Twig blight disease is one of the main diseases on bayberry (Myrica rubra Sieb.& Zucc). We deve-loped an effective method to detect and quantify the pathogens Pestalotiopsis versicolor and P. microspora by using conventional PCR and SYBR Green real-time PCR. A primer set, Pvm1L/Pvm1R, was designed based on a conserved sequence of the internal transcribed spacer (ITS1-5.8S rDNA-ITS2) region of the ribosomal DNA gene of P. versicolor (JN861773) and P. microspora (JN861776). The 188 bp DNA fragments were amplified from 30 isolates of P. versicolor and 30 isolates of P. microspora, and no product was amplified from isolates of 11 other fungal genera. Conventional PCR was able to detect the pathogens on symptomatic and artificially infected bayberry plants at 21 days after inoculation, and the detection limit was 0.6×10⁵ copies of the Pestalotiopsis DNA. In addition, the Pvm1L/Pvm1R primer set were successfully adapted to SYBR Green real-time PCR, which had a limit 100 times lower (0.6×10³) than that by conventional PCR and was able to detect the pathogens in symptomless, artificially inoculated, and naturally infected plants. The conventional and SYBR Green real-time PCR developed in this study were simple, fast, sensitive, and specific, and can be used to detect Pestalotiopsis spp. from infected bayberry in the field.

Citrus leaf blotch virus (CLBV) is type species of the genus Citrivirus. In this study, CLBV was detected in kiwifruit (Actinidia spp.) plants grown in China for the first time by reverse transcription PCR (RT-PCR). CLBV was tested in kiwifruit plants with a positive rate of 13.3%. The complete cp gene of five CLBV isolates was 1 092 nucleotides (nts) in length and shared 86.6%-99.7% nt and 96.4%-99.4% amino acid (aa) sequence identities with each other, respectively. However, they shared only about 83% nt identity with a kiwifruit isolate M3-A reported in New Zealand, and 84%-86% identity with CLBV isolates infecting citrus and its relatives. In the phylogenetic tree basing on nucleotide sequences of their cp genes, CLBV isolates identified in the present study clustered into a group with New Zealand kiwifruit CLBV isolates except for isolate M3-A. Our studies would be favorable to understand the occurrence of this disease in China and to establish a more efficient RT-PCR method for rapid detection of the CLBV infecting kiwifruit trees.

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating fungal diseases of rice worldwide. In eukaryotes, there are two kinds of GTP binding proteins, one is heterotrimeric G protein, and the other is small GTPase protein. Small GTPase proteins have GTPase hydrolysis activity with a molecular weight of 20~30 KD, and play as a molecular switch in multiple cellular responses. Small GTPase proteins are active when bound to GTP and can thus activate the downstream proteins. When GTP hydrolyzes to GDP, small GTPase protein returns to its inactivated status. There are many proteins such as GEF and GAP, controlling the activity of small GTPase proteins in the organisms. In this study, we identified and characterized a GAP protein in M. oryzae, named MoGcs1. Targeted gene deletion of MoGCS1 showed that MoGCS1 plays crucial roles in asexual development, conidial morphology, appressorium formation and stress responses, but the gene showed no roles in growth and pathogenicity. The results indicated that MoGcs1 is a key GAP protein of the rice blast fungus.

The regulatory relationship between the stringent response related gene spoT_Xcc and type III secretion system (T3SS) related genes in the bacterial phytopathogen Xanthomonas campestris pv. campestris was investigated by using reversed genetics method and semi-quantitative RT-PCR. A non-polar mutant of spoT_Xcc (XC_0956) was generated by suicide plasmid pK18mob mediated homologous integration and various phenotypes of the mutant were studied. It was found that the spoT_Xcc mutant delayed the induction of hypersensitive response on the non-host plant pepper ECW-10R. The expression of T3SS genes were down-regulated in spoT_Xcc gene mutant detected by GUS activity tests, semi-quantitative RT-PCR and staining of tissue methods, indicating that the stringent response related gene spoT_Xcc positively regulated the T3SS related genes in X. campestris pv. campestris.

Most species of Xanthomonas genus in γ-Proteobacteria are plant pathogenic bacteria, causing hypersensitive response (HR) on non-host plants and resistant host plants and pathogenicity on susceptible host plants via highly conserved type-III secretion system (T3SS) through which T3SS effectors (T3SEs) are injected into plant cells. However, it remains unclear which Xanthomonas species lacking of T3SS and T3SEs that can be the targets to understand pathogenicity factors and to design specific probes for quarantine detection. Here, seven Xanthomonas species were collected and detected. PCR and Southern blot results showed that X. campestris pv.musacearum ICMP287 and ATCC49084 strains (the causal agents of banana bacterial wilt), X. axonopodis pv. vasculorum ATCC13901 strain (gumming in sugarcane), and X.axonopodis pv. allii LMG576 and LMG578 strains (bacterial blight of onion) lacked the tale genes which encode transcriptional activator-like effectors, of these three species X. axonopodis pv. vasculorum ATCC13901 possesses neither hpa1 nor xopQ and hrcC genes for the T3SS; and X. campestris pv. phaseoli ATCC49119 strain (common bacterial blight in phaseolus beans) possesses xopQ and hrcC genes but hpa1. In contrast, 2 to 12 putative tale genes were detected in the other strains including X. axonopodis pv. glycines ICMP5732 and ATCC43911 (bacterial pustule of soybean), X. axonopodis pv. vignicola ATCC11648 (Cowpea Bacterial Blight and Pustule), X. campestris pv. malvacearum ATCC12131 (cotton bacterial blight), and X. c. pv. phaseoli ATCC49119. HR assays on tobacco demonstrated that only X. axonopodis pv. glycines ATCC43911 strain did not trigger HR, despite that hpa1, xopQ, hrcC and tale genes were detected in this strain. These results provided fundamental basis for further understanding the differences of pathogenic determinants and designing specific quarantine probes among them.

