16S rDNA and ribosomal protein gene (rp) of JWB-Hubei phytoplasma isolates from jujube trees collected in Sui county, Suizhou city, Hubei Province were amplified by Nested-PCR. The PCR products were purified, cloned and sequenced. The size of cloned fragments from 16S rDNA and rp gene were 1 247 bp and 1 196 bp, respectively. The phylogenetic analysis on nucleotide (nt) sequences of 16S rDNA and rp were constructed. The results of 16S rDNA gene nucleotide sequences analysis showed that the JWB-Hubei phytoplasma isolates had the higher 99% nucleotide sequence identity with the isolates from Shandong, Henan Province and so on, and were grouped into a separate sub-branch of the same sub-group. Virtual RFLP analysis results showed that JWB- Hubei belonged to a member of 16SrV-B subgroup, which was in accordance with the phylogenetic tree. The nucleotide sequences based on rpl22-rps3 gene from JWB-Hubei phytoplasma isolates showed that the higher 99% nucleotide sequence identity with the isolates from Shandong, and Shannxi Province. The phylogenetic tree analysis indicated that JWB-Hubei phytoplasma isolate was clustered into a separate sub-branch of the same sub-group with the JWB phytoplasma, belonging to rpV-C subgroup. The above results identify the classification status and genetic evolution relationship of the JWB-Hubei isolate, which lay theoretical foundation to further study on classification and genetic evolution of the phytoplasma.

Fusarium species are important fungal pathogens of maize (Zea mays L.) and other cereals worldwide. A total of 163 Fusarium isolates were recovered from maize basal stalks collected from 14 districts (42 counties) in Henan Province in 2011 and 2012 in order to analyze the population distribution of Fusarium spp. on maize stalk. Fusarium isolates were identified based on morphological characters and species-specific PCR assays. DNA sequence-based identification of some unsure isolates was achieved using the translation elongation factor 1-α (TEF-1α) gene region. The results showed that Fusarium graminearum was the most prevalent species representing 44.2% (72 of 163) of the isolates, followed by Fusarium proliferatum and Fusarium verticillioides, representing 28.8% (47 of 163) and 27.0% (44 of 163), respectively. Our results also indicated that F. verticillioides was predominant in Jiaozuo (47.6%) and Luoyang (50.0%) in northwest Henan, as well as Xuchang (45.5%) in central Henan, F. proliferatum dominated Shangqiu (42.9%) and Kaifeng (57.1%) in east Henan, and F. graminearum was dominant in Anyang (66.7%), Puyang (71.4%) and Xinxiang (62.5%) in north Henan, Nanyang (57.1%) and Zhumadian (45.0%) in south Henan, and Zhengzhou (57.1%) and Luohe (66.7%) in central Henan.

In order to know the pathogens of maize ear rot in Gansu Province, samples were collected from 4 ecological zones in september, 2009. The pathogens were isolated by tissue isolation method from cankered maize ear on standard medium. Fusarium colonies obtained were identified according to the taxonomic system of Leisle after being subcultured and single-spored . The results showed that the total 271 Fusarium isolates were obtained from the sampling sites, and the dominant species were F. verticillioides and F. culmorum. According to Koch's Postulate, the pathogenicity of isolate GSYC17-2-1 was tested with mixed isolates on maize varieties Shendan 16 and Jinsui 96832 and verified that F. verticillioides was pathogenic to maize ear. The two F. verticillioides isolates were selected randomly for sequence analysis of EF-1α (tef) gene. The PCR products of the three isolates were collected, purified and sequenced. The sequences were aligned in GenBank. It was showed that a very close relationship of isolates GSLT4-3-2 with the FN179339, FN179345 and FN179338; and that of GSTS24-2-1 with FN179343, FN179346, FN179340, FN179344 and FN252384 downloaded from GenBank, their max similarity were 100% similar with F. verticillioides. The results also indicated that The Fusarium could grow under15～35℃, pH 4～10, with optimal condition at 28 ℃, pH 8, and the largest conidia germination at pH 7. Vegetative growth of F. verticillioides was found to be significantly influenced by nitrogen source compared with carbon source. The best cultural condition for mycelial was 24 h under dark while the largest sporulation quantity was alternating light and dark. The lethal temperature was 70 ℃ for 10 min for hypha of F. verticillioides.

