73 combination produced a large number of mature perithecia and ascospores. Collectively, we used the ascospore cultures to set up mating experiment in the laboratory and obtained the optimized conditions and high fertile strains which provide methods and material for future study on gene functions at the sexual reproduction stage.

Meloidogyne graminicola is an important soil-borne pathogen of rice that causes significant damage on rice. This study aims to uncover the effect of imbalance of rice mitogen-activated protein kinase gene 5 (MPK5) homeostasis on M. graminicola-rice susceptible interaction. Firstly, according to the result of nematode infection test, it was found that the imbalance of MPK5 homeostasis caused by overexpressing and silencing MPK5 in rice led to the inhibition of M. graminicola-rice susceptible interaction. Quantitative RT-PCR assay indicated that MPK5 was up-regulated by M. graminicola infection in both MPK5-overexpressing and silencing rice plants but not in rice wild-type. Meanwhile, upon M. graminicola infection chitinase went down in MPK5-overexpressing rice plant, and the pathogenesis-related genes PR5 and PR10 went up in MPK5-silencing rice plant. These results suggest that the imbalance of MPK5 homeostasis in rice leads to the expression of MPK5 induced by M. graminicola infection, and the imbalance of MPK5 homeostasis results in rice resistance to M. graminicola through different pathways in both MPK5-overexpressing and -silencing rice plants.

In this study, we aimed to develop an efficient protoplast transformation system in Alternaria alternata. Several main parameters for isolation of protoplasts, such as the mycelial age, enzyme system, and digesting time, were analyzed and optimized for the strain LI1 of A. alternata with strong pathogenicity. The combined conditions for highest efficiency of protoplast isolation for LI1 strain were as below: the mycelia cultured in CM liquid medium for 20 hours, 0.7 mol·L^-1 NaCl as the osmotic stabilizer, enzyme mixture including 1% Driselase, 1% Lysing enzyme and 1% Snailase for 4 hours of incubation with the mycelia at 28 ℃ at 110 r·min^-1. Moreover, the regeneration of protoplasts was tested with transformation of the plasmid pCT74 DNA containing GFP reporter and the Hyg B resistant genes into LI1 by using PEG/CaCl₂-mediated method. The following confirmation of transformants by PCR and growth phenotypes indicated that the GFP gene was successfully integrated into the LI1 strain genome. In summary, we successfully developed the protoplast transformation system and generated the GFP transgenic strain of A. alternata, which would benefited for understanding of the infection, host colonization, and pathogenic mechanism of this fungus.

In order to identify the pathogen of strawberry red leaf root rot in Jiawang District of Xuzhou City, Jiangsu Province, we conducted experiments on isolation and purification, pathogenicity determination, morphological identification and molecular phylogenetic analysis. The results showed that Neopestalotiopsis roase was the pathogen. It is the first report of N. roase infecting strawberry in China.

In September 2019, an unknown rot disease was found on passion fruit of the yellow type that was marketed in Yulin, Guangxi province. The pathogens were isolated by tissue separation method from tissues of diseased passion fruits, and their pathogenicity were verified through Koch′s postulates. According to the morphological characteristics and combined with rDNA-ITS and TEF1-α gene sequences, the pathogen was identified as Lasiodiplodia theobromae. To our knowledge, this is the first repot of postharvest fruit rot in passion fruit caused by L. theobromae.

In November 2019, the typical anthracnose fruits were collected from a papaya orchard in Danzhou City, Hainan Province. The isolate 23 was isolated by tissue isolation method and purified using single-spore isolation approach. According to morphological characteristics and phylogenetic analysis of ITS, GAPDH, TUB, and ACT gene sequences, the pathogen was identified as Colletotrichum okinawense. This is the first report of C. okinawense caused anthracnose on papaya in China mainland.

The pathogen causing blast disease on Zizania latifolia in Dandong city, Liaoning province is identified as Xenopyricularia zizaniicola by the Koch’s postulate, morphological characteristics, and gene sequences analysis on actin, β-tubulin and calmodulin. A representative pathogenic isolate is similar to X. zizaniicola on conidium morphology and is clustered into one clade with X. zizaniicola in phylogenetic tree. Taken together, our findings indicate that X. zizaniicola is the pathogen of blast disease on Z. latifolia in Dandong city.

