The fourteen Alternaria isolates were obtained from muskmelon seeds using washing tests and potato dextrose agar (PDA) plate methods. After the morphological observations on colony morphology, sporulation pattern, conidia, and conidiophores, combined with sequences analysis of ITS-rDNA and the partial histone 3 gene, the results showed that Alternaria species associated with muskmelon seeds were A. tenuissima, A. alternata, and A. obovoidea. Furthermore, the pathogenicity of these Alternaria isolates on the detached leaves of muskmelon and the effect of their spore suspensions on the muskmelon seed germination were also studied. Except for A. obovoidea, the other two Alternaria species including A. tenuissima and A. alternata were pathogenic to the detached leaves of muskmelon. The conidia suspensions of A. tenuissima and A. alternata showed inhibitory effects on muskmelon seed germination. To our knowledge, this is the first detailed report about species identification and pathogenicity tests of muskmelon seedborne Alternaria spp. in China.

The research on the classification of Alternaria has made rapid progress in recent years, while the exact pathogen species responsible for Alternaria brown spot and Alternaria black rot remains to be identified. In this research, 49 strains were isolated from citrus fruits with symptoms Alternaria black rot,while 8 representative strains from citrus leaves with symptoms of Alternaria brown spot. Phylogenetic trees were constructed on the basis of the combined sequences of Alta1, endoPG, LSU, OPA10-2, OPAl-3 and OPA2-1. The study showed that both Alternaria black rot and Alternaria brown spot could be caused by more than one species of Alternaria in which Alternaria alternate is the predominant. The pathogen species of these two diseases cannot be distinguished by the phylogenetic tree, but the pathogenicity as well as ACT toxin synthesis gene can be used to differentiate isolates into groups. The two pathogenic species associated with the diseases were proposed to be named after their predominant species. The pathogen causing Alternaria brown spot should keep the original name, the tangerine pathotype of A. alternate. Accordingly, the pathogen causing Alternaria black rot should be called as A. alternata.

To understand the types of viruses infecting pepper samples in Shihezi, Xinjiang, pepper samples exhibiting significant wrinkles, chlorosis and leaf rolling were collected for LncRNA (Long noncoding RNA) sequencing. According to LncRNA sequencing data and RT-PCR detection, Bell pepper endornavirus (BPEV), Pepper cryptic virus 2 (PCV2) and Pepper mild mottle virus (PMMoV) were found from pepper samples. RT-PCR data showed that peppers in Shihezi were generally infected with BPEV and PMMoV. The sequence simila-rity and phylogenetic analysis were performed based on the BPEV RdRp nucleotide sequence and the PCV2-RNA1 containing RdRp nucleotide sequence. The results showed that the similarities between BPEV-XJ and PCV2-XJ identified in this study and the counterpart of other isolates deposited in GenBank were 89.1%-97.3% and 96.9%-99.7%, respectively. BPEV-XJ was closer to the counterpart of BPEV-TW isolate; PCV2-XJ showed the closest relationship with the counterpart of PCV2-DR. Detection of pepper-infecting BPEV and PCV2 was reported first time from Xinjiang.

In our study,four species of root lesion nematodes including Pratylenchus bolivianus, P. convallariae, P. coffeae and P. speijeri were identified by morphology and molecular biology from Ligularia rumicifolia and Convallaria majalis in the Netherlands, Ficus altissima in Thailand, and Musa sp. in Myanmar, respectively. The morphological characteristics of these four nematode species were described in detail. Morphological characteristics, including head ring, stylet, lateral field, reproductive system and tail shape, etc., are of great importance in terms of species classification. The sequences of the 28S rRNA gene D2/ D3 were obtained by using the primer pair 28S-D2A/ 28S-D3Br. The sequence analysis revealed that the genetic distance variation among the different populations of P. bolivianus from both Europe and America was small (0-0.007). By contrast, significant genetic variation of different individuals in the same population of P. convallariae was identified (0.006-0.029). The average genetic distance of different populations of P. coffeae was 0.013, while the average genetic distance between the different populations of P. speijeri was 0.010. In addition, the genetic relationship between P. coffeae and P. speijeri was close with each other, with the average genetic distance between them being only 0.026. Moreover, our study demonstrated that the D2/D3 gene sequence of the 28S rRNA gene of nematode could be used as a basis for interspecific identification of root lesion nematodes.

Glomerella leaf spot of apple (GLSA) caused mainly by Colletotrichum gloeosporioides has become a major apple leaf disease, which threatens apple growth. The CMK1-MAPK pathway plays an important role in the pathogenesis of plant pathogens. In this study, the homologous gene of C. lagenarium CMK1 in Colletotrichum gloeosporioides was cloned and designated as CgCMK1, which is 1 068 bp in ORF length, which encodes 355 amino acids. The CgCMK1 is not essential for vegetative growth, melanin pigmentation and lipid transport. The ΔCgCMK1 mutant that causes a significant decrease in conidiation is unable to form appressoria, exogenous cAMP couldn’t induce ΔCgCMK1 mutant form appressorium-like globular structures at the tips of germ tubes, protein kinase catalytic subunit CgCPK1 expressed continuously in ΔCgCMK1 mutant cannot restore the defect of appressorium formation. CgCMK1 is involved in the response to oxidative stress, but not in the response to ionic stress. The ΔCgCMK1 mutant is fully non-pathogenicity on apple leaves, even wounded leaves. Fluorescent signal showed that the fused protein CgCMK1-mCherry is distributed to the cytoplasm. These results demonstrated that CgCMK1 is involved in regulations of vegetative growth, conidium production, oxidative stress response and pathogenicity of C. gloeosporioides.

