Two pathogenic strains including FJ-85 (producing black spot) and FJ-11-2 (producing necrotic lesion) of Colletotrichum fructicola infecting pear were studied. Results showed that these two strains could produce plus and minus monospora strains by single ascospore culturing. The plus monospora strains formed light white colonies and their ascospores produced plus and minus monospora strains. The minus monospora strains formed dark gray colonies and their ascospores only produced minus monospora strains. Of the monospora strains from these two strains, the proportions of the minus types were significantly higher than the plus ones. By paired culturing with two types of monospora strains, perithecia were observed in their colonies as well as at the junction of two colonies. However, the perithecia were observed only in their colonies during paired culturing with the same type of monospora strain. The results indicated that homothallism happened in the same monospora strains, while there was still heterothallism between plus and minus monospora strains. The pathogenicity of the plus monospora strains were similar to that of the original strains, while the pathogenicity of minus monospora strains were weaker than the original ones. The symptoms caused by two types of monospora strains on the leaves of Pyrus pyrifolia cultivar Cuiguan were the same as those of their original strains. The results provide new useful information for the analysis of the relationship between sexual reproduction and pathogenicity.

In order to determine the Verticillium species caused potato Verticillium wilt in Ningxia, China, 27 samples with symptom of Verticillium wilt were collected from Guyuan and Xiji in Ningxia. A total of 11 Verticillium spp. strains were recovered from the samples by tissue isolation method. All isolates were identified as Verticillium nonalfalfae based on morphological characteristics and based on multigene phylogenic analysis of the partial sequences of actin (ACT), elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GPD) and tryptophan synthase (TS) genes. Pathogenicity test indicated that V. nonalfalfae could infect potato seedlings and caused typical symptoms of Verticillium wilt. Pathogenicity test of V. nonalfalfae to 13 dicotyledonous crops of 6 families including Solanaceae, Asteraceae, Leguminosae, Cruciferae, Cucurbitaceae, Malvaceae showed that V. nonalfalfae exhibiting high pathogenicity to summer squash, cucumber, eggplant, tobacco, cowpea, cotton and sunflower, and moderate pathogenicity to tomato, pepper, lettuce, and cabbage. However, V. nonalfalfae has no pathogenicity to broccoli and lucerne.

The conserved region of RdRp (RNA-dependent RNA polymerase) gene of Odontoglossum ringspot virus (ORSV) was amplified by RT-PCR from the Chenopodium amarantcolar leaves infected with ORSV Guizhou (China) isolate. DNA sequencing showed that the RdRp gene fragment was 516 bp in length and encoded 171 amino acid residues. The RdRp gene fragment was subcloned into a prokaryotic expression vector pET32a(+). By inducing with 0.4 mmol·L^-1 IPTG at 25℃ the recombinant RdRp gene was expressed in E. coli BL21(DE3) and its product identified as a specific band of 36 kDa by SDS-PAGE. Polyclonal antibodies against the recombinant RdRp were obtained by hypodernal injection of mice. The titer of the prepared polyclonal antibodies was 1∶102 400, and the antibodies reacted specifically with ORSV infected leaf extract, but not with other 4 virus-infected samples.

Ustilaginoidea virens secretes a large number of effectors during infection. We identified a potential effector UvSix1-1 from U. virens, which is the ortholog of Six1 in other pathogens containing conserved amino acid resides by sequence alignment and phylogenetic analysis. A signal peptide predicted in UvSix1-1 was confirmed to be functional in yeast system. Transient expression of UvSix1-1 in Nicotiana benthamiana could suppress Bax-, XEG1- and NF1-induced cell death and promote the colonization of Phytophthora capsici in host cells. Sequence analysis of UvSix1-1 in different strains of U. virens revealed no sequence polymorphism. All the results indicate that UvSix1-1 plays a role in the infection process of U. virens and would be a useful model for the study on effectors of U. virens and other pathogens.

Sclerotinia sclerotiorum (Lib.) de Bary is a typical filamentous homothallic fungus which is a se-rious plant pathogenic fungi and has a wide range of hosts. MADS-box protein family genes are widely present in a variety of organisms and participate in the regulation of cellular functions, including cell recognition, metabolism and cell cycle. The transcription factor SsMCM1 is involved in the fruiting body formation and virulence. To clarify the role of SsMCM1 in S. sclerotiorum mycelial growth and pathogenicity, this study considered the wild-type strain uf-70 cDNA as a model and obtained SsMCM1 gene through PCR specific amplification. The resulting gene fragment was ligated into the prokaryotic expression vector pET-28a (+) and the recombinant plasmid was transformed into E. coli BL21 (pLysS) expression strain. The protein SsMCM1 was purified after IPTG induction and affinity chromatography and used as antigen by using the combination of muscle subcutaneous immunization to obtain the appropriate antiserum and complete potency. The specific antiserum was found to have a specific combination with total protein and cytoplasmic protein in S. sclerotiorum. However, no specific response was found in cell wall proteins. It is speculated that the protein is not located in S. sclerotiorum cell wall. On the other hand, by constructing the subcellular localization vectors pCG-1301 and expressing it transiently via the Agrobacterium infusion. The result shows that SsMCM1 is located in the nucleus, which acts as a transcription factor to regulate the fruiting body and pathogenicity in S. sclerotiorum.