NF-Y (nuclear factor-Y) family genes play an important role in plant growth, development and response to stress. Related studies on NF-YC subunit genes of maize have not been reported. In this study, 17 NF-YC subunit genes in silicon cloning and bioinformatics methods were divided into three subfamilies, encoding product of CBFD_NFYB_HMF domain by conserved domain analysis. During maize different developmental stages and different tissues, NF-YC subunit genes exhibited significantly different expression levels. The maize NF-YC subunit genes were detected with obvious differences in their expression level under different abiotic stresses and Fusarium Verticillioide infection. Based on qRT-PCR results, the expression levels of NF-YC subunit genes were significantly changed after SA, JA and ET hormones treatments. Collectively, NF-YC subunit genes display conserved functions in maize growth and development as well as resistance to abiotic and biotic stresses, laying a foundation for elucidating the mechanism of NF-YC subunit genes in maize.

Rehmannia glutinosa plants with diseased stems were collected from a R. glutinosa plantation in Wen County, Henan Province, and 4 strains were isolated from the diseased stem samples. Morphological observation, pathogenicity test and phylogenetic analysis of internal transcribed spacer (ITS), elongation factor 1-α (EF1-α), β-tublin (TUB), calmodulin (CAL) and histone H3 (HIS) genomic loci were conducted to identify the pathogen. The results indicated that one of the strains was identified as Diaporthe amygdali, which was a new pathogen of R. glutinosa stem diseases. The study of biological characteristics showed that PDA was the most suitable medium for the growth of D. amygdali. The optimal temperature for the mycelium growth was 25 ℃, and the optimal pH value was 6. D. amygdali was not sensitive to light. The optimal carbon and nitrogen sources for mycelium growth were maltose and peptone, respectively. The bioassays were carried out and the results showed that all of the ten tested fungicides had inhibitory effects on the mycelium growth of D. amygdali, where 500 g·L^-1 Fluazinam SC had the highest activity (EC₅₀=0.092 5 mg·L^-1). The results presented herein provide potentially useful information toward developing effective management strategies to control R. glutinosa stem diseases caused by D. amygdali.

Mulberry vein banding associated virus (MVBaV), the main pathogenic virus of mulberry viral disease in Guangxi Zhuang Autonomous Region, belongs to the genus Orthotospovirus in the family Tospoviridae. The nucleocapsid protein (N) gene of MVBaV was cloned into prokaryotic expression vector pET-30a and the construct was used to transform Escherichia coli strain BL21 (DE3). The His-tagged fusion protein with an apparent molecular weight of 36 kDa was expressed after induction by IPTG. The Ni-NTA resin purified fusion protein was used to immunize Japanese big-ear rabbits. The prepared polyclonal antibody had a titer of 1∶256 000 as tested with the indirect enzyme-linked immunosorbent assay (ELISA). Western blot analysis showed that the antibody in dilution of 1∶1 000 reacted specifically with prokaryotic expressed fusion protein and protein sample prepared from the MVBaV-infected mulberry leaves, indicating that the antibody had a relatively high efficiency and specificity. In ELISA assays with the antibodies in dilution of 1∶2 500, MVBaV infected mulberry could be detected. The successful development of polyclonal antibody against the N protein of MVBaV provides an important reagent for diagnosis and surveillance of MVBaV in the field, and also for the study of interaction between MVBaV and mulberry.

Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most important fungal diseases of wheat. F. graminearum produces deoxynivalenol (DON) during plant infection which is harmful to human and animal health. In this study, we functionally characterized a histone acetyltransferase FgHat1 and found that it was localized in the nucleus to acetylate histone 4. Although the Fghat1 deletion mutant was normal in vegetative growth, asexual and sexual development, and pathogenicity, it had severe defects in DON production. Not only TRI genes expression, but also DON associated cellular differentiation was affect by deletion of FgHATI. Exogenous treatment of cyclic adenosine monophosphate (cAMP) rescued the defects of Fghat1 deletion mutant in DON production, indicating a relationship between FgHat1 and cAMP signaling. Taken together, FgHat1 is associated with cAMP signaling to regulate DON biosynthesis in F. graminearum.

Pokkah boeng disease, causing agent Fusarium sacchari, is a serious disease on sugarcane and causes substantial reduction in the yield and the quality. It is of great significance to elucidate the pathogenic mechanism, which can provide insights for sugarcane disease resistance breeding. Our studies have found that NLP (Nep1-like protein) genes play important roles in fungal infection and colonization. Based on genome sequence of the pathogen, four NLP genes (Fs_00548, Fs_03159, Fs_06646, and Fs_11062) were selected out through sequence similarity using BLASTP. Among these genes, only Fs_00548 induce cell death in Nicotiana tabacum leaves by transient expression, as consistent with the Bax-induced apoptotic cell death. Moreover, yeast invertase secretion assay showed that Fs_00548 was a secreted protein with a signal peptide that played a key role in the pathogenesis. Infectivity rate of Fs_00548 was further confirmed through qRT-PCR analyses, which reported the maximum expression of Fs_00548 at 72 hours post infection. Altogether, these results suggest that Fs_00548 plays an important role in the infection process of F. sacchari and provide understanding for further research on the interaction between sugarcane and F. sacchari.