In order to explain the anti-bacterial mechanism of methyl gallate (MG) in Ralstonia solanacearum strain Rs-T02, two-dimensional gel electrophoresis (2-DE) was used to seperate the differential protein of the pathogen with MG treatment. The results showed that 29 differential protein spots were detected. Twenty two proteins, which included 11 newly-emerged, 5 disappeared, 1 up-regulated and 5 down-regulated proteins,were identified by LC-MS/MS. These proteins were related to energy metabolism, signal transduction and translocation of solute or DNA repair. We hypothesized that newly-emerged protein spot 9 and disappeared protein spot 65, which were related to the energy metabolism, might involve in the anti-bacterial mechanism of MG in R. Solanacearum.

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules which play important roles in plant growth and development, signal transduction, transcription regulation, metabolism and various stress conditions. In this study, target genes and their functions of miR159 and miR858 were predicted and analyzed by bioinformatics. The results indicated that most of the target genes of miR159 and miR858 were members of MYB transcription factor gene family which performed a variety of functions in plant growth, development processes and disease resistance. The expression characteristics of cucumber miR159 and miR858 were analyzed in different periods and tissues after Cucumber green mottle mosaic virus (CGMMV) infection using real-time quantitative RT-PCR. It was found that miR159 and miR858 responding to CGMMV infection were significantly and differentially expressed in different tissues, and their expressions showed temporal and spatial specificity, as well as tissue specificity.

ORF1 sequence of Tobacco bushy top virus (TBTV) was amplified and cloned into prokaryotic expression vector pEHISTEV. The recombination plasmid containing ORF1 of TBTV was transformed into E.coli Rosetta and performed to express ORF1 protein under the induction of IPTG. The purified ORF1 protein from SDS-PAGE gel was used to inject the rabbits as antigens to get antiserum with high titer at 1∶24 3000 by indirect ELISA.Through the antigen affinity purification, the polyclonal antibody against ORF1 protein with higher sensitivity and specificity was purified from whole antiserum. The polyclonal antibody could be used to detect the ORF1 of the TBTV-infected tobaccos in the field as well as in vivo (refer to Arabidopsis protoplasts) and in vitro translation system (refer to WGE), in which TBTV full-length RNA was incubated. In addition, ORF2 was identified to be expressed as ORF1-extended protein, possibly through translational frameshift mechanism based on the over-lapping status of ORF1 and ORF2, potential hepta-nucleotide slippery sequence and downstream stable stem-loop structure. This project provided substantial serological support for the consequent research on the detailed mechanism of the multi-levels of translation of ORF1 and ORF2 in Tobacco bushy top virus.

A fungus named HBF1 was isolated from female of M. incognita on tobacco in Hubei province, China and was identified as Trichoderma longibrachiatum based on morphological characteristics and molecular analysis of rDNA-ITS coupled with translation elongation factor 1-alpha. To identify virulent factors and elucidate the parasitic mechanism on M. incognita eggs, the egg parasitism of M. incognita eggs by T. longibrachiatum HBF1 was investigated in vitro. The percentage of parasitized eggs was 80.45% at the 10 th day after inoculation. From this isolate, the novel genomic and cDNA clones encoding chitinase have been cloned using the degenerate PCR primers and RACE techniques. The analysis of the gene and the deduced amino acid sequence along with its phylogeny analysis was by biology software. Results showed that HBF1 was able to penetrate nematode egg and produce chitinase, maximum activity of chitinase was 24.88 μmol/h/mL recorded at the tenth day after inocu-lation. We have cloned a novel chitinase gene TlChi46 from T. longibrachiatum HBF1 for the first time. The gene is 1 793 bp in length and contains three putative introns. The ORF of TlChi46 is 1 272 bp in size with encoding protein of 423 aa , molecular mass of 45.9 kDa and pI of 5.23. Phylogeny analysis reveals that this chitinase and other reported Trichoderma spp. chitinases are clustered together and are phylogenetically distant from other entomopathogenic fungi. These results demonstrated that the amino acid sequence coded by chitinase gene TlChi46 from T. longibrachiatum HBF1 possesses promising functional domains for the further research, T. longibrachiatum HBF1 with the the strong chitinolytic and egg-parasitic activity has a great biocontrol potential against M. incognita.