Sesame Fusarium wilt (Fusarium oxysporum f. sp. sesami, FOS) is an important disease in sesame production. In order to estabolish an effective evalutating method for FOS pathogenecity, 15 strains were tested on Zhengzhi 98N09 and other three cultivars of sesame at young seedling stage. The results indicated that the inoculated seedlings showed wilt symptom in seven days after inoculation with 1×10⁶ microconidia/mL suspension mixed with aseptic vermiculite and soil under the ratio of V1∶V2∶V6 (i. e., the final concentration of 1.4×10⁵ microconidia/g soil). The optimal date for investigating pathogenicity was the 25-28 th days after inoculation. Among the tested 15 FOS strains, eight strains showed high pathogenicity (DI>50)and five strains showed low or no pathogenicity (DI<20) on the four cultivars. The results also showed that different cultivars exhibited the various resistance levels to 15 strains. This technique could be applied for the evaluation of the FOS pathogenicity and sesame resistance to FOS, and give the technological supports for further exploration of these mechanisms.

Thirty-two Fusarium oxysporum isolates obtained from wilt bitter gourd in different regions of China were identified by morphology and formae speciales test. The results showed that all isolates were Fusarium oxysporum f. sp. momordicae (FOM). These isolates could only infect the seedlings of bitter gourd and bottle gourd, but not the other cucurbit species. Amplifying and sequencing results of the rDNA-ITS of isolates showed that the total length of rDNA-ITS (ITS1, 5.8S and ITS2) of FOM isolate was 456 base pairs. The clustering analysis of rDNA-ITS indicated that FOM and F. oxysporum f. spp. isolates could be gathered into one group. Meanwhile the results of the genetic diversity analysis with RAPD molecular marker showed that the genetic similarity coefficient of FOM isolates and the other formae speciales of F. oxysporum isolates from cucurbit hosts was ranging from 0.59 to 0.99; forty-eight isolates could be divided into ten RAPD groups (G_1-10) when the genetic similarity coefficient reached 0.85. In RAPD dendrogram, all the FOM isolates were gathered into one phylogenetic branch (group G₁) with the genetic similarity coefficient ranged from 0.92 to 1.00, which indicated a high genetic similarity existed in these isolates, and classification of phylogenetic group was related to geographic origin in some extents.

Acireductone dioxygenase (ARD) is an enzyme catalyzing the penultimate step of the methionine salvage pathway (MSP) in ubiquitous prokaryotic and eukaryotic organisms. Here the counterpart gene in Xanthomonas oryzae pv. oryzae (Xoo), named xard was identified. The nucleotide sequences of xard shared the entire identities in Xoo strains PXO99^A, MAFF311018 and KACC10331. The strain with an inactive form of xard could not grow normally when methylthioadenosine (MTA) as the exclusive sulfur source. This result confirmed that Xard functioned in MSP. Post inoculation of xard mutant and wild type strain PXO99^A on rice line IR24, the lesion length data of 15 days showed that mutation of xard gene had no effect on virulence of Xoo in rice plant IR24.

As one of the largest transcription factor families in plant, many WRKY mumbers were reported to play essential roles in the developmental process and responses to biotic/abiotic stress. To better understand the function of rice WRKY proteins, the expression profile of eight WRKY proteins in leaf growth and Xa21-mediated Xanthomonas oryzae pv. oryzae (Xoo) resistance response was surveyed by western blotting (WB). The results demonstrated that OsWRKY12, 43, 55 and 86 were expressed in a low level at seedling stage and increased gradually in later stages. OsWRKY50 and 84 were expressed in a high level at seedling stage, while decreased in the later stages. However, the expression level of OsWRKY40/64 and 52 was not detected in rice leaves at all stages. In the rice-Xoo incompatible interactions, the expression of OsWRKY40/64, 50 and 52 was induced dramatically, and that of OsWRKY84 was also increased. While the expression levels of OsWRKY12, 43, 55 and 86 were stable. Comparison analysis among rice-Xoo incompatible, compatible and mock treatments revealed a parallel expression pattern between the incompatible and compatible interactions. Based on these experiments, we postulated that OsWRKY12, 43, 55 and 86 might play a role in rice leaf growth, OsWRKY40/64 and 52 might be essential in the interaction between rice and Xoo, while OsWRKY50 and 84 might be involved in both processes.