In this study, the gene UeRbf1 (GenBank No. MW375682) was cloned based on the whole genome sequence of Ustilago esculenta. The UeRbf1 ORF is 1 281 bp without intron, encoding 426 amino acids containing three C₂H₂ zinc finger domains, suggesting a potential transcriptional regulatory function. We deleted the UeRbf1 gene, which the knockout mutants showed no detectable alterations in phenotype, growth rate, along with the cell fusion and hyphal formation ability of the compatible haploid △UeRbf1 in vitro. However, the filamentous growth of the △UeRbf1 was severely reduced in vitro fusion test and the invasive test, leading to no swollen stem formed after inoculation. Above results support that UeRbf1 is required for pathogenesis of U. esculenta, including filamentous growth and the virulence. In addition, the expressions of the genes Biz1, Clp1, Hdp1, and UeKpp6, involving in the regulation of filamentous growth and the virulence, were decreased during fusion in vitro and the infection of the △UeRbf1, in which the expression of Biz1 was almost no detectable. These data implied that UeRbf1 function on filamentous growth and the virulence in U. esculenta might be through regulating of the factor Biz1.

In order to determine whether the Pouzolzia zeylanica plants with yellow and mosaic symptoms in Yunnan Province were infected by begomoviruses, PCR amplification, cloning and sequencing were conducted to determine the types of pathogens in these samples. The results showed that the pouzolzia golden mosaic virus (PouGMV) was identified from these diseased plants. The PouGMV isolate had a genome organization typical of monopartite begomoviruses and was most closely related to the PouGMV (KC857508) identified from Vietnam (with the highest sequence identity of 94.6%), and relatively distant from other PouGMV isolates (with the highest sequence identity of 88.9%-93.2%). Alphasatellite detection showed that the alphasatellite molecule was also identified from these samples, and its full-length nucleotide sequences were most similar to tomato yellow leaf curl Yunnan alphasatellite (TYLCYnA), with the highest sequence identity of 92.3%. The results of our study showed that PouGMV was isolated from Pouzolzia zeylanica plants, and heterologously associated TYLCYnA satellite molecules. This is the first report of PouGMV associated alphasatellite infecting Pouzolzia zeylanica plants.

Meloidogyne enterolobii is a type of root-knot nematode, recently found in China. It is known to spread rapid, infecting a wide host range and has strong pathogenicity as well. In order to explore the pathogenicity of M. enterolobii, we used M. enterolobii and M. incognita to inoculate VFNT and Rutgers as nematode-resistant and susceptible tomato varieties, respectively. After 30 days, the cumulative invasion rates of VFNT and Rutgers by M. enterolobii were 14.1 % and 19.2 %, while the percentage of gall was 62.5 % and 81.6 %, respectively. At the same time, the cumulative invasion rates of M. incognita to VFNT and Rutgers were 0 and 18.8 %, while the percentage of gall was 0 and 64.5 %, respectively. These results indicated that the infection and pathogenicity of M. enterolobii were remarkably stronger than that of M. incognita. Through the root tissue staining and pathological section of the resistant VFNT as well as the early callose and reactive oxygen species staining of the infected root tip, it was revealed that the specific resistance induced by Mi-1 gene was not activated by the infection of the M. enterolobii. Furthermore, at the early stage of infection, the amount of callosum deposition caused by M. incognita infection was the largest at 72 h, which was 2.0 times that of M. enterolobii infection. In addition, in vitro H₂O₂ stress test displayed that M. enterolobii was more tolerant to H₂O₂ with the 10-80 μmol·L^-1 concentration range than M. incognita. The results of the study indicated that the strong pathogenicity of M. enterolobii might be attributed to its stronger infectivity and tolerance to reactive oxygen species. Additionally, its infection does not activate the specific resistance induced by Mi-1 gene and causes a lower accumulation of callose.