A series of molecular methods have been developed for the detection of Phytophthora species. Development of molecular markers and sequence analysis of specific loci has been proved to be the most accurate technique for identification at generic and species level. The SNARE-related gene YKT6 possesses conserved flanking coding regions, which are appropriate for the design of Phytophthora genus-specific PCR primers, with sufficient polymorphism for the development of molecular markers for almost all Phytophthora species. The Phytophthora genus-specific primer set P-YKT6-F/P-YKT6-R were able to specifically amplify a single product of about 600 bp in 31 different Phytophthora species. With no product from Pythium spp. or fungi tested. The specific primers sets Ps-YKT6-F/Ps-YKT6-R or Pc-YKT6-F/Pc-YKT6-R generated specific products of 399 bp and 282 bp from isolates of P. sojae and P. capsici, respectively. For both species, the sensitivity achieved was 100 pg for stan-dard PCR and 10 fg for nested PCR. These primers also proved to be efficient in detecting pathogens from soil and diseased plants. In addition, real-time quantitative PCR assays coupled with the species-specific primers were developed to detect and quantify the two species. The results demonstrated that the YKT6-based molecular marker and assays have clear potential for detection and quantification of Phytophthora spp. in a range of contexts from pathogen surveys to statutory testing.

A yeast cDNA library of woodland strawberry infected with Strawberry vein banding virus (SVBV) was constructed, and 15 SVBV P1-interacting host factors were screened through a yeast two-hybrid system. Bioinformatics analysis showed that these genes were involved in various biological processes such as jasmonic acid pathway, ubiquitination, photosynthesis, defense response, protein modification, protein transportation and oxidation-reduction process. In addition, these factors also possessed other molecular functions, including oxidoreductase activity, protein disulfide isomerase activity and metal ion binding activity, etc. This study preliminarily discussed the interaction mechanism between P1 and the host factors, providing a theoretical basis for revealing the molecular mechanism of SVBV infection and transport in woodland strawberry.

Sensitivity of Bipolaris maydis to propiconazole, genetic diversity and pathogenicity of different propiconazole-sensitive populations of B. maydis in Fujian Province were studied using methods of measuring the mycelial growth on the fungicide-amended media, inter-simple sequence repeat (ISSR) molecular markers and conidial suspension inoculation, respectively. Sensitivity test results indicated that propiconazole-resistance has been developed among isolates of B. maydis in Fujian Province, and resistance factors among resistant isolates ranged from 2.1 to 9.4. A total of 153 loci were detected in 55 isolates of B. maydis using 10 screened ISSR pri-mers, and as high as 93.46% of these loci were polymorphic loci. The percentages of polymorphic loci in the population of propiconazole-sensitive, -intermediary and -resistant were 77.12%, 69.93% and 81.70%, respectively. The values of observed number of alleles, effective number of alleles, Nei’s gene diversity, and Shannon’s information index for the resistant population were higher than those for the sensitive population, suggesting that genetic diversity in the resistant population of B. maydis was more diverse than that of sensitive population. Clustering analysis indicated that genetic diversity of different propiconazole-sensitive populations had highly correlations with the level of fungicide resistance and geographical origin. Pathogenicity assays revealed that the different propiconazole-sensitive populations of B. maydis had highly virulent to 11 sweet corn cultivars. Nevertheless, the frequencies of high pathogenic isolates in the propiconazole-sensitive population on 9 corn cultivars were considerably less than those in the resistant population. These results provide a theoretical basis for the further study of genetic structure and the fungicide resistance monitoring of B. maydis.

Barley spot blotch, initiated by the fungal pathogen , happened frequently in most barley-grown areas in the world, and caused serious barley yield losses and also decreased grain quality. Development and wider application of resistant barley cultivars was the most effective control strategy for spot blotch. However, few barley resistance genes or genotypes to spot blotch were available in breeding programs. In this study, 233 representive barley germplasm accessions originating from China were screened for spot blotch resistance by artificial inoculation in adult-plant stage. Only 10 accessions among them such as Kenpimai 5 showed high resistance to all three isolates used, accounting for 4.3%. Another 37 spot blotch resistance barley lines from domestic and foreign were also evaluated for resistance levels to B.sorokiniana isolates in adult-plant and seedling stages respectively. The results showed that 41%-46% of the 37 accessions were resistant to spot blotch in adult-plant stage, but in seedling stage the resistance percentage ranged from 50% to 64%.Eleven barley lines such as ND 17293 were resistant to all three isolates used both in adult-plant and seedling stages. These resistance lines can be still utilized as resistance sources. Based on the spot blotch resistance identification results with 3 isolates, resistance percentage of the barley accessions in seedling stage was usually higher than in adult-plant stage, indicating that there is great difference between barley seeding and adult-plant resistance to spot blotch. In addition, barley accessions showed resistance specificity to pathotypes of B. sorokiniana.

Leymus racemosus is a wild relative of wheat and has high resistance to wheat Fusarium head blight (FHB). The transfer of FHB resistance gene from L. racemosus to Triticum aestivum is of great significance for broadening the resistance of wheat germplasm. To obtain T. aestivum-L. racemosus translocation line with FHB resistance, the pollen of T. aestivum-L. racemosus disomic addition line DA5Lr was irradiated by ⁶⁰Co-γ-rays 1200 R (100R·min^-1) and then pollinated to emasculated T. aestivum cv. Chinese Spring. One plant with one T. aestivum-L. racemosus translocation chromosome was detected from the M₁ plants by genomic in situ hybridization (GISH). This plant was self-pollinated, and the progenies with two translocation chromosomes were analyzed for chromosome pairing behavior at meiotic metaphase I of their pollen mother cells (PMCs). One ring bivalent was observed at meiotic metaphase I in one of the progenies, indicated that the plant was translocation homozygote. Using Oligo-pAs1-2 and Oligo-pSc119.2-2 as probe, the translocation line was further identified as T5AS/5LrL translocation line by C-banding and sequential GISH-FISH analysis. Three EST-STS markers, BE591127, BQ168298 and BE591737, were selected and could be used for tracing the translocation chromosome. Three years’ FHB resistance tests of T5AS/5LrL showed that the proportions of scabbed spikelets were 7.69%, 10.29% and 8.66%, respectively, which was obviously lower than that of susceptible varieties Chinese Spring and Mianyang 85-45 .The translocation line will provide a new source for FHB resistance in wheat breeding