cDNA-amplified fragment length polymorphism(cDNA-AFLP) was employed to analyze the gene expression patterns of Chinese wild melon ‘Yuntian-930’ in response to Podosphaera xanthii race 2F. A total of 188 transcript derived fragments (TDFs) were generated with 256 pairs of primers, of which 109 were up-regulated while 79 down-regulated. 60 of differentially expressed sequence tags (ESTs) were produced after cloning and sequencing. Blastx analyses and functional annotations were then performed and the results revealed that most of them were involved in substance synthesis and metabolism(48%), others were related to transcription(12%), disease/defense(12%), transcriptional regulation(8%), energy metabolism (8%) and signal transduction(4%). Seven others did not show any homology to sequences with known functions. Four differentially expressed genes related to metabolism, disease-defense, signal transduction and protein trafficking were chosen for further qRT-PCR expression, and the results fully confirmed to their cDNA-AFLP profiles. Meanwhile, the results also indicated that these genes might play roles in interaction between melon and powdery mildew pathogens.

College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, China)
Abstract:The cereal cyst nematode has become one of the serious diseases of wheat in recent years in China. The basic work for disease control is to clarify the infection dynamics of pest nematodes. In this study, the infection dynamics of two species of cereal cyst nematode, Heterodera avenae and H.filipjevi were assessed under field conditions in Zhengzhou of Henan. The results indicated that the wheat roots were infected by the second stage juveniles (J2s) two weeks after planting. A few of J2s developed into J3 subsequently. The first infection peak of J2s appeared at the sixth weeks after planting, and a few of the fourth stage juveniles were found in wheat roots at the same time. As the temperature decreased after 60 days of planting in the winter, the number of juvenile at different stages remained stable. As the temperature increased in the spring at 120 days after planting, the number of the second stage juveniles in roots started to increase and reached the second peak in late March and early April (150 days after planting), with fewer nematodes than those in the first infection peak. Duration of the second peak is also much shorter than the first one. Finally the larva gradually developed into white females and cysts. The infection dynamics of two CCN species are almost same, but J3, J4 and white female of H.filipjevi appeared one week earlier than H.avenae. These results provided the essential information for the control of cereal cyst nematode of wheat.

The sensitivities of 100 Magnaporthe oryzae isolates from the main rice production areas in China to SYP-1620 were determined by colony diameter assay. The results showed that the EC₅₀ values were distributed in 0.011 1-0.295 6 μg·mL^-1. The mean EC₅₀ value was (0.078 6±0.056 1) μg·mL^-1. The sensitivity frequency of M. oryzae to SYP-1620 distributed as a unilateral peak curve, which showed that there was no resistant subpopulation among these strains, and could be used as baseline-sensitivity for field resistance monitoring of M. oryzae to SYP-1620. Two highly resistant mutants and five low resistant mutants were obtained by fungicide adaptation at a mutation frequency 1.11×10^-4. The highly resistant mutants showed stable resistance factors were above 1 000 folds, but the low resistant mutants which resistance factors were 2.05 to 4.55 folds and unstable. As compared to parent isolates, the virulence on rice leaves of the two highly resistant mutants exhibited significantly decreased. However, there was no significant difference of fitness between the five low resistant mutants and their parent isolates. Cross-resistance studies showed that there was positive cross resistance between SYP-1620 and azoxystrobin, but no correlationship between SYP-1620 and iprobenfos, isoprothiolane. Based on the significantly impaired fitness, the field resistance risk of M. oryzae to SYP-1620 might be low to moderate. The cytb gene was cloned from the mutants and parent isolates. One single-point mutation was found in CYTB at amino acid position 143 from glycine to serine (G143S), which might be the resistant molecular mechanism of M. oryzae to SYP-1620. The AS-PCR was developed to detect the G143S mutants in field. There was no mutation in cytb gene of the low resistant mutants.