Peronophythora litchi is an oomycete, a group that shares the kingdom Straminipila with photosynthetic algae, and causes severe economic losses due to litchi downy blight disease. Two-component signal (TCS) transduction system is involved in a variety of life activities of microbes, but to date, little is known about the TCS in oomycetes. In this study, bioinformatics approaches were used to search the TCS-related proteins in Pe. litchii based on known protein sequences, and two hybrid histidine kinases (PlHK1、PlHK2) and one response regulator (PlRR1) were identified. In the phylogenetic trees, the histidine kinases and the response regulator in oomycetes were conserved and distantly evolved compared to fungi. Conserved domain analysis indicated that the histidine kinases of Pe. litchii comprised a histidine-containing phosphotransfer protein (Hpt) domain within the C-terminus, which is significantly different from the orthologs of fungi. Furthermore, real-time PCR analysis revealed that three TCS-related genes from Pe. litchii showed a relatively higher expression level during infection stages and under H₂O₂ or osmotic stress. These results could provide basic data for clarifying the function of TCS in oomycetes.

Argonaute (AGO) proteins are conserved in eukaryotes and prokaryotes, which were guided by non-coding small RNA or DNA for degradation, translation repression, or chromatin modification of the completely or partially matching targets. In this study, we analyzed AGO family genes in 127 oomycete and fungal species via bioinformatic approaches on amounts of AGO genes, functional domains, phylogenetic relationships and transcriptional patterns. We found that 51% of the analyzed species contained two AGO genes, while some species, such as those in Phytophthora genus and Chytridiomycetes class, contained at least four AGO genes. Phylogeny of AGO genes was distinct between oomycetes and fungi and the multiple copies of AGO genes might be the results of gene duplication before or after specie evolution. Nearly all AGO proteins had 6 predicted domains, i.e. N, Linker 1, PAZ, Linker 2, MID, and PIWI. Conservation of residues corresponding to the 5' and 3' nucleic acid binding sites and catalytic sites in PAZ and PIWI domains were observed in oomycete and fungi, despite of few varied individual loci. Similar transcription patterns and high levels during the infection implied conserved functions of the two paralleling orthologous AGO genes from Phytophthora sojae and Pythium ultimum. The results will provide fundamental understanding for the AGO proteins on RNA interference mechanisms and biological functions.

Magnaporthe oryzae is a devastating fungal pathogen that attacks rice and other cereals thus seriously threatening their safe production globally. Endoplasmic reticulum-associated protein degradation (ERAD) is one of the main pathways for the organisms to respond to endoplasmic reticulum (ER) stress. The ERAD pathway also plays an important role in the process of organismal growth and development. The HRD (HMG-COA reductase degradation) complex as a crucial component of ERAD is mainly composed of Hrd1, Hrd3, lectin Yos9, and other proteins. It is responsible for the recognition, transportation, and ubiquitination of misfolded proteins in the ER before their degradation in the proteasome. These cellular processes effectively modulate ER functions and promote the normal physiological metabolism of cells. Previous studies have shown that Hrd3 belongs to a single transmembrane protein, which is bound to Hrd1 and Yos9 in the ER cavity, and is responsible for substrate recognition and plays a role in stabilizing Hrd1. However, the biological function of HRD3 in M. oryzae is still unknown. In this study, we identified and characterized HRD3 in the rice blast fungus. Deletion of MoHRD3 caused defects in vegetative growth and sporulation. Furthermore, the DMohrd3 mutant exhibited a significant reduction in virulence toward both rice and barley. Moreover, deletion of MoHRD3 increased the tolerance of M. oryzae to external salt stress and osmotic stress, but it was more sensitive to ER stress and showed no significant changes to cell wall stress. Loss of MoHRD3 also led to activation of the unfolded protein response (UPR). Taken together, our results show that MoHRD3 is involved in the regulation of vegetative growth, asexual reproduction, pathogenesis and tolerance to different stress conditions.

A mass of virus-derived small interfering RNAs (vsiRNAs) are caused by the conserved antiviral mechanism of RNA silencing during the process of virus infecting plants. The vsiRNAs target the transcripts of plants to defense the RNA silencing, a conserved antiviral mechanism, for the viral successful infection. Barley yellow dwarf virus-GAV (BYDV-GAV) resulted in an important viral disease in China with yellowing and dwarfing symptoms in winter wheat. Susceptible variety of wheat ‘Xiaoyan 6’ was infected by BYDV-GAV and the vsiRNAs derived from BYDV-GAV were analyzed using deep sequencing. A total of 11 384 vsiRNAs were found, and 37 784 host target transcripts were obtained based on the vsiRNAs information. The vsiRNAs number distributed on the sense strand of BYDV-GAV genome was approximately equal to that on the antisense strand. The predominated of vsiRNAs were 21nt and 22nt in length, and the nucleotides of A and C were preferred at the 5'- terminus. Functional analysis suggested that the potential target genes of vsiRNAs were involved in a wide range of biological functions, especially the host-pathogen interaction. 25 resistance-related genes involved in host-pathogen interaction were screened for qRT-PCR verification. Among of them, 15 potential target genes of vsiRNAs were significantly down-regulated, 6 were up-regulated, and 4 were weakly down-regulated after the inoculation of BYDV-GAV. The result of qRT-PCR illustrated the reliability of deep sequencing and target gene prediction. Our data revealed that vsiRNAs involved in the host resistance gene expression and signal transduction in the BYDV-GAV infection, which is helpful to understand the molecular mechanism of interaction between BYDV-GAV and wheat.