In this study ,we mapped and characterized quantitative trait loci(QTL) associated with the resistance to Phytophthora infestans in a tetraploid potato variety. A total of 190 F₁ individuals obtained by crossing between foreign cultivar ‘Atlantic’ and local cultivar ‘Longshu No.6’and used to construct the map containing 171 SSR markers. By using interval mapping, 7 QTLs for resistance to P. infestans were identified. These QTLs were mapped on linkage group 5, 6, 7, 10 and 11, respectively. The phenotypic variations explained by each QTL ranged from 17.37% to 65.68%, and their LOD values varied from 2.70 to 10.32. Of the three QTLs with LOD value higher than 3.5 were considered major QTLs. The two tight linkage SSR markers S183-210 and S148-460 would be used in fine mapping of the resistance to P. infestans in tetraploid potato.

Two hundred single spore isolates of Magnaporthe oryzea from nine counties/cities in Sichuan and Chongqing were amplified with 16 pairs of virulence gene-specific primers. The results showed that the target bands could be clearly amplified with all the primers, and the percentage of polymorphic loci (P) reached 93.75%. Those isolates were classified into 70 different haplotypes and 27 genetic lineages at the genetic similarity coefficient level of 0.86 by cluster analysis. Among the 70 haplotypes, SCH13 was the dominant haplotype. The twenty-seven genetic lineages included 1 advantage lineage, 3 subdominant lineages, 14 secondary lineages and 9 minor lineages. At population level, it was showed that there existed rich genetic diversity among 200 isolates, and the diversity existed among fungal populations from different geographic regions (H=0.324 4,I=0.484 2). The 9 populations were clustered into 4 groups at 0.05 genetic distance level, and the genetic lineages of the populations from different areas are related to their geographic distributions. Meanwhile, certain genetic differentiation existed in the same fungal population. The genetic diversity of within-population was higher than that of among-population (Hs=0.179 6,Dst=0.140 4), and accounted for 56.13% of the total variation existed in within-population (Gst=0.438 7). The gene flow was less in within-population (Nm=0.639 6). The study reveals the genetic structure, genetic diversity of M. oryzea populations and the relationship of the geographic distributions and the fungal populations from Sichuan and Chongqing, which could provide the basis for rice blast resistance breeding and rational arrangement of rice varieties in these regions.

Tibet Province is an independent epidemic area for wheat stripe rust in China. The wheat stripe rust is one of the most destructive diseases on wheat, affecting safe production of wheat in Tibet. Linzhi prefecture has been considered an area where wheat stripe rust occurs frequently and is serious epidemic. It was verified recently that Berberis spp. serve as alternate host for Puccinia striiformis f. sp. tritici (the causal fungus of wheat stripe rust, Pst), playing a role in resulting in variation in pathogenicity to produce new Pst races and in occurrence of the disease in China. Linzhi has a distribution of plenty of Berberis spp., however, it is lack of studies on Berberis spp. severing as alternate host for Pst in Linzhi prefecture so far. In this study, whether Berberis spp can serve as alternate host for Pst were identified based on investigations on Berberis spp. in Linzhi, Tibet where wheat stripe rust occurs frequently. The results showed that six Berberis spp. are very general in geographic distribution, and were identified to serve as alternate host for Pst. This provides a basis for further studying on occurrence and epidemics of wheat stripe rust, pathogenic variation of Pst, and perfecting integrated control of the disease in Tibet.

Conidia of Blumeria graminis f. sp. tritici(Bgt)in the air were monitored by Burkard volumetric spore samplers in 2012 and 2013. Conidia concentration of Bgt within the canopy was positively correlated with above the canopy and was significantly higher than that above the canopy. Conidia concentration raised along with time, reached the maximum concentration at filling stage of wheat, and then declined. Time series analysis showed that conidia concentration in the fields was fitted with ARIMA (1, 1, 0) models. A model was constructed based on the significant correlation between conidia concentrations in the air and temperature. Two models for prediction of disease index were established by inoculum variable only, and by both inoculum and weather va-riables, resprctively. The model based on inoculum only has more universal applicability to predicting disease index in comparison with the model based on both inoculum and weather variables.

A new black spot disease was observed on the banana cultivar 'Williams'(AAA, Cavendish) in Guangxi province, China. The pathogen was isolated from the infected banana leaves and cultured. Based on the analysis of its morphology, biological characteristics, pathogenicity and ITS sequence, the pathogen was identified as Guignardia mangiferae A. J. Roy. Colonies of the pathogen on PDA were dark olive, and the ascus was clavate with stipe, ascospores were fusiform and conidia were obpyriform.G. mangiferae could grow with an optimal condition at 28℃，pH 6. The best carbon sources for the pathogen growth were fructose and arabinose, and the best nitrogen sources were peptone and yeast extract. The best cultural condition for mycelial growth was 24 hunder light. The lethal temperature was 54℃ for hypha of G. mangiferae. To the best of our knowledge, this is the first report of black spot on banana caused by G. mangiferae in the world.

Gray mold of strawberry caused by Botrytis cinerea Pers. is one of the most serious diseases on strawberry. In order to understand the resistance of B. cinerea from strawberry to azoxystrobin, a systemic fungicide commonly used to protect plants and fruits/vegetables from fungal diseases, in China, 134 isolates were collected from 10 provinces or cities in 2012 and their resistance to the fungicide was detected using PCR with specific primers BcAR-F/BcAR-R. The resistant isolates were confirmed on PDA medium supplemented with the fungicide. The biological characteristics of the azoxystrobin-resistant and -sensitive isolates were also compared. Our results indicated that 54 isolates (40.3%) from Beijing, Hubei, Jiangsu, Hebei, Liaoning and Sichuan, were moderate resistant to azoxystrobin. It was found that a glycine was replaced by an alanine at position 143 in cytb gene in all the 15 resistant isolates. There were no significant differences in optimum growth temperature, colonial growth rate, and spore germination rate when 4 azoxystrobin-resistant and 2 azoxystrobin-sensitive isolates were randomly selected and compared. However, sporulation and virulence to strawberry leaves of resistant isolates were significantly decreased when compared to the azoxystrobin-sensitive isolates.