The sensitivity to fluopicolide of 42 Phytophthora capsici strains collected from Taian, Pinggu, Hangzhou and Kunming were determined by mycelia growth inhibition method. The results showed that EC₅₀ values ranged from 0.618-0.927 μg·mL^-1 with a mean of 0.743±0.067 7 μg·mL^-1. The sensitivity frequency of P. capsici to fluopicolide distributed as a unimodel curve, which showed there was no resistant subpopulation among these isolates, so this sensitivity baseline was suitable for resistance monitoring of P. capsici to fluopicolide. The two fluopicolide-resistant mutants were selected by UV-irradiating and fungicide-selection. Genetic stability, mycelium growth rate and cross-resistance to other fungicides of the resistant strains were determined by measuring the colony diameters, differences of virulence and sporangium production between the sensitive and resistant strains were measured by leaf disk in vivo. The results showed that the resistance of the induced P. capsici isolate TA-R to fluopicolide reached 58.0 folds after 48 times selection compared with the sensitive strain, while the resistance ratios of the mutant that inducted by UV-irradiating reached up to 260.6. The resistant levels of two resistant mutants were stable. The TA-R and TA-UV exhibited positive cross resis-tance to metalaxyl and cymoxanil, while negligible cross resistance to dimethomorph, chlorothalonil, mancozeb and propineb. The differences in virulence and sporulation between resistant strains and TA weren't significant, however, the growth rates and dry-weights of mycelium of two resistant mutants were lower than that of the sensitive(P＜0.05).

Arbuscular mycorrhizal fungi (AMF), as one kind of the environmental functioning organisms, have the dual role of biocide and fertilizer, which not only enhance nutrient absorption and utilization by host plants, but also antagonize soil-borne pathogens and improve plant disease resistance. In recent years, cereal cyst nematode (CCN) disease on wheat (Triticum aestivum) become serious and the new biocontrol approach needs to be explored. The purpose of this study was to clarify the interaction relationship between AMF and CCN; evaluate the effects of different AMF suppressing CCN and reducing disease. Pot experiments were carried with 12 treatments: inoculation with AMF Gigaspora margarita (Gi.m), Glomus mosseae(G.m), Glomus intraradices (G.i), Glomus versiforme (G.v), Gi.m+G.m+G.i+G.v, CCN, CCN+Gi.m, CCN+G.m, CCN+G.i, CCN+G.v, CCN+Gi.m+G. m+G.i+G.v and non-inoculation control (CK). The results showed that the treatments with AMF reduced CCN infection rate, cyst numbers in the soil and J2 numbers in the root and Gi. m treatment was the best. CCN reduced the numbers of AMF entry points and spores. The arbuscule colonization percentage in Gi.m and CCN +Gi. m treatments was the highest. The activity of superoxide dismutase(SOD), phenylalanine ammonia-lyase (PAL), and catalase(CAT) in roots inoculated with CCN+Gi.m were significantly higher than that in the other treatments, while the content of malondialdehyde(MDA) was lower than that in the other CCN+AMF treatments. The plant height, stems and leaves dry weight of plants inoculated with Gi. m or G. i were higher than that in the other treatments; the weight per ear and yield per plant treated with Gi. m, or CCN +Gi. m were higher than other treatments. That showed AMF could inhibit CCN, enhance wheat growth and increase yield in some degree, and Gi.m was superior. It was suggested there was negative interactions between AMF and CCN; and AMF can antagonize CCN through inducing defense reactions.

Inhibition of Hypocrellin A (HA) against 18 pathogenic fungi by mycelium growth rate in light and dark conditions was evaluated. The results showed that 50 mg·L^-1 HA exhibited antifungal activities against all of 18 pathogenic fungi under luminous intensity of 12 000 Lx. The inhibition rates of HA to Lecanosticta acicola, Fusarium graminearum, Valsa mali and Botryosphaeria dothidea were all higher than 90%. The EC₅₀ of HA to Botryosphaeria dothidea and Lecanosticta acicola was 0.60 mg·L^-1 and 0.77 mg·L^-1 respectively. However, HA showed weak and even no antifungal activity in dark condition. The study suggested that the potential of HA could be as a new kind of photoactivated biopesticide.