Cotton verticillium wilt caused by Verticillium dahliae Kleb. is a soil-borne fungal disease, which is very difficult to control. We found that broccoli residues retention showed promising control effect on the disease, and the control effect reached to 62.82%. To understand the control mechanism of broccoli residues against cotton verticillium wilt, we generated a green fluorescent protein (GFP)-labeled strain, gfp-wx-1, of V. dahliae by the method of Agrobacterium transformation. The localizations of the GFP-labeled strain in cotton plant grown in growth substrate amended or un-amended with broccoli residues were observed under a confocal microscope. Results showed that there was no significant differences on biological characteristics between the strain gfp-wx-1 and a wild-type strain wx-1. But we found that the broccoli residues significantly inhibited the spread ofV. dahlia. In the cotton plant grown in growth substrate un-amended with broccoli residues, V. dahliae infected the surface and interior of the root tip on the second day after inoculation and penetrated into the stem vascular bundles on the third day. A limited amount of the pathogen was found in the first cotyledon on the 5th day, and a large number of the pathogen was found in the first cotyledon on the 7th day. It showed a rapid spread of V. dahliae in cotton plant in the late stage. However, in the cotton plant grown in growth substrate amended with broccoli residues, V. dahliae only infected the root tip surface layer on the 2nd day. The pathogen started to infect the interior of root tip on the 3th day, and it infected the stem vascular bundle on the 5th day. A limited amount of pathogen could be found in the first cotyledon on the 7th day. The pathogen expanded very slowly in the cotton plants at the late stage, and the sizes of the lesions on cotton leaves were smaller. Colonization of V. dahliae in cotton grown in growth substrate amended or un-amended with broccoli residues was detected by fluorescence quantitative PCR, and the results showed that the broccoli residues significantly reduced the colonization of V. dahliae in cotton roots. This study suggests that inhibition of the colonization of V. dahiliae in cotton plant is probably the mechanism responsible for suppression of cotton verticillium wilt by broccoli residues.

The pathogen Didymella glomerata is included in the list of Chinese quarantine pests. The diseased plant materials are the important carrier of organism for the disease transmission. The accurate, sensitive and rapid detection method at ports of entry and land border in comprehensive quarantine system would be the powerful measure for the disease prevention and control. In this study, a pair of primers and a TaqMan-MGB probe based on the actin gene sequence of D. glomerata and its similar species were designed and synthesized for real-time fluorescent PCR assay. The optimal primer concentration and probe concentration were 0.6 μmol·L^-1 and 0.6 μmol·L^-1, respectively. The detection limit of this method was 20 pg of total DNA in 20 μL reaction mixture. The real-time PCR method was rapid, sensitive and completed within a single tube, without post-PCR handling of the amplification products, which provides a valuable tool for early rapid detection and identification of D. glomerata.

In July 2017, a new shoot dieback disease, caused brown necrotic spots with black granules on the shoots in the late stage, was found in several apple orchards in Yantai, Shandong Province. The fungal pathogen was isolated on PDA plate from the infected shoots using the tissue isolation, which formed white to grey colonies bearing abundant fruiting bodies on Czapek solid media with lactose as carbon source. The purified fungal isolates La1-La3 were obtained from single spore isolation after spore tendrils exuding from pycnidia generated on the plates. This study was to identify and characterize the causal agent . Based on morphological characteristics and analysis of rDNA internal transcribed spacer (ITS), β-tubulin (TUB) and translation elongation factor 1-α (TEF1) nucleotide sequences, the fungal isolates were identified as Lasiodiplodia pseudotheobromae, a new pathogen on apple tree. The test of Koch's rule confirmed that L. pseudotheobromae was the pathogen of this disease. Effects of different temperature, pH value, carbon and nitrogen sources were tested against the growth of isolated fungus in vitro. The results showed that the optimal growth temperature and pH value were at 30℃ and pH 6-9, respectively, while the optimum carbon source was sucrose, and favourable nitrogen sources were sodium nitrate, ammonium nitrate and peptone.

To confirm the causal pathogen and damage degree of a new disease of kiwifruit-black spot in Shaanxi Province, a field investigation was conducted in Zhouzhi County and Huyi District in 2018. The pathogen was isolated from the diseased fruits and identified based on morphological characteristics, pathogenicity testing, sequence alignment and phylogenic tree analysis. The results showed that the average incidence of black spot was 65%, and the early fruiting rate was 33%. Fruits infected with black spot disease were soft-rotted after being stored for two months under low temperature conditions. The pathogenic fungi were isolated from the under skin of Cuixiang kiwifruits and the strains could produce two types of conidia, including oval α-type conidia and curved β-type conidia. Through sequences alignment of 18S rRNA, ITS, reference gene (EF1-α) and phylogenetic tree clustering analysis, it was identified for the first time that the causal pathogen causing black spot disease of Cuixiang kiwifruit in Shaanxi Province was Diaporthe eres.

Through sequencing and assembly of small RNAs, a Camellia chlorotic dwarf-associated virus (CaCDaV) was identified on a camellia plant showing chlorosis and distorting leaves from Hangzhou (CaCDaV-HZ1). The full genome of CaCDaV-HZ1 was determined and compared with that of a CaCDaV isolate from Chongqing (CaCDaV-CA1). Results showed that they shared 85.7% overall nucleotide sequence identity. Insert and delete mutations were mainly distributed in the non-coding region, whereas the coding regions mainly contained synonymous substitution. All genes except V3 have identical length among the two isolates. Additionally, the small RNA profile of CaCDaV-HZ1 was also analysed and results showed that 24 nt is the major class of viral-derived siRNA indicating that methylation is a major resistance mechanism of camellia plants against geminiviruses.

The total DNA was extracted from strawberry leaves infected with Strawberry vein banding virus (SVBV) by CTAB method. Specific primer pairs were designed to amplify the gene ORF I of SVBV-Shenyang isolate by PCR, gene ORF I was cloned into modified prokaryotic expression vector pET-32a (+)-GST, the recombinant plasmid pET-ORF I was transformed into E. coli DH5α, then the positive clones were screened and sequenced. The recombinant plasmid was extracted and transformed into Escherichia coli BL2l (DE3), the fusion protein with an approximate molecular weight of 56 kDa was obtained with IPTG induction and Ni²⁺-NTA affinity column purification. The purified fusion protein was used to immunize the rabbits to prepare the specific antiserum. The result of ELISA showed that the titer of the prepared antiserum is up to 1:520 000, Western blot analysis indicated that the prepared antiserum could reacted specifically with purified recombinant fusion protein. Not only the expression of P1 protein in SVBV infected-strawberry leaves, but also the expression of P1 protein in P1-infiltrated Nicotiana benthamiana leaves could be detected, using 2 000 times diluted antiserum.