Heat shock proteins are widely present in various organisms and respond to a variety of biotic and abiotic stresses. Previous studies showed that over-expression of the small heat shock protein gene OsHsp18.0-CI can increase the resistance to bacterial leaf streak by enhancing rice basic defense response. This study found that the OsHsp18.0-CI gene can also be induced by PXO99, a strain of Xanthomonas oryzae pv. oryzae (Xoo) which caused bacterial blight in rice. Over-expression of OsHsp18.0-CI could enhance resistance to several Xoo strains in rice. Transcriptome sequencing analysis showed that OsHsp18.0-CI gene-mediated resistance to bacterial blight is not only partly similar to its resistance pathway to bacterial leaf streak, but also a large number of new unknown function genes are involved. The above results indicate that the OsHsp18.0-CI gene can increase resistance to bacterial blight by enhancing the basic defense response of rice infected with pathogenic bacteria of Xoo.

Phenotypic characteristics of resistance to rice blast, sheath blight, bacterial blight, and bacterial leaf streak of seven different populations of Oryza offcinalis from Yunnan Province were identified. Detection of the presence of cloned resistant genes of rice blast and bacterial blight was carried out. The results showed that six O. offcinalis populations except Menghai (7) had resistance to rice blast. The Mengzhe (4) and Jingnashanggou (1) did not contain Pib and Pi2, respectively, and all of other five O. offcinalis populations contained Pib, Pi2, Pi9, Pid2, Pikp, Pis, and Pi56. All tested O. offcinalis populations showed high resistance to sheath blight and bacterial leaf streak isolates RS105 and RS1-20, except Mengzhe (5) was susceptible to isolate RS1-20, Mengwang (13) and Lancangmengkuang (14) was susceptible to isolate RS105. Strong virulent isolate of Yunnan Xoo CX30-1 and Philippines Xoo isolates PXO99 and PXO86 had strong pathogenicity to Jingnashanggou (1) and Lancangmengkuang (14), but other wild rice populations showed resistance or moderate resistance to these three isolates. Seven O. offcinalis populations contained Xa5, Xa13, and Xa21 genes. This study is the first report that O. offcinalis populations of Yunnan had different characteristics of resistance to four diseases, which will be beneficial to laying foundation for deeply digging resistance genes from O. offcinalis in Yunnan Province.

Some mycoviruses have effect on growth rate, sporulation ability, pathogenicity biological characte-ristics of fungi host. Aims at detection of dsRNA mycoviruses infecting Botryosphaeria dothidea strains collected from Hubei province, which are to be explored as bio-control resources. The obtained results are the followings. 13 strains were isolated and cultured from pear branches with symptoms of ring spot disease to some extent in Wuhan city of Hubei province. The growth rate showed differences among 13 B. dothidiea strains, which exhibit different virulence. The patterns of dsRNAs were detected. The results show that 12 B. dothidiea strains have different sizes of molecular weight. The dsRNA profiles analysis indicate that the dsRNAs from HBWH-14 strain contain 9 bands with a molecular weight range of 1-7 kb, meanwhile the other strains exhibit one or more than dsRNA bands in accordance with that of HBWH-14 strain. In total, single or mixed infection with BdCV1, BdPV1, BdRV1 and a new member of Totiviridae (designated as BdTV1) are positive for the above one or more strains by RT-PCR detection; There was no correlation between mycoviruses and pathogenicity for 13 strains. dsRNAs from HBWH-14 regenerated strains are 100% positive by protoplast isolation.

Brown rot caused by Monilinia fructicola is an important fungal disease in peach production. In order to demonstrate the resistance mechanism and explore the molecular target of fungicide, MfSre1 was knocked out by using the Split Marker method and PEG-mediated protoplast transformation. Compared with the parental demethylase inhibitors (DMI) fungicide-resistant strain Bmpc7, there was no significant difference in the mycelial growth, sporulation and pathogenicity of knockout transformants (P>0.5). Also, there was no significant difference in the sensitivity to DMIs fungicides, hydrogen peroxide, and glycerol osmotic stress. However, the sensitivities to metal ions, saccharides compounds, cell wall and cell membrane damage agent were significantly decreased, and the resistance to dicarboximide fungicides (DCFs) increased significantly. These results indicate that MfSre1 is involved in regulating the exogenous stress and DCFs fungicide resistance.

Grey mould, caused by Botrytis cinerea, is one of the most important diseases of vegetables, flowers, and fruits in the greenhouse-grown crops, but the germination conditions and features of sclerotium of B. cinerea have not been reported previously. To control the preliminary infection of B. cinerea, the sclerotium germination on different environmental conditions was measured in this study. The result showed that the germination rate was not decreased when sclerotium were cultured at 50℃ for 12 h or at 55℃ for 4 h. The highest level of germination was detected when inoculated at 57% of the relative water content, while lower germination when waterlogged or less water incubation. The highest value of germination rate was observed at pH 6 in all treatments, while no germination at pH less than 3. In addition, the common fertilizers had no significant effect on the germination of sclerotium. Therefore, high soil temperature treatment and modulation of soil pH value might be the effective ways to reduce the preliminary infection in the greenhouse.