In 2014, a disease of black spot was found on leaves of Avicennia marina (one of the mangroves) in Donghai island of Zhanjiang, Guangdong Province. By the method of pathogen-containing tissue isolation and pathogenicity test, we obtained a pathogenic isolate bgr1. Based on morphological characteristics and sequence analysis of rDNA-ITS and glyceraldehyde-3-phosphate dehydrogenase (gpd) gene, the isolate was identified as Stemphylium lycopersici. This is the first report of leaf black spot on Avicennia marina in China.

Tar spot of Dalbergia odorifera, caused by Phyllachora dalbergiicola, is a common disease, and seriously affected the rate of photosynthesis. Here we developed a species-specific Nested-PCR approach for rapid and accurate detection of P. dalbergiicola, based on the differences in internal transcribed spacer (ITS) sequences of P. dalbergiicola and another P. spp., an endophytic fungus, from which a pair of species-specific primers P1/P2(P1:5'-CGAGGTCAGAATCAAACG-3', P2:5'-TGAAGAACGCAGCGAAAT-3'), was designed. P1/P2 amplified only a unique 273 bp band from the genomic DNA of P. dalbergiicola. A Nested-PCR procedure using ITS4/ITS5 as the first-round primers, followed by P1/P2 primers, increased detection sensitivity 10 000 fold to 100 ag. Using the Fast DNA-kit to extract DNA from the diseased plant tissues, the detection of the pathogen by Nested-PCR assay could be completed within 1 day. The results suggested that the PCR-based methods here could simplify both plant disease diagnosis and pathogen detection

Toxin-producing culture medium and culture time for Phomopsis asparagi were optimized, and the effect of the pathogen toxin on asparagus callus was investigated in the study. It was found that the toxin produced from culture for 12 days in the Fries medium showed the strongest inhibition effect on asparagus radicle. Toxin caused callus of ‘Apollo’, ‘Champion’ and ‘NJ987’ browning. Resistance of the callus of the three varieties to toxin could be induced by toxin treatment. Toxin concentration for resistant callus selection was 30%-40%.

Rice false smut has become one of the major diseases in Northeast China and in the areas of the middle and lower reaches of Yangtze River. However, its infection type remains to be controversial. In this study, we constructed a simple and quick approach for the detection of Ustilaginoidea virens and revealed the distribution of U. virens inside different tissues of rice samples in both the seedling stage at 35 days after transplantation and the filling stage. The primers designed from the sequence of U. virens mitochondria showed qualified specificity and sensitivity for 1 pg U. virens DNA. With this detection approach, U. virens was detected in root and sheath tissues in the seedling stage samples and stem tissues in the filling stage samples. The results not only directly offered molecular evidence of the distribution of U. virens in various rice tissues in the seedling and filling stages, but also may be beneficial to develop novel strategies to control rice false smut.

Mycoviruses are widespread in filamentous fungi and yeast; some of them are related to impaired growth, abnormal sporulation and pigmentation of their hosts. In this study, 120 M. oryzae isolates were obtained from rice blast lesions on leaves by using single-conidium isolation technique, and about 52% of these isolates contained dsRNA fragments. One of these strains QSP5 is infected by two mycoviruses, namely M. oryzae chrysovirus 1-C (MoCV1-C) and M. oryzae virus 3 (MoV3) according to their high similarity to M. oryzae chrysovirus 1-A (MoCV1-A) and M. oryzae virus 2 (MoV2). Besides, a single-conidium isolate QSP5-9, only infected by MoV3, was obtained from the conidia of strain QSP5. Comparative analysis of sporulation production, mycelial and colonial morphology between QSP5 and QSP5-9 revealed that MoCV1-C may be mainly rela-ted to abnormal sporulation, mycelial growth and pigmentation. Meanwhile, to test the stability of MoV3 in M. oryzae, 100 single-conidium isolates of M. oryzae strain QSP5 were obtained and MoV3 was detected in all the 40 randomly selected isolates, however, MoCV1-C only existed in part of these isolates. In addition, 94 single-conidium isolates of M. oryzae strain QSP5-9 were obtained and 45 randomly selected isolates were proved to carry the same dsRNA genome as strain QSP5-9. These results indicated that MoV3 is stable in M. oryzae.

Phytophthora pathogens secrete large amounts of RxLR effectors to interfere with host plant cell physiology and function. Here, we identified 9 PiAvr3a-like genes in P. capsici by BLAST searching and demonstrated that this gene group was also widely distributed in P. sojae, suggesting their potential importance in virulence. We cloned 5 PiAvr3a-like genes from P. capsici, three of which exhibited high expressional levels at early infection stage, suggesting that they may play important roles in this stage. Among the 5 genes, transient expression of PcAvh128 in Nicotiana benthamiana could promote P. capsici infection whereas PcAvh132 could reduce the infection. None of them could suppress plant cell death that are induced by INF1 or effectors, and induce hypersensitivity reaction in N. benthamiana. These results together suggest P. capsici PiAvr3a-like effectors have divergent functions during infection and lay a fundament to study interactions of Phytophthora pathogens and their hosts.