The diseases caused by Phytophthora lateralis were risky and quarantinable. A species-specific PCR assay for rapid and accurate detection of the pathogenic oomycete P. lateralis in diseased plant tissues was developed in the study. Based on differences in tRNA sequences of P. lateralis and other Phytophthora spp., a pair of species-specific primers T1/T2 was synthesized. More than 15 species of Phytophthora and other species of pathogens were used to test the specificity of the primers; T1/T2 amplified only a unique 192 bp band from P. lateralis. The detection sensitivity with T1/T2 was 10 pg of genomic DNA in 25 μL PCR reaction solution. A nested PCR procedure using T3/T4 as the first-round primers and followed with T1/ T2 increased detection sensitivity 10 000-fold to 1 fg. The detection sensitivity for the zoospores in distilled water was 0.5 zoospore. The duplex PCR method combining primers (T1/T2 and T3/T4) was used to detect the pathogen in the plant tissues inoculated with pathogens. Furthermore, Real-time fluorescent quantitative PCR assays were developed to detect and monitor the P. lateralis from the diseased plant tissues.

The primers CM1/CM4 and PSM1/CM3 were designed for specific and sensitive detection for Clavibacter michiganensis subsp. nebraskensis(Cmn). The primers PSM1/CM3 could amplify 4 strains of Cmn and get an expected 208 bp product, but no target band for other 36 strains of related species. The nested PCR by primers CM1/CM4 and PSM1/CM3 could detect as low as 40 fg DNA or 6.8 CFU bacteria of Cmn with a better sensitivity than conventional PCR with primers PSM1/CM3. The results of detection with 100 corn samples imported from USA showed 24% positive by nested PCR and 8% for conventional PCR. These detection methods for Cmn exhibited potential role in the routine detection for imported corn sample.

A multiplex PCR-based method was designed for accurate and rapid identification of Colletotrichum orbiculare, Sclerotinia sclerotiorum (Lib.) de Bary and Erwinia tracheiphila simultaneously in the early stages of the diseases. Three-level designs for six factors (three primers, Taq DNA polymerase, dNTP and Mg²⁺) were constructed to optimize the multiplex PCR system by using the orthogonal design method. The annealing temperature of the PCR reactions was also optimized. A triplex PCR system for simultaneous detection of these three pathogens of cucumber was successfully established. The reaction volume was 25 μL and the annealing temperature was 63^oC. The reaction solution contained 0.24 μmol·L^-1 CY1/CY2, 0.72 μmol·L^-1 SSFWD/SSREV, 0.336 μmol·L^-1 ET-P1/ ET-P2, 1 U Taq DNA polymerase, 0.15 mmol·L^-1 dNTP and 1 mmol·L^-1 Mg²⁺. The triplex PCR system could detect C. orbiculare, S. sclerotiorum and E. tracheiphila in infected plant samples and soils rapidly with the sensitivity at 10 pg DNA·μL^-1.

Take-all is the most damaging root disease of wheat and the economically important disease in Henan Province. Eighty-two strains were isolated from wheat root samples which collected from 10 cities, Henan Province. The strains were identified by morphological characteristics, pathogenicity determination and molecular identification. The hypha of 47 strains showed “∧” form at branch. The perithecium was produced immerge or semi-immerge, and the mature ascus was clavate. Ascospores were linear and slight curving and the mature ascospore had 5-10 clear membrance and the size was 68-94×2-4 μm.The rDNA-ITS region of 82 strains were amplified by PCR and then sequenced. By BLAST analysis, rDNA ITS of 47 strains revealed 97%-99% nucleotide identity to that of Gaeumannomyces graminis. Specific fragment of Gaeumannomyces graminis var.tritici was 870 bp in length and which was amplified using the specific primers of 4 variants, therefore the 47 strains were identified as Gaeumannomyces graminis var.tritici.