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense race 1 (Foc1), is one of the most important diseases that causes the greatest reductions in banana yield worldwide. Secreted proteins can act as pathogenicity factors and play important roles in the Foc-banana interactions. In this study, a refined Foc1 secretome was predicted by combining several bioinformatic approaches, including SignalP, WoLF PSORT, TargetP, TMHMM and big-PI Predictor. Among the 15438 protein sequences of Foc1, a total of 988 classically secreted proteins are predicted, representing 6.40% of the total proteins. The characteristics of these proteins showed that the length of amino acids was concentrated between 101 to 500 (71.26%), the length of the signal peptides was concentrated between 17 to 20 amino acids (61.94%), signal peptide cleavage sites mainly belongs to SPase I-cleaved signal peptides (92.91%). Among the 988 classically secreted proteins, 281 carbohydrate-active enzymes were also predicted, in which glycoside hydrolases superfamily was the most numerous. In addition, 378 effector candidates were predicated with amino acid length ≤ 400 and cysteine residues ≥ 4. Quantitative PCR analysis showed that the expression of seven genes encoding the effector candidates increased significantly at transcriptional level induced by banana root extract, which further showing that these effector candidates predicted from secretomes are true effectors by experimental validation. To our best knowledge, it is the first attempt to predict Foc1 secretome and effectors on genome-wide scale, which will help to understand the mechanism of the Foc-banana interactions.

Barley yellow dwarf viruses (BYDVs) belong to the family Luteoviridae, which are mainly transmitted by aphids, causing crop losses or failures in many crops. In this study, movement protein (MP) gene of Barley yellow dwarf virus (BYDV)-PAV Qinghai isolate was integrated into a prokaryotic expression vector pDB-MBP-His, and transformed to the Escherichia coli strain Rosetta (DE3). The fusion MP (61 kDa) was induced by IPTG, and the ‘New Zealand’ rabbits were used to prepare the specific antiserum by immunization. The PAV-MP-3Flag fusion protein which was transient expressed in Nicotiana benthamiana. Western blot results showed that the titer of antiserum reached 1∶32 000 and the detection sensitivity was 1∶256. Moreover, the antiserum can produce strong serological reaction with the MP of PAV015 isolate and other BYDVs (PAS、MAV、Ker Ⅱ and Ker Ⅲ) belonging to the genus Luteovirus. The polyclonal antibody against the movement protein of BYDV-PAV Qinghai isolate prepared in this work provides the basis for exploring the function and mechanism of BYDV-PAV movement protein.

Pepper virus disease showing severe and multiple symptoms broken out in Wenchuan county of Sichuan province. Systemic mosaic,withered leaves and stem tip necrosis were observed in pepper and partial necrosis in Petunia hybrida and Nicotiana tabacum cv. Samsun NN through the method of mechanical inoculation.We found that Tomato spotted wilt virus and Pepper mild mottle virus were presented in all samples tested by small RNA deep sequencing. The two viruses were detected in all peels and some seeds of the tested samples and re-inoculation of the host followed by RT-PCR further verified that viral disease in this area was caused by co-infection of these two viruses. Pepper-infecting TSWV was reported for the first time from Sichuan region.The phylogenetic tree constructed based on TSWV N gene and PMMoV CP gene sequence respectively indicated that TSWV-WC(MK468469) was more closely related to Guizhou isolate(KP684518), whereas PMMoV-WC(MK408614) remained closest to Beijing isolate(AY859497). We supposed that outbreak of pepper virus disease in the region was related to seeds imported from other regions.

Wheat stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) seriously threatens the wheat production in our country. Therefore, to elucidate mechanism of pathogenesis is crucial to develpoment of effective disease control techniques and strategies. Pathogens are known to have secreted effectors as virulence factors and to dissect the molecular basis of regulating host immunity system is benefit for understanding the pathogensis. In our previous screening for the candidate genes encoding secreted proteins, we obtained a highly expressed gene Pst30 which encodes a protein without obvious functional domain except a signal peptide in N-terminus. qRT-PCR analysis showed that Pst30 was induced in wheat at 12 h after infection and reached the peak at 72 h; The agroinfiltration-based transient expression of Pst30^ΔSP-GFP fusion protein in tobacco, demonstrate that Pst30 localizes in the cytoplasm; This protein also could inhibit Bax-induced cell death in tobacco. Transiently expressing Pst30 in wheat via the Type Ⅲ secretion system, inhibited not only the deposition of callose caused by Pseudomonas fluorescens but also reduced the necrotic area and reactive oxygen species accumulation caused by the avirulent Pst pathotype CYR23, which resulted in the hyphal area and length increased. Taken together, it is proposed that Pst30 inhibited PAMP-triggered immunity (PTI) and Effectors-triggered immunity (ETI) of host plants to promote infection.

Wheat stripe (yellow) rust is a typical cool-ecological airborne fungal disease. It was found that after oversummering, Puccinia striiformis f. sp. tritici (Pst) at the autumn determines the primary inoculum sources in the disease winter-increasing areas. The epidemic outbreaks of wheat stripe rust are greatly influenced by the environmental key factors such as temperature and humidity. The disease investigations in recent years found that the minimum altitude of oversummering regions of Pst become lower year by year and its oversummering areas extended more widely, indicating that the thermotolerant capability of Pst has been improving greatly. Thus, wheat stripe rust epidemics study and the development of control strategy would face new challenge. The gene VosA, a member of fungus-specific Velvet transcription factor family, involves in regulation of fungal development and secondary metabolism, but little is known about its roles in regulating fungal thermotolerance. Real-time quantitative PCR analysis indicated that PstVosA1 expression was induced under high temperature during the infecting course both in thermotolerant isolate A4 and temperature sensitive isolate Peng 9, and the significantly higher expression level was detected in the tolerant one. Silencing of the PstVosA1 in the thermotolerant isolate A4 with host induced gene silencing (HIGS) approach led to significant reduce on average disease severity, uredium density and fungal biomass when inoculation at high temperature (21℃). These results showed that the PstVosA1 positively regulated thermotolerance responses, and increasing the expression level could promote thermotolerant capability in Pst.