Propiconazole is a triazole fungicide with broad-spectrum activities. In this study, baseline sensitivity of Fusarium fujikuroi to propiconazole was firstly determined using 80 isolates collected from young diseased rice of different fields in Shaoxing of Zhejiang Province, Huaian of Jiangsu Province and Shangzhi of Heilongjiang Province by mycelial growth assay. Physiological effect of propiconazole against F. fujikuroi and its safety on rice were measured. EC₅₀ values ranged from 0.029 8 to 0.211 0 μg·mL^-1 with the average value of (0.106 7 ± 0.004 4) μg·mL^-1 for mycelial growth. After 0.1 μg·mL^-1(the average EC₅₀) or 3 μg·mL^-1(the average EC₉₀) propiconazole treated, conidial production decreased significantly and cell membrane permeability increased markedly. 3 μg·mL^-1 propiconazole did not affect the distribution of cell nucleus of F. fujikuroi but could constricte the septum, dilate the tops of the hyphae and damage the membrane and organelle structure of F. fujikuroi. When the concentration of propiconazole was less than 200 μg·mL^-1, the germination rate and plant height of rice seeds were not significantly affected, but the fresh weight of rice was increased. At the concentration of 500 μg·mL^-1, propiconazole had no significant effect on the germination rate, plant height and fresh weight of rice seeds. When the concentration reached 1 000 μg·mL^-1, the germination rate of rice decreased significantly, but there was no significant difference in plant height and fresh weight. The germination length of rice seeds decreased with the increase of propiconazole concentration. These results provide useful information for the management of rice bakanae disease by propiconazole and further increase our understanding about the mode of action of propiconazole against phytopathogens.

Several species of Ilyonectria are the main pathogens causing root rot of American ginseng,which are responsible for significant economic loss on the production of American ginseng. In this paper, four species of Ilyonectria, including I. mors-panacis, I. robusta, I. vredehoekensis and I. communis from diseased American ginseng in Jilin and Shandong provinces, were selected to compare their biological characteristics and fungicide sensitivities. Mycelial growth and sporulation of the four species were measured under different media, temperatures, carbon and nitrogen sources, and light conditions by plate culture method, respectively. The sensitivities of the tested strains to six common fungicides were determined by mycelial growth rate method. The optimum medium for mycelial growth of the four Ilyonectria species was Czapek while PDA, OA and CMA were more favorable for sporulation. The optimum carbon and nitrogen sources for mycelial growth were soluble starch and nitrate nitrogen, respectively. The optimum carbon source for sporulation were different among different species, and the optimum nitrogen source was (NH₄)₂SO₄. The optimum temperature for mycelial growth of I. vredehoekensis was 25 ℃, and that of I. robusta was from 20-25 ℃. It was 20 ℃ for the other species. While the optimum temperature range for sporulation was 15-20 ℃. All the tested strains were favorable to grow under dark conditions, and light had effects on sporulation. There was different effect of the same fungicide on the four tes-ted species. Among the six tested fungicides, all the tested Ilyonectria species were more sensitive to carbendazim with EC₅₀<0.5 μg·mL^-1, followed by tebuconazole with EC₅₀<15 μg·mL^-1. The inhibitory effect of hymexazol was worst with EC₅₀>960 μg·mL^-1. The present results give some new knowledge about characteristics of the four tested Ilyonectria species and provide reference on fungicides selection for the control of American ginseng root rot.

In order to find natural, environmental-friendly and safe biological control microorganisms against Rice Bacterial Blight (RBB), a total of 165 actinomycete strains were isolated from the rhizosphere soil of plants. Five strains of actinomycete with antagonistic resistance to Xanthomonas oryzae pv. oryzae (Xoo) were screened by coculture and Oxford cup methods. Among them, the Sl-10 strain exhibited the strongest antibacte-rial activity (inhibition zone diameter 62.1 mm ± 1.5 mm). According to morphological characteristics, physiological and biochemical experiments as well as 16S rDNA sequencing and phylogenetic analysis, the Sl-10 strain was identified as S. lavendulae. The S. lavendulae Sl-10 fermentation broth showed biological activities against 7 kinds of plant pathogenic bacteria including P. syringae pv. glycinea, P. syringae pv. tabaci, X. campestris pv. campestris, X. axonopodis pv. glycines, X. oryzae pv. oryzicola, R. solanacearum and P. syringae pv. tomato. The S. lavendulae Sl-10 fermentation broth possessed light resistance, acid-base resistance and heat stability. The result of SDS-PAGE revealed that the S. lavendulae Sl-10 may exert an antagonistic effect by inhibiting Xoo protein synthesis. Four rice varieties (Yongyou 15, Xiangliangyou 900, Jiafeng 2 and Yongyou 1540) which have been sprayed with the fermentation broth of S. lavendulae Sl-10 could effectively prevent the occurrence of RBB, and the leaf disease spot inhibition rate of 4 rice varieties reached 82.27%-91.78%. The above results show that S. lavendulae Sl-10 has broad application potential for the biological control of RBB.