Retrotransposons are critical components of plant genome and have vital impact on the size, structure, function and the evolution of plant genome. Studies have shown that the transcription of retrotransposons could be activated by biotic and abiotic stresses, but their regulation mechanism and function are unknown. Three retrotransposon related genes were selected based on the microarray results of Yunnan landrace rice variety Acuche inoculated with Magnaporthe oryza and the impact of different stresses on their expression levels were analyzed by RT PCR. The results showed that all the ways of M. oryza, SA, 2, 4 D and high salt could induce the quick expression of the genes, suggesting that they could respond to both biotic and abiotic stresses and that they were ideal candidates for investigating expression and regulation of retrotransposons to stresses. Bioinformatics analysis showed that the 3 genes had conserved structure in Acuche and reference genome; their paralogs were related to disease resistant. These results implied that their expression probably contributed to resistance, their expression pattern and functions in biotic and abiotic stresses were valuable to investigate in detail.

The β-glucosidase is an important component of the cellulase complex. It not only hydrolyzes cellobiose and cello-oligosaccharide, but also prevents accumulation of cellobiose and reduces the inhibition of cellulase. By using of solid fermentation, fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE-52 and Sephadex G75 chromatography, β-glucosidase from antifungal Streptomyces spp.R₁₅ obained from the chinese cabbage rhizosphere in the suburb of Taian, Shandong Province was purified. The results showed that the purification fold and yield were16.28 and 14.59%, respectively. The molecular mass of the enzyme was estimated to be 63.89 kDa by 12% SDS-PAGE. To make an analysis of the enzyme reaction by TLC, β-glucosidase can react with salicin to form D-glucose and salicyl alcohol, which proved that the protein was β-glucosidase. By using of salicin as substrate, the Km and Vmax values for β-glucosidase were 10.644 mmol/L and 0.525 μmol·L^-1·min^-1, respectively. The optimum temperature was 65℃, the β-glucosidase was more stable when temperature was lower than 70℃. The optimum pH was 5, the relatively activity was above 75% when pH was between 3 and 7.Cu²⁺, Hg²⁺ and Fe²⁺ could inhibit the activity of β-glucosidase, the relatively activity were 0, 3.60%, 62.98% respectively. The inhibitory effect of Hg²⁺, Cu²⁺ were the strongest of them, the activity was completely lost. Ca²⁺, Mn²⁺ could stimulate the activity, the relatively activity were 137.18% and 119.52% respectively. Fe³⁺, Ba²⁺, Mg²⁺, K+, Al³⁺ and Zn²⁺ had no significant effect on the activity at the level P＜0.05.

To understand the correlation between lifestyle and extracellular CAZymes, secretomes and CAZymes were predicted and annotated in phytopathogenic fungi, followed by comparative analysis. The results indicated that the ratio of secreted proteins to total putative proteins in hemibiotrophic and necrotrophic fungi was higher than that in biotrophic fungi. The CAZymes especially glycoside hydrolases (GH) and polysaccharide lyases (PL) subfamilies were expanded significantly in hemibiotrophic and necrotrophic fungi. Functional analysis of secreted CAZymes indicated that the plant cell wall (PCW) degrading enzymes involved in degradation of cellulose, pectin and xylan, were more abundant in hemibiotrophic and necrotrophic fungi. The distinct expansion of PCW degrading enzymes indicated the high correlation between fungal lifestyle and CAZymes, meanwhile revealing their potential roles in pathogenesis in hemibiotrophic and necrotrophic fungi.

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot disease in cruciferous plants. Few effectors of Xcc have been characterized genetically and biochemically. In this study, a putative effector coding gene Xc_2994 in Xcc 8004 was characterized. In order to explore the virulence function of XopXccP, a XopXccP deletion mutant was generated by gene knockout. The pathogenicity of △XopXccP was tested on cabbage, cauliflower and Arabidopsis (Col-0). The results indicated that pathogenicity of △XopXccP was significantly declined. To test the function of XopXccP in vivo, an inducible promoter driven XopXccP was transferred into Arabidopsis. Pathogenicity test indicated that transgenic Arabidopsis appeared lesion mimic cell death after the induced expression of XopXccP. These results suggest that the effector XopXccP is a virulent factor on host plants and is required for pathgenicity on cruciferous plants.