Phytophthora infestans is the pathogenic microbe of late blight, which secrets numerous of RxLR effectors into potato during the infection. However, the function and mechanism of most RxLR effectors remain unclear. In this study, we cloned a RxLR effector of PITG_16427.2 from P. infestans. It can suppress the hypersensitive response (HR) caused by six elicitors including INF1, BAX, PsojNIP, SIF2, Avh238 and Avh241 after co-expressing in Nicotiana benthamiana. Furthermore, the expression of PITG_16427.2 is up-regulated at the early stage of infection. Homologous sequences of PITG_16427.2 were cloned from all 15 isolates of P. infestans and 3 isolates of Phytophthoras which shared over 93% identity at amino acids level. Interestingly, all homologous sequences of PITG_16427.2 could suppress the HR caused by expressing INF1 or Avh241 as well as PITG_16427.2^HLJ, indicating that they are highly conserved in both sequence and function among on the Phytophthoras. Transient expression of PITG_16427.2 in Désirée and N. benthamiana promote the colonization of P. infestans. By using qRT-PCR, we identified that the expression of ethylene signaling pathway related gene ERF1 is significantly up-regulated and the salicylic acid signaling pathway related gene PR1b is down-regulated respectively. The results imply that PITG_16427.2 may enhance the plant susceptibility by suppressing the SA signaling pathway. Overall, we concluded that the RxLR effector of PITG_16427.2 is an important pathogenic factor for late blight in P. infestans.

As transcriptional cofactors, VQ motif-containing proteins (VQ proteins) play essential regulatory functions in multiple physiological processes such as plant growth, development and resistance to stresses. The intact cDNA sequence of rice VQ37 gene was cloned by reverse transcription-PCR from leaf tissues. The whole sequence of VQ37 cDNA is 622 bp in length, with an open reading frame (ORF) of 546 bp, encoding a polypeptide of 181 amino acid residues. Motif analysis indicated that the deduced VQ37 contains a VQ motif variant: FxxxVHxVTG. Phylogenetic analysis showed that VQ37 is closely related to other Gramineae plants such as Oryza brachyantha, Hordeum vulgare and Dichanthelium oligosanthes. Except its paralogue VQ39 in rice, VQ37 shares the highest identity of amino acids with XP 015699121 from O. brachyantha. Protoplast transient expression experiment revealed that VQ37 is localized into the nucleus. Fluorescence quantitative PCR results showed that the organ-specific expression characteristics and induced expression profiles of the VQ37 gene is generally consistent with the prediction of cis-elements in the promoter. The transcript levels of VQ37 are relatively high in leaves, followed by leaf sheaths, stems, panicles, roots and flowers, whereas no transcripts were detected in embryos and endosperms of seeds. VQ37 transcription was significantly induced by blast fungus (Magnaporthe oryza), sheath blight fungus (Rhizoctonia solani) and fungal pathogen-associated molecular pattern (PAMP) — chitin oligosaccharide. Expression of VQ37 was also induced by methyl jasmonate and ethephon. However, bacterial blight pathogen (Xanthomonas oryzae pv. oryzae), bacterial PAMP flg22 and salicylic acid exerted no obvious effects on its expression. These findings suggest that VQ37 may be involved in the regulation of rice defense responses to sheath blight and blast fungi through jasmonic acid/ethylene-, but not salicylic acid -depen-dent signaling pathway. This research provides not only a foundation for elucidating the roles of VQ37 genes in the regulation of defense responses in rice, but also a gene material for reasonable utilization of pathogen-resistance.

Post-translational prenylation pathway (CAAX modification) mediates many subcellular localization of critical proteins and protein-protein interactions in eukaryotes. The rice blast fungus Magnaporthe oryzae causes one of most important diseases and results in dramatic yield loss of rice around the world. To further understand the pathogenesis and to better control the rice blast disease, we examined whether the post-translational prenylation pathway plays a role in the fungal development and pathogenicity. We identified a CAAX prenyl protease MoRce1 from M. oryzae genome database. Domain prediction revealed that the conserved domain of MoRce1 is significantly distinct to Rce1 homologs from different species, suggesting that Rce1 may evolve into different functions. Deletion of MoRCE1 through homologous recombination exhibited growth defects under cell wall stress conditions, while the vegetative growth, conidiation, germination, and pathogenicity show no significant defects in the gene deletion mutant. In summary, our results suggested that MoRce1 may participate in the modification of cell wall synthesis-related proteins but it does not play a role in the pathogenicity of M. oryzae.

Cotton leaf curl Multan virus (CLCuMV), a typical monopartite geminivirus associating with a betasatellite, is one of the major causal agents of the cotton leaf curl disease. In this study, the seven viral proteins encoded by CLCuMV and its betasatellite were expressed in leaf epidermal cells of Nicotiana benthamiana using the Agrobacterium-mediated transient expression system. The subcellular localization of the seven transiently expressed proteins was observed under a confocal laser scanning microscope (CLSM). The results showed that V1, C2, and C3 were localized mainly to the nucleus. C1 and βC1 were distributed both in the nucleus and the cytoplasm or the cytomembrane and formed filamentous structures in the cytoplasm. V2 and C4 were localized predominately in the cytoplasm or the cytomembrane, while both V2 and C4 were also found occasionally in the nucleus. In addition, V2 formed granular aggregates of varying sizes in the cytoplasm, while C4 could form punctate structures in the cytomembrane. Besides all the above studies, the transcriptional and expression levels of viral genes were analyzed by RT-PCR and Western blot. The data obtained in this study provide an important theoretical basis for further functional studies on the functions of the proteins encoded by CLCuMV.