An isolate 1314 was obtained from imported Canadian pea seeds with semi-selective MT(milk tween agar) medium, and identified by PCR, 16S and 23S rRNA sequence analysis, multilocus sequence analysis (MLSA), Biolog test, hypersensitivity reaction on tobacco and pathogenicity. With the specific primer AN7F/AN7R of Pseudomonas syringae pv. pisi (Ppi), 272 bp products (100% sequence identity) were yielded with this isolate and the Ppi strain ATCC 11043, and the sequence was 99.57% identical to the Ppi isolate (X97405) in GenBank. Sequence analysis revealed that partial sequences of 16S and 23S rRNA and 16S-23S sequence of the isolate 1314 shared 100% identities with the Ppi strain ATCC 11043 in correspondence. MLSA and phylogenetic analysis were carried out with the sequences from four housekeeping genes including gap1, gltA, gyrB and ropD. The isolate 1314 was clustered with Ppi strains in the phylogenetic tree. The result of Biolog test showed that this isolate was Ppi with the similarity (SIM) of 0.72 and probability (PROB) of 0.898.The isolate 1314 can trigger hypersensitivity reaction on tobacco by artificial injection inoculation and cause typical water-soaked symptom on the stem of inoculated young pea plant. Based on these results above, this isolate was identified as Pseudomonas syringae pv. pisi.

In order to elucidate the cause of cotton red leaf disease, a series of experiments were conducted to study correlation between sink/source (boll/leaf) ratio and the disease severity. The results showed that the grades of the diseased plants were positively correlated with their boll/leaf ratios significantly. The early-maturing cotton variety had an earlier flowering date, higher boll/leaf ratio and a more serious disease severity than those of the late-maturing one. The air temperature before full squaring date was found more important on cotton growth and the occurrence of red leaf disease than soil moisture. Compared to the normal temperature treatment, increasing air temperature from the 3^rd leaf date to full squaring date promoted cotton vegetative growth and reduced the sink/source ratio, thus, the peak stage of the disease was postponed by about 45 days, the average disease index decreased by 72.9% and the average biological yield of cotton increased by 123.8%. Decreased boll/leaf ratio by removing squares and delayed topping could reduce the incidence of red leaf disease. The treatment of removing all squares from cotton plants resulted in the least disease severity. Foliar application of mepiquat chloride increased cotton boll/leaf ratio and was thus favorable to the development of red leaf disease. Those findings indicate that the imbalanced sink/source ratio is the main cause of red leaf disease in cotton. The growth of metabolic source (leaf) before flowering date determines the onset and the severity of the disease.

To understand the effects of different concentrations of methyl jasmonate (MeJA) on the resistance of wheat seeding against stripe rust caused by Puccinia striiformis f.sp. tritici (Pst), wheat seedling was inoculated with Pst after spraying with different concentrations of MeJA. The inducing effect was observed in the resistance to stripe rust pretreated with MeJA at the concentration of 2.00 mmol·L^-1 with the effect reached to 35.87%, MeJA could inhibit the germination of urediniospore significantly at the concentration of 0.50 mmol·L^-1 after 4 hours inoculation. Ultrastructure of intercellular hyphae, haustorium, and haustorial mother cells were significantly inhibited. A series of changes associated with resistance to infection were reported. Our results indicated that the resistance of wheat seedling against Pst was induced after MeJA treatment.

Bacterial fruit blotch caused by Acidovorax citrulli is a world-wide disease that causes serious damages on the cucurbits plants. luxR₄₂₂₉ is a LuxR family member gene in A. citrulli Ac-5 isolated from watermelon. The luxR₄₂₂₉ deletion mutant was constructed by using two-step homologous recombination. The pathogenicity, biofilm formation and the swimming ability of the wild-type Ac-5 and its mutant strains were investigated. Meanwhile, the expression of the genes fliR and fliC encoding flagella proteins and the genes hrpE and hrcN involved in Type III secretion system were also determined by using qRT-PCR. The result showed that the mutant strain had lower pathogenicity, weaker swimming ability, but stronger ability of biofilm formation compared to its wild-type parent. The expressions of fliR, fliC, hrpE and hrcN genes were decreased in the mutant, indicating a positive regulation of luxR₄₂₂₉ to these genes. This study revealed that the luxR₄₂₂₉ gene plays an important regulatory role in the pathogenicity, swimming ability and biofilim formation of A. citrulli.

AvrXccC is a type III secretion systems (TTSS) effector protein of Xanthomonas campestris pv. campestris (Xcc) strain 8004. Previous studies have shown that AvrXccC acts as a virulence factor in Arabidopsis rar1 mutant, and suppresses flg22-induced innate immunity. However, it remains unclear about the virulence target of AvrXccC in plants. Here, we found AvrXccC specifically interacted with the cytoplasmic kinase BIK1 in vitro and in vivo. Furthermore, AvrXccC did not suppress flg22-induced BIK1 migration in protoplast, indicating that AvrXccC is not a regulator of BIK1 phosphorylation. Interestingly, we found that AvrXccC was phosphorylated by BIK1 in vitro, suggesting that AvrXccC probably acts as a substrate of BIK1 to interfere with the plant innate immunity.