Grapevine leafroll-associated virus 13(GLRaV-13) was reported in Japan in 2016 as a new member of genus Ampelovirus, and have not yet been found in other countries. In this study, we adopted small RNA sequencing to detect potential viruses in an amur grape cultivar “Zuoshan Ⅱ” exhibiting chlorotic mottling. By assembling the obtained small RNAs, 112 contig sequences showed 87.5%-100% nucleotide identity and 64.9% coverage with the Japanese GLRaV-13 isolate a177 (GenBank ID: NC_029783). Two sets of primers, designed from the contig sequences, were used to amplify the cp and hsp70h gene sequences of the GLRaV-13 isolate, and the sequencing results showed the obtained sequences were the specific fragments of GLRaV-13. In addition,thirty amur grapevines and 90 other grapevines were used to detect GLRaV-13 using two pairs of primers, and the results showed that only amur grapevines tested positive for GLRaV-13. This is the first report of GLRaV-13 in China, and the study could help improve the sanitary status of grape cultivars in China.

Data changes have been analyzed between the ICTV 9th report and the ICTV 2017 release. In addition, data of ICTV 2017 release have been analyzed comprehensively from the aspects including species number and ratio of different types of viruses, and those of plant viruses as well as several special issues in virus taxonomy of ICTV 2017 release.

In order to confirm the occurrence of maize ear rot, pathogenic composition and the regional variation, the survey was conducted before harvesting in 2016 and 2017, and the diseased samples were randomly collected for pathogen identification. The results of field survey indicated that the corn planting area and the incidence of maize ear rot had decreased in 2017, compared with 2016. Identification results showed that the predominant pathogen of maize ear rot in summer corn region of Hebei Province was Fusarium verticillioides with isolation frequency of 63.49%, and the other pathogens included F. proliferatum, F. graminearum, F.incarnatum, Aspergillus niger, A. flavus, Penicillium oxalicum and Trichoderma harzianum with frequency of 19.05%, 6.35%, 1.59%, 6.35%, 3.17%, 9.52% and 1.59%, respectively. To clear the role of fumonisin gene for identification of fumonisin-producing Fusarium species, the phylogenetic trees of F. verticillioides, F. proliferatum and F. fujikuroi were generated based on the sequences of EF-1α and FUM1 gene, respectively. The similar topology in the two phylogenetic tree indicated that the fumonisin gene could be used for the purpose of identification in which the interspecific genetic distance based on FUM1 gene was greater than that of EF-1α gene, while the intraspecific genetic distance was opposite.

Citrus Huanglongbing, caused by "Candidatus Liberibacter asiaticus" (CLas), is one of the most destructive diseases in citrus production. Early studies showed that CLas and phytoplasma ("Candidatus Phytoplasma asteri", CPa) can be simultaneously detected in CLas-infected citrus, however, the relationship between CLas and CPa remains unknown. In this study, periwinkle (Catharanthus roseus) was used as host materials to explore the relationship between CLas and CPa by different grafting combinations. The result revealed that the periwinkle plant showed the phyllody and shortened internode when only infected with CPa, but didn’t cause the death of plant. The CLas-infected periwinkle showed mottled leaves and died in 2 months after grafting. When the periwinkle was infected with CLas and CPa, the plant showed phyllody along with mottled leaves and died in about 3 months after grafting, however, this was longer than the survival time of periwinkle only infected with CLas. In addition, our results showed that the movement speed of CLas was faster than CPa, but the propagation of CLas was slower than CPa. This study provided a theoretical basis for studying the difference of symptoms caused by CLas and CPa and interaction between CLas and CPa in periwinkle.

During the virus detection of kiwifruit cultivars in Shaanxi province, Citrus leaf blotch virus was found to occur at high frequencies. In order to detect CLBV accurately, a specific RT-qPCR assay based on SYBR Green I fluorescent dye was established. The results showed that the RT-qPCR assay had good specificity and sensitivity for CLBV detection. The slope of the standard curve and determination coefficient R² were -3.378 and 0.997 9, and the amplification efficiency reached 97.7%, which was 100 times more sensitive than conventional RT-PCR. This method was suited to batch inspection of field samples and low-abundance virus samples (such as dormant shoots of kiwifruit). The RT-qPCR assay laid the foundation for the early diagnosis of CLBV-infected seedlings, forecast and control of CLBV in orchard.

Intense extracellular protease activity, usually contributed to the virulence of Xanthomonas axonopodis pv. glycines (Xag), is unclear whether xanthomonappepsin precursor (Xpp) encoded by xpp is involved in. To elucidate Xpp biological functions, we generated the xpp deletion mutant (NΔxpp) by allelic exchange. Compared with the wild-type, the resulting xpp mutant exhibited significantly reduced on reproduction or growth and virulence in soybean host. The lost properties in NΔxpp were completely restored by the wild-type xpp. Protease assays showed that NΔxpp displayed the protease activity similarly to that of the wild-type, and the hetero-logous expression of xpp also did not take on the protease activity. qRT-PCRs confirmed that the transcription of xpp was significantly negatively regulated by DSF (diffusible signal factor) signals pathway and Clp (Crp-like protein). The expression of xpp was also inducible in soybean juice rather than in NYG medium, and positively regulated by HrpG and HrpX. Our results suggest that xpp is required for pathogenicity to soybean independent of the protease activity.