Based on the artificial inoculation tests and the investigations in natural conditions for many years, it is found that the most rice varieties used in China were susceptible to rice false smut pathogen, Villosiclava virens. However, the disease severity in different rice varieties is quite different in paddy fields in natural conditions. Based on the number of rice false smut balls in a panicle, rice varieties can be divided artificially into the high susceptible with multiple smut balls and the relatively resistant varieties with few smut balls. To evaluate the relationship between the features in rice panicles at rice booting stage and the disease resistance, the panicle features in various rice varieties were measured and compared here. The results showed that there were significant differences in panicle size, floret density, panicle sheath closure and sealing, flag leaf area between high susceptible varieties with multiple balls and those with relatively resistant phenotype with few balls.The closure of panicle sheath in the resistant varieties was better than that in the susceptible ones.

To identify pathotypes different of Xanthomonas oryzae pv. oryzicola and rice varieties resistant to bacterial leaf streak from Yunnan Province. The virulence levles differentiation of 86 isolates of Xoc collected from 8 rice-growing areas in Yunnan province were identified, by using 6 rice varieties ( IRBB4, IRBB5, IRBB14, IRBB18, IRBB21, IR24) which each containing a single resistance gene. The cluster analysis using UPGMA method to Xoc virulence levels showed that 86 strains were classified as 9 pathotypes I-Ⅸ, Among the 9 pathotypes, the pathotype I is the dominant group and most of the pathotype I strains are SSSSSS disease phenotype. The pathotypes Ⅰ was dominant Xoc pathotype in various rice areas of Yunnan. Further research, resistance evaluation of 80 rice varieties from Yunnan Province to pathotype I strain NO.19, pathotype II strain NO.14 and pathotype VI strain NO.16,only 30.0%, 35.0% and 57.5% exhibited resistance to 3 pathotype strain respectively. 9 rice varieties showed resistance to three pathotype strains, especially Deyou16 and Changgui2 showed high resistance levels, this result will be one of the perfect ways to develop bacterial leaf streak disease resistance breeding.

Matrix stiffness is an important physical signal for the formation of infection structures in plant pathogenic fungi. In order to explore effects of matrix stiffness on the infecting ability of Setosphaeria turcica, PDA media with different stiffness was used to mimic the simulation. The results were further verified on corn leaves with different developmental ages. There are less aerial hyphae and more hyphal branches on hard matrix than those on soft one. On the hard matrix, the number of conidia was the highest, which was 29.93 and 6.82 folds of the soft one and the moderate one, respectively. The rate of appressorium formation on the hard matrix was significantly higher than that on the soft one. Moreover, the colony color was deeper and the melanin content of the hyphae was higher on the hard matrix. Real-time fluorescence quantitative PCR was used to analyze expression levels of genes involved in the melanin synthesis pathway of S. turcica. The results showed that the expression levels of StMR1, StPKS, St4HNR, StSCD, St3HNR, StLAC1, StLAC2 and StLAC4 were higher on the hard matrix than that on the soft one. The above results indicated that the matrix stiffness affected the morphological structure of S. turcica, which led to the change of melanin content and the development of infection structures. It also provided a theoretical basis for the research on infection mechanism of S. turcica and the prevention and control of this disease.

Gray mold, caused by Botrytis cinerea, is one of the main diseases on cherry flowers, leaves, and fruits in the middle and late growth period and the storage period with high incidence rate. Based on the internal transcribed spacer (ITS) sequence, a pair of primers were designed and used to develop recombinase polymerase amplification combined with a lateral flow dipstick (LFD-RPA) assay. The results showed that the optimal reaction temperature and time were 37℃ for 30 min for specific detection of B. cinerea with the sensitivity at 100 fg·μL^-1, which was slightly lower than conventional PCR at 10 fg·μL^-1 and real-time quantitative PCR at 7.43 fg·μL^-1. However, the detection process was relatively simple and rapid, just including RPA reaction in 30 min and LFD detection in 10 min. This method can be of great value for the surveillance of pathogen and early diagnosis of the disease.

Black spot disease caused by Alternaria species is an important and common disease on Panax notoginseng. In this study, 82 isolates were obtained from diseased P. notoginseng plants sampled from Yunnan and Guangxi provinces. ITS region and partial coding sequence of histone 3 gene of all isolates were amplified and sequenced to do the phylogenetic analysis. Based on molecular identification and morphological traits, the isolates were identified as Alternaria tenuissima, A. alternata and A. panax separately. This study firstly confirmed that A. tenuissima was the dominant pathogen population (62 isolates, 75.6%) associated with black spot disease in main producing areas of P. notoginseng, followed by A. alternata (12 isolates, 14.6%) and A. panax (8 isolates, 9.8%). Among these species, A. panax had the highest pathogenicity to P. notoginseng.

Virus-infecting leaf samples of Atractylodes macrocephala Koidz. were collected from Lin′an District, Hangzhou city, Zhejiang province. Small RNA (sRNA) library construction and deep sequencing were conducted with samples holding mosaic, chlorosis and dwarf symptoms. The Cucumber mosaic virus (CMV) was specially identified in the samples after assembly of the overall sRNAs. And the complete genomic fragments of CMV (CMV-ZJAm) were verified by PCR and sanger sequencing. The results showed the 3 363 nt fragment of RNA1 sharing 99.01% nucleotide similarity with that of CMV isolate JC (CMV-JC, GenBank accession number MH594044), the 3 053 nt fragment of RNA2 sharing 99.04% similarity (CMV-JC, MH594045), the 2 217 nt RNA3 sharing 97.52% similarity (CMV isolate Anhui, KX660758). Phylogenetic analysis with complete genomic sequences indicated that CMV-ZJAm, CMV-JC, CMV-Am (CMV isolate Am, JX993910) and CMV-Anhui were clustered into one group belonging to CMV subgroup I B. In conclusion, this is the first report of CMV from A. macrocephala in Zhejiang province.