Phytophthora is the highest concern of plant pathogenic fungi. Four candidate genes of ITS, CO1, EF-1α and β-tubulin were tested with 122 strains from 80 species of Phytophthora genus to investigate the feasibility of serving as a DNA barcode. The results showed that the highest successful rate of PCR amplification and sequencing were from ITS and CO1 with 100% and 96.7%. Even the barcoding gaps of ITS, CO1 and β-tubulin were obvious. A smaller overlap between the frequency distribution of intra- and interspecific existed. The Wil-coxon rank test of intraspecific showed the equal distinguish effect, while the ability to distinguish interspecies was ITS>CO1>β-tubulin. ITS fragment and CO1 gene should be combined as barcodes for the eleven quarantine Phytophthora species, with assist of β-tubulin gene.

Rice blast causes serious damage to rice growth and great economic losses in rice production yearly. Compared to the wild type plants, OsDCL1-RNAi knock-down mutant plants showed enhanced resistance to rice blast. To investigate the resistant mechanism of the mutant to rice blast, we analyzed and compared the transcriptome profiling in the wild type and OsDCL1-RNAi mutant plants upon challenge with the rice blast pathogen. Out of 7 129 differentially expressed genes (DEGs) responsive to the infection of rice blast pathogen, 5 382 and 5 180 were identified in NPB and OsDCL1-RNAi mutant, respectively. The genes involved in 4 pathway groups including biotic stress response, signaling, protein metabolism, and RNA regulation were significantly identified in the DEGs. Moreover, we identified 1 318 DEGs in OsDCL1-RNAi mutant compared to the wild type plants without pathogen infection. Of those DEGs in OsDCL1-RNAi mutant plants, 70% were up-regulated and most of them were involved in two pathway groups, i.e., biotic stress response and signaling. Interestingly, the genes encoding various families of pathogenesis related (PR) and cell wall related proteins were frequently identified in biotic stress response genes. We also identified 10 jasmonic acid and 11 salicylic acid related genes that were down- and up- regulated in OsDCL1-RNAi mutant plants, respectively. The genes encoding transcription factors, e.g., WRKY and MYB families, and some receptor kinases were also abundantly identified in OsDCL1-RNAi mutant plants. Some differentially expressed genes were selected for qRT-PCR validation, the result is consistent with the DEGs. The altered transcriptome profiling of OsDCL1-RNAi mutant plants documented in this study could provide new insights into the enhanced resistance mediated by the silencing of OsDCL1 in rice.

Based upon the plasmid pSVBV-E3 containing the full-length Strawberry vein banding virus (SVBV) genomes, infectious clone of SVBV was constructed. Fragments of 0.5-mer SVBV and 1.0-mer SVBV were obtained by digesting plasmid pSVBV-E3 with restriction enzymes, respectively, and then sequentially ligated to the plant expression vector pBINPLUS to generate the infectious clone pBIN-1.5SVBV. Plasmid pBIN-1.5SVBV was transformed into Agrobacterium tumefaciens, and then innoculated onto Fragaria vesca and 4 species of Nicotiana plants to validate its infectivity, respectively. The result showed that typical vein banding and yellowing symptoms appeared on F. vesca plants inoculated with SVBV infectious clone 8 weeks later, and cp gene of SVBV could be detected from symptomatic F. vesca plant by PCR, and the presence of SVBV genome was further confirmed by Southern blot. In contrast, no symptoms could be observed on 4 species of Nicotiana plants inoculated with SVBV infectious clone 8 weeks later, and cp gene of SVBV could not be detected by PCR, either. Taken together, successful establishment of infection of SVBV in F. vesca through agroinoculation of the infectious clone would provide a basis for further study the pathogenic mechanism of SVBV in F. vesca.

Typical tobacco bushy top disease was mainly caused by mixed infection of Tobacco bushy top virus (TBTV) and Tobacco vein distorting virus (TVDV). Coat protein (CP) encoded by TVDV could package the genome of TBTV and TVDV respectively, resulting in forming of virions and mediating the transmission by aphid. However, some disease samples in the field contain the TVDV in the absence of TBTV. In this study, cp genes of two types of TVDV (TVDV-MD isolate with the coexistence of TBTV and TVDV-BS isolate without the coexistence of TBTV), were cloned by RT-PCR and compared. The lengths of TVDV-MD and TVDV-BS cp genes are 618 nt and 621 nt, respectively. Moreover, nucleotide and amino acid identities of cp gene between TVDV-MD and TVDV-BS were 90.02 % and 89.81 %, suggesting that TVDV-MD and TVDV-BS CPs belong to different types. However, sequence identities of TVDV cp gene with the same length were 98.5 % -100%. In addition, phylogenetic analysis also showed that these two types of cp gene respectively belong to two groups with far distance. Although TVDV-MD CP and TVDV-BS CP have about 10% amino acid divergence, their structures predicted by SWISS-MODEL did not present remarkable difference with only slight alteration in minor elements and linker regions. Our results suggested that mutation on amino acid sequences rather than the change of structure of CP in TVDV-BS might cause the inability of packaging of TBTV and result in missing the TBTV during the transmission of tobacco bushy top disease.