Single factor test and response surface analysis were used to optimize the fermentation process of Bacillus amyloliquefaciens ZF57. Results showed that the optimal medium and culture condition for strain ZF57 were as follows: sorbitol 10 g·L^-1, cottonseed cake powder 17 g·L^-1, NaH₂PO₄ 0.5 g·L^-1、Na₂HPO₄ 0.4 g·L^-1, pH 7.0, 80 mL medium in 250 mL flask, inoculum concentration 2.1%, 30 ℃, 180 r·min^-1, 32 h. Under the optimized condition, the yield of strain ZF57 reached to 3.8 × 10⁸ CFU·mL^-1, increased by 80% in comparison with the initial fermentation process. Screened a nutritional auxiliary and the optimum formulation of Bacillus amyloliquefaciens ZF57 micropowder. 50% of satrin ZF57 mother powder as active ingredient, 10% silica as second carrier, 1% sodium ligninsulfonate as dispersant, 4% sodium dodecyl sulfate as surfactant, 1% carboxymethylcellulose as protector, diatomite makes up to 100%. The B. amyloliquefaciens ZF57 micropowder has a particle size of 8.31 μm and a dispersion index of 98.16%. The planktonic index is 85.37, the water content is 1.42%, The slope angle is 68°. These results were in line with formulation standard. And the control effect of Bacillus amyloliquefaciens ZF57 powder on cucumber coryneform leaf spot was as high as 97.12%.

Gray mold (Botrytis cinerea) is an important disease of tomato and causes huge economic loss to tomato production. Microbial fungicide is proved as an alternative and environmentally friendly method to suppress the gray mold. In this study, bacterial strain HMB19198, a potential biocontrol agent for tomato gray mold was obtained based on the inhibitory test against B. cinerea in vitro and suppressing ability against gray mold in the greenhouse. Using 16S rDNA sequences, strain HMB19198 was preliminarily identified as Bacillus genus and further identified as B. subtilis species based on the phylogenetic tree constructed using multiple genes sequences of gyrA, gyrB, rpoB, and rpoC. The lipopeptide extract of strain HMB19198 showed strong inhibitory effect against the growth of B. cinerea in vitro as well as in vivo. The active compounds were isolated from the lipopeptide extract using high-performance liquid chromatography (HPLC) and identified by UPLC-Triple TOF-MS/MS. Results showed the lipopeptide extract contained fengycin and surfactin, and only the fengycin showed strong inhibitory effect against B. cinerea and caused abnormality of the hypha. The fengycin produced by strain HMB19198 contained isoforms of fengycin A (C15-C18) and fengycin C (C19-C20). Bioinformatic analysis further revealed that the genome sequence of strain HMB19198 carries a gene cluster for the synthesis of fengycin.

To study the effects of compound microbial fertilizer on replanted apple trees, the mechanisms of compound microbial fertilizer were explored based on the enzyme activity, structure and function of fungi community from the rhizosphere soil. In this study, the replanted apple trees were applied with Kimidori microbial manure (KMM) and organic fertilizer control (CK) in spring and autumn. The growth of apple trees, changes of enzyme activities as well as structure and function of the fungal community in rhizosphere soil from replanted apple trees were studied systematically by agrochemical analysis of soil and high-throughput sequencing of Illumina MiSeq. The results showed that plant height, stem diameter, chlorophyll, branch number, and branch length of replanted apple saplings applied with KMM were significantly increased compared with CK. Also soil enzyme activities (neutral phosphatase, urease, sucrase, and catalase) increased. However, soil fungal community richness was decreased and soil fungal community structure was also changed. There were 21 fungi genera with the relative abundance of the higher than 1% related with five growth parameters of replanted apple trees. The abundance of Lophistoma, Plectosphaerella, Nectria, and Vishniacozyma increased significantly, while Angullospora and Pseudeurotium decreased significantly. Based on the analysis of the function of the sequenced fragments, the biosynthesis of amino acids, nucleosides, and nucleotides in the fungal community were significantly reduced after the application of KMM. Electron transfer, fermentation, respiration, and the relative abundance of functional sequences of pyrimidine deoxyribonucleic acid biosynthesis by CTP were also reduced. The application of compound microbial fertilizer could change fungal community structure, increase soil enzyme activity, decrease the fungal community’s metabolic function, and promote the growth of replanted young apple trees.