Post-harvest diseases cause great losses to citrus industry. In this study, pathogen was isolated and identified from diseased fruits collected from Chenggu County, Shannxi Province in 2018. Strain CBS1 could infect Satsuma mandarin, Ponkan, Orah and Lane Late navel orange fruits and cause rot disease. Strain CBS1 and the strains recovered from the inoculated materials were identified as Rhizopus stolonifer based on the morphologi-cal characteristics and ITS sequence analysis. This is the first report that R. stolonifer infects citrus fruit, and the results will help to control post-harvest decay of citrus.

In 2018, a new spot blight disease on leaf of Paris polyphylla Smith var. chinensis was found with the incidence of about 35% or the whole leaf collapsed at favorite whether conditions in Wenchuan county, Sichuan Province. The causal agent, was isolated by tissue isolation method, and pathogenicity was tested follo-wing reisolation of the pathogen from the diseased leaves to satisfy Koch′s rule. The pathogen was identified based on morphological characteristics and sequence analysis of rDNA-ITS region and RPB2 gene. The results showed that the colony morphology, pycnidia and conidia of the isolates were similar to those of Didymella sp.. The sequences of ITS-RPB2 genes showed 100% identities with D. glomerata (No. FJ427013 and GU371781). Therefore, D. glomerata was identified as the pathogen causing the spot blight on leaf of Paris polyphylla Smith var. chinensis.

Tree canker disease is quite common on forest and fruit trees in Xinjiang and has been becoming more serious in recent years. We investigated Valsa disease on apple, walnut and poplar trees in Aksu, Khotan, Ili, Kashgar and Korla, which occurred to a certain degree, and collected the diseased samples from 14 locations for pathogen isolation. The results showed that the main pathogens identified on apple tree samples were Valsa mali var. mali (isolation frequency was 64.5%), V. mali var. pyri (25.8%), V. malicola (3.2%) and V. nivea (6.5%). The V. sordida (83.3% and 96%) and V. nivea (16.7% and 4%) were obtained from walnut and poplar tree samples, respectively. In pathogenicity test on detached branches and fruits, all strains could infect their corresponding original host plant. In conclusion, Valsa mali var. mali is the main pathogen caused Valsa canker on apple, while V. sordida is the major one caused Valsa canker on walnut and poplar in partial areas of Xinjiang. Meanwhile, we speculate a potential risk of cross infection among these three kinds of trees because the diseased poplar branches are used as support wood in orchard.

Sorghum is a major cash crop in Sichuan province, it is often seriously infected by anthracnose disease. Colletotrichum graminicola was even considered as the pathogen of sorghum anthracnose in previous literatures. In recent literatures, C. sublineola, rather than C. graminicola was identified as the causal pathogen of sorghum anthracnose based on molecular biology and genomic analysis. In order to classify the causal pathogen of sorghum anthracnose in Sichuan province and other sorghum-growing areas in China, 198 samples were collected from 11 regions such as Luzhou Lu County in Sichuan Province and 6 other provinces and cities such as Chongqing, from which 225 fungal strains were isolated. Based on morphological observation, ITS and GAPDH gene sequence analysis and adjacent tree construction, 215 strains isolated from 17 regions belong to C. sublineola with crescent shaped spores, and the resting 10 strains from other two regions belong to C. cliviicola with rhabdoid spores. It is further proved that 17 representative strains of anthracnose generated from different regions have the similar morphological characteristics and belong to C. sublineola by phylogenetic of polygenes (CHS, ACT, TUB2). These data suggest that the morphology of conidia and appresoria and the genes of ITS, GAPDH CHS, ACT and TUB2 can be used for identification of sorghum anthracnose. Finally, all of the C. sublineola strains, rather than C. cliviicola, showed pathogenic on three common sorghum varieties. The results showed that C. sublineola was the pathogen of sorghum anthracnose in Sichuan province. Furthermore, C. sublineola strain ZG-FS-1, the most infectious pathogen in 19 representative strains, was used to screen the resis-tance resources of sorghum, and we found that variety 402B show the strongest resistance against sorghum anthracnose, indicating that it has application potential in sorghum anthracnose resistance breeding.

During 2015 and 2016, severe viral disease occurred in watermelon fields in Kaifeng and Zhongmu, Henan Province. Complex symptoms include yellowing, fern leaves, shrinking, curling, topmost upwarping, fruit shrunking and fuzziness of fruit pattern, which lead to difficulty for identifying pathogens. In this research, we combined small RNA sequencing and bioinformatic analysis to reveal the viruses in collected symptomatic watermelon samples. Ultimately, eight viruses were discovered including Zucchini yellow mosaic virus (ZYMV), Melon aphid-borne yellows virus (MABYV), Watermelon mosaic virus (WMV), Cucumber green mottle mosaic virus (CGMMV) and Citrullus lanatus cryptic virus (CiLCV) which have been reported in China. Other three new viruses, Watermelon virus A (WVA), Watermelon crinkle leaf-associated virus 1 (WCLaV-1) and Watermelon crinkle leaf-associated virus 2 (WCLaV-2) reported by our laboratory in recent years, were also detected in the samples. After testing of 66 samples collected from different fields by RT-PCR, we found WMV, CiLCV and ZYMV as the three most common viruses in samples, with frequency 61%, 56% and 52%, respectively. Meanwhile, we also found most samples infected by the complex viruses with 68% of infection rate.