Rice false smut disease is an increasingly concerned problem in both rice research and production. Diverse resistance germplasm is the most important factor in resistance breeding program for controlling this di-sease. After three years of screening in our disease nursery in Sichuan Province, out of 843 accessions we identified 179 that showed no occurrence of false smut disease and two accessions that were highly susceptible with diseased panicle percentage more than 50%. The rest accessions had diseased panicle percentage ranging from 0.1% to 48.8%. More than half (55.1%) of the infected panicles formed only one smut ball, but the number reached up to 38 in the most susceptible accession Pujiang 6. Then we selected 36 accessions for further evaluation of disease severity variation at two locations with 2-3 planting times with Pujiang 6 as a susceptible control. Three of the 36 accessions showed no disease in all these experiments, and 18 accessions were occasionally infected and thus were highly resistant to false smut disease. In order to find accessions with the highest polymorphism to the susceptible accession Pujiang 6, we performed polymorphism analysis between 35 accessions and Pujiang 6 by using 450 simple sequence repeat (SSR) markers. Twelve accessions showed polymorphism to Pujiang 6 in more than 10% of these markers. IRAT144 was at the first place with 16.1% of polymorphism rating, followed by Luxiang 90-2 and Domsia-2 with 14.8% and 14.4%, respectively. Thus the 12 accessions were selected to cross with Pujiang 6 to construct gene mapping and cloning populations.

Xanthomonas campestris pv. campestris (Xcc) causes black rot on cruciferous plants and is one of the model bacteria for studying the plant-microbial interaction. The XC_3605 gene of Xcc was deleted by using a suicide plasmid pK18mobsacB to generate the mutant D3605. The exopolysaccharide production, mobility and virulence on the host plant were decreased in mutant D3605, while biofilm formation was stimulated. The altered phenotypes of the mutant could be restored by the intact XC_3605 gene carried in pLAFRJ. These results indicated that XC_3605 gene plays an important role in the pathogenicity of Xcc.

Epidemic processes and yield losses of early leaf spot (Cercospora arachidicola) and web blotch (Phoma arachidicola) of peanut were analyzed in the field plots. The results showed that there were no significant correlations between the two diseases at the early stage when they occurred together, but the negative correlation between the two diseases gradually increased with the development of the disease. This phenomenon showed that there was an inhibitory effect between the two diseases. Disease severity was different when C. arachidicola and P. arachidicola were inoculated in the different growth stage, early leaf spot and web blotch were more serious when C. arachidicola and P. arachidicola were inoculated in the early flowering stage and the flowering stage than in the late flowering stage, and the results demonstrated the inhibitory effect. The yield loss when two diseases occurred together was less than the sum of that caused by each individual disease. Yield loss of Baisha1016 caused by the two diseases occurring together was about 77.2%-85.7% of the sum of yield loss that caused by the corresponding individual disease. Yield loss of Silihong caused by the two diseases occurring together was about 76.1%-79.6% of the sum of yield loss that caused by the corresponding individual disease. The yield losses of two cultivars were both reduced gradually with the delay of inoculation period at the different growth stage.

Based on biological property and 16S rDNA sequence anslysis, actinomycetes strain SC11 was identified as Streptomyces spectabilis. Strain SC11 could establish a stable colonization in cotton rhizosphere and cotton root with populations of 1.38×10⁵ CFU/g and 2.95×10³ CFU/g 30 d after sowing, respectively. Field experiments in 2011 and 2012 showed that the powder formula of strain SC11 exhibited control efficacies (CE) of 42.51% and 36.70% against cotton Fusarium wilt, which was as effective as that of the 50% carbendazim (WP). During 90-150 dpi, the CE of the strain SC11 was significantly higher than that of the carbendazim treatment control (P<0.05). The area of the top 3 leaves treated by the SC11 increased by 52.6% compared with the water treated control, and the yields of seed cotton and ginned cotton increased 21.9% and 23.0% (P<0.05), respectively. Results of above research suggest that the strain SC11 has a potential biocontrol ability against cotton Fusarium wilt.

Populus alba is widely planted in northeast China and seriously infected with a rust fungus. In this study, the causal rust fungus of Populus alba was identified as Melampsora laricis based on its morphological characteristics and life cycle. Its spermogonial and aecial stages on Larix kaempferi and L.olgensis were confirmed by inoculations with basidiospores produced on P. alba.

It should be taken seriously to monitor powdery mildew (PM) of wheat quickly and accurately in continuous space since the outbreak of this disease may cause substantially reduction of output and lower the quality of wheat. A new method was presented to monitor the external environment of the PM pathogen based on the remote sensing information including greenness wetness and land surface temperature (LST) and the meteorological information including temperature and the number of rainy days. Three kinds of models were established using algorithms Fisher liner discriminant analysis (FLDA) and support vector machine (SVM), respectively. And these models were individually used to supervise the incidence of PM in the western regions of Guanzhong Plain, Shaanxi Province. The results showed that the integrating models (integrating the remote sensing information and the meteorological information) reached at a satisfactory accuracy (FLDA: 74%, SVM: 77%). The integrating models performed better than the meteorological models (FLDA: 60%, SVM: 65%) and the remote sensing models (FLDA: 65%, SVM: 70%). The SVM method outperformed the FLDA method. The results indicated that the integration of remote sensing information and meteorological information can promote the monitoring accuracy of plant diseases.