The objective of this study was to develop a simple and rapid method for detection of Bipolaris sorokiniana by loop-mediated isothermal amplification (LAMP). LAMP primers were designed based on the internal transcribed spacer (ITS) sequence of nuclear ribosomal DNA. After a set of specific LAMP primers was screened from the designed primers, a method for rapid and accurate detection of B. sorokiniana was established successfully. The effect test for betafin showed that amplification systems with and without betafin has no obvious effects on amplification results. The specificity test showed that the LAMP method was able to specifically detect B. sorokiniana from 14 species of plant pathogens. The sensitivity test showed that the detection limit of the LAMP method was 10^-3 ng·μL^-1, which was 100 times higher than that of the conventional PCR detection. The utility test showed that the LAMP method was able to accurately detect B. sorokiniana isolates from different regions, including those collected from Luoyang, Anyang, Kaifeng and Handan. Under artificially inoculated conditions, B. sorokiniana could be detected accurately by the LAMP method from diseased wheat tissues at 12 h post inoculation or longer. The results indicated that the developed LAMP method was sensitive, specific and reliable, and it was suitable for early diagnosis of wheat root rot caused by B. sorokiniana.

From 2018 to 2019, we carried out the occurrence and pathogen identification of strawberry root rot in Beijing. Samples were collected for tissue isolation and purification, and three single-spore strains (JZB3310013, JZB3310014 and JZB3310015) were obtained. According to the morphological characteristics, three strains were primarily identified as Dactylonectria sp.. The phylogenetic tree showed that JZB3310013 and JZB3310014 were clustered with Dactylonectria alcacerensis (CBS 129087), and JZB3310015 was clustered with D. torresensis (CBS 129086). Based on both morphological characters described and phylogenetic analysis, JZB3310013 and JZB3310014 were identified as D. alcacerensis, and JZB3310015 were identified as D. torresensis. The assay of Koch’s postulates confirmed that both D. alcacerensis and D. torresensis were the pathogen of this disease. To our knowledge, this is the first report of D. alcacerensis causing root rot on strawberry worldwide, and D. torresensis causing root rot on strawberry in China.

During August-October 2020,a new leaf Fusarium wilt of welsh onion was found in Moxi Welsh Onion Planting Base in Shaanxi Province. Small pieces of symptomatic tissue were taken from welsh onion leaves using a tissue isolation method. Based on morphological characteristics, and phylogenetic analyses of ITS and TEF gene sequences, the pathogens were identified as Fusarium equiseti and F. proliferatum. To our knowledge, this is the first report of F. equiseti and F. proliferatum causing Fusarium wilt disease on welsh onion leaves in China as well as worldwide.

In order to explore the pathogenic bacteria of walnut black spot in Luoyang, a field survey was carried out in Luoyang walnut orchard in 2020, typical disease samples were collected, and the pathogenic bacteria were isolated by tissue separation method. After purification and culture, the pathogens were connected to healthy leaves by acupuncture method, and the pathogenicity was tested and Koch′s rule was verified. The isolated strain YSYB was identified by morphological characteristics, physiological and biological identification, and the results showed that the strain YSYB was very similar to Pantoea. Using the total DNA of the strain as a template, the 16S rDNA universal primer was used for PCR amplification. Agarose gel electrophoresis showed a fragment of about 1 500 bp in length, and sequencing results showed that the amplified 16S rDNA fragment was 1 450 bp in length. The results showed that the 16S rDNA sequence of strain YSYB was 100% similar to Pantoea agglomerans by comparison with the known sequence in GenBank, and the phylogenetic tree was clustered into the same branch. The results showed that the pathogen was P. agglomerans.

In 2018, some Impatiens balsamina plants exhibiting viral symptoms like chlorosis and mosaic were observed in the herb garden of Changchun University of Chinese Medicine in Changchun city, Jilin province. Systemic mosaic symptoms of Chenopodium amaranticolor and Nicotiana glutinosa mechanically inoculated with the sap of the diseased I. balsamina leaves were found. To further identify the pathogen of the diseased I. balsamina, high-throughput siRNA sequencing was performed and further verified by RT-PCR. The results showed that the diseased I. balsamina plants were mainly infected by alfalfa mosaic virus (AMV). The sequences of p1, p2, mp and cp genes of AMV I. balsamina (AMV-Ib) isolate were obtained by RT-PCR. Phylogenetic analysis based on the nucleotide sequence of the coat protein genes of AMV-Ib isolate showed that it is closely related to the subgroup I of AMV isolates. To our knowledge, this is the world’s first report of natural AMV infection on I. balsamina.