Like many other filamentous fungi, cyclic AMP (cAMP)-dependent signaling pathway is one of the major signal transduction pathways in Fusarium graminearum. It has been reported that cAMP-PKA pathway plays an important role in DON biosynthesis. FAC1 acts upstream the cAMP-dependent signaling pathway. Lack of FAC1 can block the DON biosynthesis. In this study, FAC1 mutant exhibited defects in the expression of TRI1 on the levels of transcription and translation. Microscopic observations revealed that Tri1 was located around the nucleus and the hyphal swelling structure of fac1 mutant had a significant increase compared with that of PH-1. In addition, the number of nuclear division in the swelling structures was more than that in normal hypha of PH-1. In the swelling structure of PH-1, the nuclear division number was more than 4, whose ratio had reached to 65.4%. Whereas the nuclear division number of fac1 mutant was only 0 to 3, in which the nuclear number with 0 to 2 was up to 90.7%. This study suggests that there is no direct connection between hyphal swelling structure and DON production, and that the DON biosynthesis is closely related to nuclear division in the hyphal swelling structure.

In order to gain new insights into molecular mechanisms of methyl gallate (MG) in Ralstonia solanacearum,transcriptome sequencing of MG-treated and non-treated R. solanacearum Rs-T02 was performed via Illumina Hiseq technology. Differentially- expressed genes (DEGs) were then sorted using GO terms and KEGG pathways analyses. As a result, 1 037 genes were up-regulated and 1 135 genes were down-regulated in a total of 2 172 DEGs. To validate the findings, 10 DEGs were selected randomly for further analyses using quantitative real-time PCR (qRT-PCR) where the results were fairly consistent with that of transcriptional sequencing. Specially, the findings showed that the genes associated with cytoarchitecture including the encoding genes of peptidoglycan hydrolase, 3-phosphate glyceryl transferase and UDP-N-acetyl muramoylalanine-D-glutamate ligase, etc. were up-regulated, and the coding gene of YeaQ, outer membrane protein W and outer membrane protein BamE, etc. were down-regulated in the Rs-T02 strain treated with MG. Genes associated with energy metabolism including the encoding genes of ATP synthetase structural protein, coenzyme Q subunit and cytochrome, etc. were up-regulated, the encoding genes of glyceraldehyde-3-phosphate dehydrogenase, glyceraldehyde dehydrogenase and isocitrate lyase, etc. were downregulated. Genes associated with pathogenicity such as gspD, gspE and gspF, etc. were up-regulated, whereas hrpY, hrpB and gspG, etc. were down-regulated. Furthermore, the encoding genes of the aminoacyl -tRNA ligase, the small subunit of ribosome RNA methyltransferase G and the multidrug efflux pump subunit AcrA etc. related to bacterial resistance were up-regulated. Genes associated with bacterial motility such as FlgB, flgC and flgD, etc. were up-regulated. It is speculated that the bacteriostasis mechanism of MG on R. solanacearum Rs-T02 strain may be related to the DEGs that associated with cytoarchitecture and energy metabolism. Meanwhile, MG affected the differential expression of T3SS and T2SS related genes, which had an impact to the pathogenicity of bacteria. Last but not least, the up-regulated expression of 3-phospho glyceryl transferase, small subunit of ribosome RNA methyltransferase G and the multidrug efflux subunit AcrA, etc. might be related to the mechanism of the bacterial resistance to MG.

Jiaobai, the second largest aquatic vegetable in China, was formed because of the interaction of Ustilago esculenta and its host Zizania latifolia. As a dimorphic fungus, the dimorphic transition of U. esculenta is closely related to its early infection, which is very important to formation of Jiaobai. In this study, the Ustilago maydis Fuz7 homologous gene UeFuz7 in U. esculenta was cloned, through homologous gene search by tBlastn based on thewhole genome sequence of U. esculenta. The cDNA of UeFuz7 is 1 308 bp, without introns, encoding 435 amino acids, which is relatively conservative in the smut fungi. It was found that UeFuz7 interacted with UeKpp2 by yeast double hybrid experiment. Besides, its expression pattern analysis showed that the relative expression of UeFuz7 was the highest during the dimorphic transition of U. esculenta. Furthermore, PEG mediated protoplast transformation was carried out to construct UeFuz7 mutant, and phenotypic analysis found that UeFuz7 disrupted mutants significantly reduced the ability of conjugation tube formation and hyphal growth during dimorphic transition. The above results indicate that UeFuz7 functions on the dimorphic transition of U. esculenta.

In this study, we developed an in vitro transcription system based on Nib (RdRp) of Wheat yellow mosaic virus (WYMV). Nib coding sequence of WYMV was amplified and inserted into the prokaryotic expression vector pMAL-C2X to construct the plasmid pMAL-WY-Nib. MBP-Nib fused protein (100 kDa) was expressed with the induction of 0.4 mmol·L^-1 IPTG. Based on the differential temperature experiment, it is suggested that 20℃ is the optimal temperature for MBP-Nib expression. The soluble ratio of MBP-Nib is about 30%, which is sufficient for MBP-tag isolation using affinity chromatography. As compared with other proteins of WYMV and viral RdRp of Tobacco bushy top virus, the purified MBP-Nib can specifically recognize the 3′-terminal region of WYMV RNA1 and RNA2 and synthesize complementary strand in vitro. This Nib-based in vitro transcription system will facilitate the study on regulation of WYMV replication.