The genes encoding extracellular cellulase are different and diverse in plant pathogenic Xanthomonas. Extracellular cellulase contributes to the virulence of Xanthomonas axonopodis pv. glycines (Xag), but its encoding genes and the underlying molecular regulatory mechanism are unclear. In order to identify the genes encoding extracellular cellulase in Xag, 6 candidate genes were screened from the whole genome of strain NEAU001. Ectopic expression assay showed that EngXCA and Egl2 hydrolyzed carboxymethylcellulose, and the enzyme activity assay showed that engXCA and egl2 played the major role in the extracellular cellulase activity in Xag. In addition, the enzyme activity assay of 10 mutants in which the core virulence regulatory gene was deleted revealed that the extracellular cellulase activity of Xag was positively regulated by diffuse signal factor (DSF) signaling pathway and the global regulator Clp (Crp-like protein). qRT-PCR experiments further confirmed that both RpfF and Clp positively regulated engXCA and egl2 at the mRNA levels. Taken together, these results suggest that the DSF signaling pathway may regulate the expression of engXCA and egl2 through Clp, resulting in the regulation of the extracellular cellulase activity in Xag.

Poplar canker disease caused by Lonsdalea quercina subsp. populi is a bacterial disease that seriously threatens the production of Populus × euramericana plants. The two-component system is one of the most important signal transduction pathways and plays a crucial role in the growth, reproduction, adaption, stress tolerance and pathogenicity of bacteria. To explore the function of two-component system encoding genes in L. quercina, this study used homologous recombination to carry out deletion mutations on the two-component system encoding genes lqp0812-lqp0813 in strain N-5-1, and studied their biological functions. Compared with the wild-type strain, the growth rate, tolerances to heavy metal stress, salt stress, osmotic stress and pathogenicity were not changed in Δlqp0812 and Δlqp0813 mutants. However, the swimming motility was significantly impaired in Δlqp0812 mutant than that of the wild-type. In addition, the biofilm formation, hydrogen peroxide and antibiotic resistance were significantly increased in Δlqp0812 and Δlqp0813 mutants compared with the wild-type. qRT-PCR analysis showed that the expression levels of the efflux pump genes acrA, mdtB and aaeB were greatly increased in the lqp0812 and lqp0813 deletion mutants. These results indicated that lqp0812 is involved in the swimming motility of L. quercina subsp. populi, and lqp0812 and lqp0813 negatively regulated biofilm formation and tolerance to H₂O₂ and antibiotic stress together.

The field-grown faba bean samples exhibiting virus-like symptoms were collected and used for RNA extraction. Then, the siRNA was separated and used for siRNA library construction. After sequencing, velvet assembly, and blastn analysis, we found that collected faba bean samples were infected by 4 different viruses, which included VCV-M, vicia cryptic virus (VCV), milk vetch dwarf virus (MDV), and clover yellow vein virus (ClYVV). Among them, there are 14 VCV-M related contigs. As we know, the Amalgaviridae is a recently recognized family of dsRNA viruses that includes four species of plant viruses. RT-PCR amplification generated the 3 434 nt genome of VCV-M-YZ, of which the 5′-UTR and 3′-UTR were 142 nt and 117 nt, respectively. The 5′-UTR and 3′-UTR have high A+U content and form higher secondary structures, as well as the 5′-UTR and 3′-UTR of southern tomato virus, which is the representative virus of Amalgavirus. All this implies the specific characteristic of Amalgavirus replication. The sequences identity of VCV-M-YZ and reported VCV-M were 98%, and phylogenetic analyses showed that VCV-M-YZ and VCV-M belong to the same species, without obvious difference related to geographical distribution. Our studies enrich the genome genetic information of the VCV-M population, and provide basics of further research and supervisory control of VCV-M.

In order to clarify the genetic diversity of Pratylenchus neglectus populations in China, the genetic structure and genetic differentiation of 9 geographical populations were analyzed using the mtCOI gene as the marker. The results showed that a total of 101 mtCOI sequences were obtained from 9 populations, 28 variable nucleotide sites were discovered, and 14 haplotypes were formed. The haplotype H1 was the most common one shared by 59 individuals from 7 populations, which was speculated might be the ancestral haplotype. All the geographical populations showed the moderate genetic diversity at the species level (H_T = 0.706±0.131), and the cluster analysis showed that they could be divided into two groups, as the Group Ⅰ and Group Ⅱ. The AMOVA analysis revealed that the genetic differentiation at the whole level of P. neglectus populations was mainly derived from the inter-populations. The Mantel test showed that the genetic distance among P. neglectus populations was positively correlated with their geographical distance, although there was no significant correlation between the genetic differentiation and the geographical distance among different populations. Both the neutrality test and the mismatch distribution test revealed that the historical dynamics of P. neglectus populations at the whole level as well as at the two-groups level were relatively stable.

To determine the distribution of Trichotylenchus changlingensis in different geographical populations, in 2018, nematodes were isolated and identified from the rhizosphere soil of maize in 118 regions of Heilongjiang, Jilin, Liaoning, Hebei, Inner Mongolia and Gansu provinces (autonomous regions). The results showed that the nematodes were isolated from 20 soil samples in 6 provinces (autonomous regions) with isolation frequency of 17.0%. Sixteen highly polymorphic and reproducible ISSR primers were screened out and were used to amplify the 20 T. changlingensis isolates. A total of 93 polymorphic bands were obtained with a polymorphism ratio of 93.55%. The genetic differentiation coefficient (Gst) was 0.4771, indicating that the inter-group component accounted for 47.71% of the total variation,while the inner-group component accounted for 52.29%. The gene flow (Nm) was 0.5479 indicating that there was a less gene flow among different geographical populations. But there was genetic differentiation of some extent among groups. UPGMA cluster analysis showed that the T. changlingensis was rich in genetic diversity and there was no significant correlation between genetic distance and geographical distance.