In August of 2019, the leaves of Rehmannia glutinosa exhibiting chlorotic patches, mosaic, and irregular yellowing symptoms were observed in Chinese planting area located in WenXian Country of Henan Province. Total RNA was extracted from R. glutinosa samples that were suspected to be infected by viruses, and the siRNAs were obtained. After siRNA library construction, high-throughput sequencing was performed. The obtained reads were assembled by Velvet Software and analyzed by BLASTn in NCBI. The deep sequencing results showed that these samples were mainly infected with cucurbit chlorotic yellows virus (CCYV). Furthermore, RT-PCR and ELISA analyses showed that CCYV could naturally infect R. glutinosa. The coat protein (CP) coding sequence of CCYV R. glutinosa isolate was cloned and sequenced. The genetic variation was analyzed by constructing the phylogenetic tree based on the CP gene sequences. This study enriched the genetic information of CCYV population and provided a basis for the monitoring and further research on CCYV infecting R. glutinosa.

In recent years, severe foliar blight appeared on watermelon in some gravel-mulched fields in Jingyuan County, Baiyin City, Gansu Province, with the disease incidence more than 70% in serious fields. In July 2018, some fungal isolates of Stagonosporopsis were isolated from the diseased leaves of watermelon in Gaowan Township, Jingyuan County, with a 100% isolation rate. The pathogenicity of isolate XG-3 was tested on watermelon and melon by artificial inoculation on detached leaves and plants. All inoculation treatments showed the diseased symptom within 24 h, while the control did not be infected. The re-isolation rate of the original isolate reached 100% in infected leaves, thereby fulfilling the Koch’s Postulates. Sporulation was not observed when isolate XG-3 was cultured on PDA and OA plates for 20 d at 20 ℃ and 25 ℃. A small amount of pycnidia was observed on the naturally infected leaves of watermelon,however abundant pycnidia produced on the diseased stems and leaves of watermelon and muskmelon by artificially inoculation. The pycnidia were sphe-rical to subspherical, (82.4-243.3) μm × (82.4-188.4) μm, with 1 to 2 ostioles, and the ostiole’s diameters were 15.7-27.5 μm. Conidia were colorless, 0-3 septa, rod-shaped, columnar, ellipsoidal, oblong, peanut-shaped and irregular shape, straight or slightly curved, constricted or not at the septum, and (5.2-28.3) μm × (2.2-6.0) μm. Type and size of conidia varied with substrates and infection conditions. BLASTn analysis showed that the rDNA-ITS sequences of isolate XG-3 (GenBank accession No. MW282128) had a 99.80% similarity with Sta. cucurbitacearum isolate 287ITS (GenBank accession No. AY293804.1). Phylogenetic analysis based on rDNA-ITS sequences placed isolate XG-3 in group Sta. cucurbitacearum, separated from Sta. citrulli and Sta. caricae. Isolate XG-3 was identified as Sta. cucurbitacearum [Basionym: Sphaeria cucurbitacearum] by morphological and molecular biological characters.

Autophagy is a highly conserved degradation and recycling process that controls cellular homeostasis, stress adaptation, and programmed cell death in eukaryotes. Emerging evidence indicates that autophagy is important for fungal growth, development, sporulation and pathogenicity. Litchi downy blight caused by Peronophythora litchii greatly threatens the healthy development of the Chinese litchi industry, which is the most serious disease affecting litchi production and storage. In this study, a total of 19 autophagy-related genes belonging to 16 different groups were identified in the plant pathogenic oomycete Pe. litchii using a genome-wide survey. Gene structures analysis revealed that the distribution, number, length and splicing phase of intron/exon organization varies significantly between orthologous ATGs of Pe. litchii. Moreover, all candidate proteins contained the conserved functional domains, while two additional ATG11 domains have been identified at the C-terminal of PlATG1a. In addition, an extra APG17 domain existed in PlATG11. Phylogenetic analysis of the multi-member subfamilies ATG1, ATG6, ATG8 and ATG18 showed that the ATG proteins are very conserved among different species with some differences in individual homologous proteins. Transcriptome data and real-time PCR analysis revealed that the ATGs are broadly expressed during the development and virulence of Pe. litchii. PlATG1a, 2, 3, 6a, 8 and 18a were specifically up-regulated in zoospores, while PlVPS34 showed a dramatically higher expression during sporangia stage. Interestingly, PlATG1b, PlATG12 and PlATG17 were up-regulated at sporangia and zoospore stages but down-regulated during infection stages. Moreover, the relative expression levels of 6 ATGs (PlATG6b, PlATG9, PlATG10, PlATG13, PlATG14 and PlVPS15) were up-regulated in different degrees during development and infection stages, whereas PlATG7, PlATG11 and PlATG18b exhibited a low expression level during infection stages. These results suggested that autophagy might play essential roles by regulating the development and infection mechanism of Pe. litchii.