Based on high-throughput sequencing and RT-PCR amplification, the genomic sequence of the ZyVX (Zygocactus virus X) Guizhou isolate ZyVX-GZ was cloned and analyzed. Using high-throughput sequencing for the red pitaya samples, which displayed the virus-like symptoms, four ZyVX-related contigs were obtained. RT-PCR amplification and assembly showed that the full-length genome of ZyVX-GZ was 6 567 nt, of which the 5′-untranslated region (UTR) and 3′-UTR were 60 nt and 70 nt, respectively. ZyVX-GZ genome contained five open reading frames (ORFs), which encoded RNA dependent RNA polymerasease (RdRP), triple gene block 1 (TGB1), TGB2, TGB3 and coat protein (CP). ZyVX-GZ genome shared nucleotide sequence identities of both 91% with P39 and B1 isolates. The nucleotide and deduced amino acid sequence identities of TGB1-3, CP with most ZyVX isolates were 95%-99% and 96%-100%, respectively. The nucleotide and deduced amino acid sequence identities of RdRP with most isolates were 80%-89% and 92%-97%, respectively. Phylogenetic analysis suggested that there was no correlation between the genetic differentiation of the current ZyVX population and host species, geographic distribution. The results of this study enriched the genomic information of the ZyVX population, and provided basis for the monitor and control of ZyVX.

To reveal vector factors in Laodelphax striatellus Fallen (small brown planthopper, SBPH) that are related to proliferation, accumulation, and transmission of Barley yellow striate mosaic virus (BYSMV), the nucleocapsid protein of BYSMV was selected as bait to screen a cDNA library of SBPH using a split-ubiquitin yeast membrane system. Firstly, the bait plasmid was constructed by fusing the full-length BYSMV N gene into pDHB1. The result of the expression and the functional assay of the bait showed that the fusion bait protein was correctly expressed and functional in the yeast. In the pilot screen, the bait vector was co-transformed into NMY51 with empty library vector pPR3-N plated on TDO and QDO-SD medium with different 3-AT concentrations to remove the slight background. Then, the QDO-SD medium with 12 mmol·L^-1 3-AT was selected for the library screening on which no growth appeared. As a result, 57 positive colonies were obtained from the library screening and 17 proteins were identified by searching against NCBI database, such as cuticular protein, polyubiquitin-B, ribosome-associated membrane protein, cytochrome b5 and trehalose transporter. The interaction between BYSMV N and the 17 putative proteins were further confirmed by retransformation assay and β- Galactosidase assay. In this study, vector proteins interacted with BYSMV N were successfully identified from the cDNA library of SBPH using the split-ubiquitin yeast membrane system, thus establishing a foundation for further exploration of the molecular interaction mechanism between rhabdovirus and the insect vector.

For clarifying the resistance level of wheat cultivars or lines developed in Heilongjiang to Puccinia graminis f. sp. tritici (Pgt) and finding out the stem rust resistance genes utilization in them, the seedling stem rust resistance of 83 major wheat cultivars or lines collected from Heilongjiang were evaluated with predominant Chinese Pgt races 21C3CTHQM, 34MKGQM and 34C3RTGQM in this study. Besides, molecular markers closely linked with genes Sr2, Sr24, Sr25, Sr26, Sr31, and Sr38 were used for molecular identification. Resistance genes possibly carried in the cultivars were analyzed based on the results of molecular identification, seedling reaction types as well as cultivar pedigrees. The results showed that all tested wheat cultivars or lines were resistant to the three tested races. Among them, 57, 53 and 60 of the 83 cultivars were immune or nearly immune to 21C3CTHQM, 34MKGQM and 34C3RTGQM, namely accounting for 68.68%, 63.85% and 72.29% of the total cultivars, respectively. The rest others remained moderately or highly resistant to the tested races. With molecular marker analysis, 12 of 83 wheat cultivars or lines possibly carry gene Sr2, Sr25 tested in Kehan 3, and Sr31 and Sr38 in 6 and 19 wheat cultivars respectively. Both genes Sr24 and Sr26 were not detected in any tested cultivar. Therefore, wheat cultivars developed in Heilongjiang Province was relatively highly resistant to stem rust. Most of them carry resistance gene Sr2, and/or Sr31 and Sr38 being resistant to all Chinese Pgt races, and/or possible other unknown resistance genes. These highly resistant wheat cultivars can be used as effective resistance resources for future wheat breeding.

Sclerotinia sclerotiorum is a worldwide pathogenic fungus causing serious diseases on many economically important crops. Studying on the genetic diversity of S. sclerotiorum isolates from different eco-geographical regions is crucially important for understanding the evolution of this fungal pathogen and disease control. In this study, DNA polymorphism of 66 S. sclerotiorum isolates derived from 17 different regions in Sichuan pro-vince were detected using sequence-related amplified polymorphism (SRAP) markers. A total of 129 scorable fragments were identified with 10 SRAP primer combinations, among which 123 were polymorphic loci (95.35%). UPMGA (Unweighted Pair Group Method with Arithmetic Mean) indicated that the dendrogram consisted of five groups (Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ) at the genetic similarity coefficient of 0.70, which included 60, 2, 2, 1 and 1 isolates, respectively. Moreover, the group Ⅰ contained three sub-groups (I-1, I-2, I-3) at the genetic similarity coefficient of 0.74, which included 21, 37 and 2 isolates, respectively. This study showed a rich SRAP polymorphism among the populations of S. sclerotiorum in Sichuan province, but genetic diversity had no significant correlation with geographical location.

Candidate effectors of Botryosphaeria dothidea were transient expressed in the leaves of Nicotiana benthamiana and then challenged with Agrobacteria tumefaciens cells carrying Bax at 24 h after the initial inoculation. Three tested candidate effectors completely inhibited Bax induced programmed cell death (PCD) of N. benthamiana. The secretional signal peptides of these effectors showed secretory activity in yeast system (pSUC2 vector). Expression patterns of the three effectors analyzed by Reverse Transcript-quantitative PCR showed that they were all highly expressed from 36 to 72 h post inoculation in apple fruit. Infiltration of Bdo_11198 could significantly increase the infection of Phytophthora nicotianae in N. benthamiana and the reactive oxygen accumulation was less than that of GFP. These results suggested that candidate effectors of B. dothidea might play important roles in the infection on apple.