Root rot caused by Rhizoctonia is one of the most important diseases in strawberry. In this study, the pathogen of strawberry root rot was identified based on morphology, nucleus fluorescence staining, hyphal anastomosis test, rDNA-ITS sequence analysis and Koch′s postulates. The isolates were obtained from the diseased roots of strawberry collected from greenhouse in Changping district, Beijing in 2014. Three isolates were identified as binucleate Rhizoctonia (BNR) based on their morphological characteristics and nuclei staining. Hyphae of the isolates fused with an AG-A tester isolate. rDNA-ITS sequence (GenBank Accession No. KP893156) of isolate CP-Z exhibited 100% similarity with four Ceratobasidium sp. AG-A isolates. Strawberry seedlings inoculated with isolate CP-Z showed black root rot and died gradually. Binucleate Rhizoctonia was re-isolated from the ino-culated strawberry roots. This is the first report of BNR AG-A causing root rot of strawberry in China. The optimum temperature for mycelial growth of the pathogen is 25℃~28℃.

For finding out the composition and aggressiveness variation in Fusarium populations causing wheat head blight in Henan province, three hundred and twenty-seven isolates of Fusarium spp. from diseased wheat spikes collected in 84 fields in 15 cities across Henan province during 2007 to 2014 were tested for species identification, deoxynivalenol chemotype and aggressiveness assay. The results indicated that F. graminearum s. str. and F. asiaticum (97%) are the main causal agents of Fusarium head blight in Henan province, whereas the remaining 2.1% of the isolates were identified as F. pseudograminearum, and F. culmorum, F. equiseti and F. verticillioids took up 0.3% of total samples, respectively. Among the isolates of F. graminearum species complex, F. graminearum s. str. isolates are prevalent in northern Henan province; F. graminearum s. str. and F. asiaticum were both distributed in its central and southern areas, and F. graminearum s. str. is a prevalent species in central Henan province, but F. asiaticum is prevalent in southern Henan province. All 291 F. graminearum s. str. isolates belong to the 15-acetyl deoxynivalenol type (15ADON), among the 26 F. asiaticum isolates, 22 isolates were of the 3-acetyl deoxynivalenol type (3ADON) while 3 isolates were the nivalenol (NIV) type and only 1 isolate was of 15-acetyl deoxynivalenol type. According to virulence tests, F. graminearum s. str. (15ADON) isolates were divided into three kinds of virulence groups (weak, moderate and strong virulence), the members in each group are 48 ,93 and 90 respectively. The ratio of them was approximately 1:2:2.

Celery bacterial soft rot has become severe in Beijing, especially in Shunyi and Tongzhou district. In order to identify the causal agent of the disease, bacterial strains were isolated from the diseased celery stems, and the pathogenicity of the isolates was then confirmed by inoculation of these isolates on celery seedling following Koch's postulation. Fifty-four pathogenic isolates were obtained. Based on morphological observation, physiological, biochemical and Biolog test, as well as multilocus sequence analysis of six concatenated housekeeping gene sequences(pgi、rpoS、mdh、proA、mtlD、icdA), the causal pathogen was identified as Pectobacterium carotovorum. Among these disease-causing isolates, 45 strains were identified as P. carotovorum subsp. odoriferum with a frequency of 83.33%,6 strains as P. carotovorum subsp. carotovorum with a frequency of 11.11%, and 3 strains as P. carotovorum subsp. brasiliensis with a frequency of 5.56%. These results indicate that the causing agents of celery bacterial soft rot in Beijing were identified as three subspecies of P. carotovorum, and P. carotovorum subsp. odoriferum was dominant.

This study was conducted to develop a TaqMan Locked Nucleic Acid (LNA) probe real-time PCR assay that had a wide range of application, high sensitivity, and stability for the detection of Wheat dwarf vius (WDV) in the field. The specific primers and LNA probe (19 bp) were designed from conserved WDV-Rep sequences obtained from GenBank. The method was established to detect samples infected with WDV in the fields by TaqMan LNA probe real-time PCR. Matrix method was used to optimize the reaction system including the best concentration of the primers (0.5 μmol/L) and the probe (0.3 μmol/L). The slope and correlation coefficient of the standard curve were -3.424 3 and 0.997 3 , respectively, and the amplification efficiency was up to 96%. The coefficient of variation ranging from 0.11%-1.34% intra group and 0.44%-1.21% inter group indicated the excellent stability and reproducibility of the method. The sensitivity compared with the conventional PCR was analyzed by ten-fold dilution method. Results showed that the sensitivity of the former (55 copies/μL) was at least 100 times higher than the latter. In addition, the designment of probe become simpler and more convenient than TaqMan probe real-time PCR due to that the greatly shortened LNA probe is not prone to forming intermolecular and intramolecular secondary structures. Our results indicated that the detection method based on LNA probe is feasible for WDV early monitoring in the field, thus providing an efficient and rapid tool for WDV epidemiological investigation.