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc race 4 (Foc4) is currently known as a major concern in global banana production. The protein domain analysis using a web resource SMART, revealed that myosin-1 gene contains myosin motor domain (MMD), myosin tail (TH1) and src homology domain 3 (SH3), which have high similarity to the myosin-5 gene in Fusarium graminearum. The split-marker strategy was applied to knockout the myosin-1 gene through PEG-mediated protoplast transformation with the recombinant fragments. The positive candidate mutants were obtained by PCR screening with four pairs of primers. The results demonstrated that the knockout mutants of myosin-1 showed slow growth, hyphal deformity, decreased conidia production, and significantly decreased pathogenicity to banana, while the Δmyosin-1 mutant lost its sensitivity to phenamacril. Foc4 mycelia showed growth retardation and reduction in cell viability in vitro cultures, while the mycelia exhibited multiple branching and typical conglobate structure 24 h post-treatment with dsRNA targeting the myosin-1 gene, which remained similar to the phenotype treated with phenamacril in vitro. The exogenous dsRNA mediated resistance to Foc4 infection was assessed through pre-treatment with dsRNA before artificial inoculation with Foc4 spores to pot culture banana plantlet. The dsRNA targeting myosin-1 can inhibit the external symptoms of Fusarium wilt disease, delay the time of plant death and confer plant resistance to Foc4. Taken together, myosin-1 gene might play an important role in the growth, sporulation and pathogenicity of Foc4, and externally applied dsRNA has potential to protect bananas against Foc4 infection.

Pseudomonas syringae pv. tomato DC3000 is a typical plant pathogen relying on the type III secretion system (T3SS) to cause disease. T3SS needs to cross the periplasm of the bacterial cell wall coated with peptidoglycan in order to inject the effectors into plant cells. As a T3SS-specialized lytic transglycosylase, HrpH was predicted to target on the peptidoglycan layer in the bacterial cell wall, but the relationship between lytic function and translocation of effectors is still unclear. In this study, HrpH and its truncated domains were expressed and purified to determine the peptidoglycan binding activity. HrpH could bind the peptidoglycan and the SLT domain of HrpH is the key functional domain in which the glutamic acid at position 148 is essential for peptidoglycan binding. Further studies revealed that HrpH inhibited the growth of Pst DC3000. The integrity of the cell wall of Pst DC3000 was impaired being treated with HrpH protein, which could be observed under the transmission electron microscope. These results indicate that HrpH has lytic transglycosylase activity to bind the peptidoglycan, thereby assisting the establishment of the type III secretion apparatus for effector secretion and translocation.

Fusarium pseudograminearum (Fp) is a predominant pathogen causing Fusarium crown rot in wheat, but there are few reports on the growth regulation and pathogenic mechanism. Pentatricopeptide repeat proteins (Ppr) are nuclear-encoded RNA binding proteins regulating the process of RNA maturation, which participate in the transcription and translation of mitochondrial genome. Many Ppr proteins have been well-studied in plant, animals (including human) and yeast systems. Nevertheless, the Ppr components and functions in filamentous fungi, especially plant pathogenic fungi remain largely unknown. To clarify the characteristics of the PPR gene family in F. pseudograminearum, a total of 8 PPR candidate genes were identified through genome-wide Blastp analysis, which are mainly distributed on chromosome 1, 3, and 4 with almost no introns. The Ppr proteins predicted were all hydrophilic based on the physical and chemical properties analysis ranging from 510 to 1 353 aa in length, with molecular weight of 59.09 to 152.23 kDa and isoelectric point of 5.23 to 9.69, respectively. FpPpr3 and FpPpr7 are stable proteins, while the others are unstable. The Ppr proteins were predicted to be mainly localized in mitochondria. FpPpr1, FpPpr3, FpPpr5, FpPpr6, and FpPpr7 folded obvious super-helical in 3D model. We analyzed the expression patterns of PPRs from transcriptome of the fungal vegetative and infection stages along with validation by qRT-PCR. FpPPR1, FpPPR3, and FpPPR5 were highly expressed at the vegetative hyphae stage, while FpPPR1 and FpPPR5 were significantly down-regulated at the later stage of infection. Collectively, our findings lay the base for deep understanding the roles of PPR genes in F. pseudograminearum.