Screening of host plants derived active small molecule substances inhibiting the type III secretion system (T3SS) of phytopathogen is one of the important strategy for development of natural biosafety pesticides. In this study, the small molecule substances were extracted by boiling extraction from Chinese radish, the host plant of Xanthomonas campestris pv. campestris (Xcc). The monomer structure of the active substance was analyzed by high performance liquid chromatography-mass spectrometry. The inhibition effect of active substances on T3SS of Xcc was detected through bacterial luciferase report system and quantitative PCR (qPCR). The biocontrol effect of the active small molecules was studied by leaf-cutting inoculation and infiltration inoculation methods. The results showed that phytosphingosine and sphinganine exhibited stronger inhibition effects on the transcriptional expression of Xcc T3SS genes, while the expression of type I, II and IV secretion system genes were not suppressed. Phytosphingosine affects the growth of bacteria on XCM1, but sphinganine does not. In addition, these two substances can significantly reduce the disease symptoms of Xcc on host plant Chinese radish and attenuate HR induction of Xcc on non-host pepper ECW-10R. The study will provide a theoretical base for further characterization of the interaction mechanism between small molecule compounds and Xcc T3SS and for the subsequent development of plant derived inhibitors.

Tomato yellow leaf curl virus and Tomato chlorosis virus were two major virus diseases in China. During 2016-2017, 262 leaf samples, which showed symptoms of stunting, yellowing, curling, chlorosis and rolling were collected from 18 main tomato-producing regions. Molecular and biological methods detection were used to detect TYLCV and ToCV. The results showed that virus diseases occurred in all the tomato-cultivated areas throughout the Hebei province. The symptoms of tomato virus disease in filed were complex. The infection rates of TYLCV, detected in every sampling site, were 68.66%, where the mixed infection rates with ToCV were 19.5%, and the rest were singly infected. ToCV was detected in all regions except for Luan cheng, Luan nan, Le ting and Yong nian. The infection rate of ToCV was 19.5%, and all the positive samples were mix-infected with TYLCV. No single infection of ToCV on tomato was found.

Two fungal isolates were isolated from diseased leaves of Philodendron selloum in Guangxi medicinal garden, Nanning, Guangxi. It indicated that they should belong to Lasiodiplodia based on morphological observation, and they were very similar to Lasiodiplodia theobromae after further comparison in morphology. Meanwhile, the multi-gene phylogenetic tree based on the DNA sequences of ITS, β-tubulin and elogation factor (EF-α) of isolates collected in this study and the ex-type strain of L. theobromae was built up. The results also supported that these two fungi were L. theobromae. The test of Koch′s rule confirmed that L. theobromae was the pathogen of this disease. This is the first report about L. theobromae causing P. selloum disease in China.

Several jujube plants with witches′ broom, little leaf, and big bud symptoms, which were likely infected by jujube witches′ broom (JWB) phytoplasma, were collected in Guangzhou, Guangdong Province. To identify the pathogen, PCR was performed using phytoplasma 16S rDNA universal primer pairs R16mF2/R1 and P1/P7 and SecA gene primer pair SecAfor1/rev3 with total DNA of the symptomatic plants as templates. Specific fragments, 1.4 kb, 1.8 kb, and 0.8 kb in length, were amplified from one of three symptomatic samples. Phylogenetic analysis based on 16S rDNA verified that the pathogen harming jujube plants in Guangzhou was jujube witches′ broom phytoplasma which belonged to 16SrV-B subgroup. Comparison results also showed that the 16S rDNA sequence of Guangzhou JWB phytoplasma shared the highest nucleotide identity (100%) with the reported jujube witches′ broom phytoplasma Japanese strain (AB442218) and JWB strain (AY197661) and shared the nucleotide identity ranging from 99.74% to 99.80% with the other JWB phytoplasma strains. In addition, phylogenetic analysis based on SecA also showed that Guangzhou jujube witches′ broom phytoplasma belonged to 16SrV-B subgroup and shared 99.28%-99.76% similarity with other phytoplasma strains. All these results suggested that jujube witches′ broom phytoplasma has infected jujube plants in Guangdong Province.

Tyrosine 137 is one of key residues in selective binding of DMIs to FgCYP51B(Fusarium graminearum CYP51B), which consists of a drug binding active pocket and plays an important role in the interaction between CYP51 and DMIs. In this study, we investigated the FgCYP51B Y137H biological phenotypes on conidiation, female fertility, colony morphology, growth rate, and pathogenicity. We found that the Y137H mutation of FgCYP51B showed a significantly reduction on conidiation and influences on development of ascospores compared with the wild-type strain. However, colony morphology, growth rate, and pathogenicity of the Y137H mutation of FgCYP51B were consistent with that of the wild-type strain. These results suggest that the FgCYP51B Y137H mutation has influences on conidiation and development of ascospores, but is not associated with colony morphology, growth rate and pathogenicity.

The fungal strain OV5 isolated from the azalea of Japan produced pycnidia contining conidia on PDA medium. The isolate grew well on two kinds of medium, including PDA and CMA, but not on MEA medium. The isolate usually produced little aerial mycelium and abundant spermatia. The spermatia were globose, 3.0-3.5 μm in diameter, produced at the tips of fusoid hyphae, usually seperating readily but sometimes adhering in short chains. Sclerotia typically of circular to elliptical outline, often irregular, distinctly cupped, smooth on the concave surface; (1.5 -6) mm × (2-8) mm × (0.5-1.5) mm; at maturity black throughout. Its rDNA ITS gene sequence was most similar to sequences of Ovulinia azaleae deposited in GenBank with more than 99% identity. The morphological characteristics and phylogenetic analysis confirmed the fungal strain as O. azaleae. The pathogenicity test showed that the strain OV5 infect azalea by forming typical symptoms of azalea petal blight. This is the first interception report of O. azaleae from the Japan in China.

Rice false smut, caused by Ustilaginoidea virens, is a major threat to rice production worldwide, but the mechanisms underlying rice resistance to U. virens remain elusive. To understand molecular bases underlying the initial interaction between rice and U. virens, transcriptional changes associated with the early stage of U. virens infection in the resistant ‘IR28’ and susceptible ‘LYP9’ rice cultivars were analyzed using the transcriptome analyses. The results demonstrated that 1 005 differentially expressed genes (DEGs) were common to ‘IR28’ and ‘LYP9’, Among common DEGs shared in both cultivars with the isolate P1 at 6 h post inoculation. More genes were up-regulated in ‘IR28’ (697) than in ‘LYP9’ (626) while fewer DEGs (308) in ‘IR28’ were down-regulated than those (379) in ‘LYP9’. GO enrichment and KEGG metabolic pathway analysis revealed that phenylpropanoid biosynthesis, diterpene phytoalexin biosynthetic genes, chitinase beta-1,3-glucan, glycosyl hydrolase and peroxidase genes were specifically induced in the resistant variety, while these DEGs were down-regulated or less induced in the susceptible cultivar, implying that these genes may play an important role in disease resistance at the early stage of U. virens infection. Our results provide a theoretical basis for further exploitation of rice resistance genes and for molecular breeding of false smut-resistance rice.

Special primers and detection approach were developed based on the DNA amplification fragment polymorphs by URP (Universal Rice Primers)-PCR in Fusarium oxysporum f. sp. momodicae causing bitter gourd wilt. The application showed that the specific 294 bp amplicon was generated with the specific primer pair FOMM-SPF/FOMM-SPR in 25 SymbolmAL of detection volume including 12.5 SymbolmAL of 2SymboltB Green Taq Master Mix,1 SymbolmAL of 10 SymbolmAmol·L^-1 each primer,1 SymbolmAL of template DNA,and sterile ddH₂O to obtain the final volume;Cycling conditions were 3 min initial denaturation at 95℃, 30 cycles of 15 s at 94℃, 30 s at 57℃, 20 s at 72℃ and final elongation 5 min. The specific primer pair for 294 bp product and the detection sensitivity is 2 ng· SymbolmAL^-1 for genomic DNA of F. oxysporum f. sp. momodicae and 50 spores·500 mg^-1 soil for the soil pathogen. The processing methods exhibited the high specificity and sensitivity F. oxysporum f.sp. momodicae that could be detected in the soil and plant samples rapidly and accurately instead of pathogen isolation and pathogenicity test, which would be beneficial in significance for the early diagnosis, early warning, and effective control of the bitter gourd Fusarium wilt disease.

Apple ring rot caused by Botryosphaeria dothidea is one of the devastating diseases in China, which has become a major threat to apple production. In preliminary experiments, we found the expression of pectin lyase gene (Bdpl1) was obviously up-regulated in diseased tissues than that in fungal mycelia. Therefore, the Bdpl1 was considered as a virulence gene involved in the pathogenic processes. In order to analyze its pathogenic function, the knockout vector of Bdpl1 was constructed by the split-marker method and the transformants were obtained through PEG-mediated protoplast transformation. One mutant, △Bdpl1-3, was confirmed with four pairs of primers. Compared with the wild-type strain, the △Bdpl1-3 morphology had no significant differences on PDA medium, whereas the colony diameter on pectin medium was much smaller than the wild-type one. The extracellular pectinase activity of the △Bdpl1-3 isolate was lower than the wild type. Nevertheless, pathogenicity of the △Bdpl1-3 isolate on excised one-year-old ‘Fuji’ apple twigs had no significant differences with the wild-type strain.Relative expression levels of several other pectin lyase (PL) family genes in the △Bdpl1-3 and wild-type strains were detected by qRT-PCR. The results showed that the expression of three other PL family genes were up-regulated (over three folds than the wild-type strain). These data indicated that the Bdpl1 had no obviously effects on fungal growth and pathogenicity, but it had significant acceleration to degrade pectin. In the △Bdpl1-3 strain, other PL family genes might compensate the functions of Bdpl1 by up-regulated expression.

Wheat stripe rust, caused by Puccinia striiformis f.sp. tritici (Pst), is an important disease on wheat. Studying the pathogenic related function of effectors is of great significance for revealing the pathogenic mechanism and new methods of prevention of wheat stripe rust. Based on analysis of the previous haustorial transcriptome of Pst isolate CYR32, we screened the candidate genes predicted coding secreted proteins and obtained a highly expressed gene HASP2 which was 240 bp in length. The N-terminus of HASP2 contained a 22-aa signal peptide with no transmembrane region and no predicted motifs. qRT-PCR analysis showed that HASP2 was up-regulated during the initial infection stage of rust CYR32. With Agrobacterium tumfaciens co-infiltrations in Nicotiana benthamiana, HASP2 was capable of suppressing cell death triggered by mouse pro-apoptotic protein BAX. Overexpression of HASP2 in wheat using the bacterial type III secretion system (TTSS) could inhibit callose deposits that related to host PTI. In contrast, when challenged with an avirulent Pst isolate CYR23, effector-triggered immunity (ETI) was weakened which accompanied by reduction of reactive oxygen species (ROS) accumulation and hypersensitive response (HR) that led to the smaller areas of cell death on wheat leaves, while the abnormal growth and development of stripe rust transformants were not observed.