PhoR/PhoP two-component is a global regulatory system in Bacillus subtilis. Under the phosphate starvation condition, the sensor protein kinase PhoR self-phosphorylates and transfers its phosphate group to the regulator PhoP. The phosphorylated PhoP impresses or activates the expression of phosphate-regulated genes. With the purpose of clarifying the function of regulator PhoP on the fengycin production in B. subtilis NCD-2, the phoP was in-frameless deleted by homologous recombination. The testing showed that the phoP mutant decreased the antifungal ability against the growth of Rhizoctonia solani in vitro. The lipopeptides were extracted from strains NCD-2 wild type and phoP mutant, then analyzed by Fast Protein Liquid Chromatography (FPLC). The results indicated that the production of fengycin, the major antifungal activity compound in strain NCD-2, was significantly decreased in the phoP mutant compared to that of strain NCD-2 wild type. All of these characteristics were restored by complementation of phoP gene in the phoP deletion mutant. It was confirmed that the phoP was a positively regulation factor for fengycin production in strain NCD-2.

Rice black streaked dwarf virus (RBSDV), a fijivirus, is transmitted by small brown planthopper (SBPH) in persistent propagative manner. P9 1 encoded by segment 9 is the minimal viral factor required for viroplasm formation and virus replication during RBSDV infection. In order to elucidate the infection route of RBSDV within its insect vector SBPH after viral acquisition, the antibodies against P9 1was prepared. Then, the distribution of P9 1 in the digestive system of SBPHs after ingestion of RBSDV was investigated by immunofluorescence microscopy with antibodies agianst P9 1 conjugated to FITC directly. At 3 days post acqurie virus (p.a.v.), P9 1 was first detected in the epithelial cells of the midgut. At 6 days p.a.v., P9 1 proceeded to the visceral muscles surrounding the midgut epithelial cells; then distributed the visceral muscles of the midgut and hindgut. At 10 days p.a.v., P9 1 distributed throughout in the salivary glands. These results indicated that RBSDV first infected in the midgut epithelium, then proceeded to the visceral muscles surrounding the midgut and hindgut, and finally accumulated into the salivary glands. This is the first report about the infection and spread of RBSDV in its insect vector SBPH, which could give support to develop effective measures for blocking the transmission of RBSDV by SBPH vector.

Bacillus cereus strain AR156, one kind of biopesticide, was cooperatively developed by Nanjing Agricultural University and Xinyi Zhongkai Agricultural Chemical Industry Co., Ltd. In order to explore the mechanism of this strain in preventing disease and promoting plant growth, biocontrol efficacy of AR156 on pepper wilt disease caused by Ralstonia solanacearum was investigated along with its growth promotion effects on pepper in the greenhouse, colonization ability, activation of the cellular defense responses such as hydrogen peroxide accumulation and callose deposition, and several plant defense related enzymes activity in pepper plants. It was showed that biocontrol efficacy of AR156 on pepper wilt disease was up to 73.31%, meanwhile improving the fresh weight of pepper plants by 22.30%. It was found that strain AR156 could stably colonize in rhizosphere at 5×10⁵ cfu per gram of root fresh weight on the 60th day after inoculation. Faster cellular defense responses were induced and the activity of plant defense related enzymes including superoxide dismutase (SOD) and peroxidase (POD) were significantly enhanced in pepper treated with AR156 and R. solanacearum. In conclusion, the broad spectrum resistance of AR156 to disease was realized by inducing cellular defense responses, enhancing plant defense related enzymes activity and forming robust colonies in the rhizosphere of pepper. In addition, it was found that AR156 treatment could increase the contents of chlorophyll in pepper leaves, which might be one reason that AR156 could promote pepper growth.

Dogwood is a valuable medicinal plant. Nanyang of Henan Province, is one of the main dogwood-producing areas in China. In recent years, Dogwood brown spots severely occurred in Nanyang. The leaf brown spots were divided into I- and R-types based on their distinctive symptoms. Five Alternaria sp. isolates from disease leaves were collected. Two representative isolates (A18 and A16) were identified as the members of Alternaria alternata based on their morphological characteristics and ribosomal DNA internal transcribed spacer (rDNA-ITS) sequences after their pathogenicity on dogwood leaves was confirmed. This is the first report of A. alternata causing brown spot disease on dogwood.

The stem canker disease of Sapindus mukorossi was investigated in Ningbo, Zhejiang Province, China. A fungus was isolated from the diseased stems and tested for pathogenicity according to Koch’s postulation. Its rDNA ITS sequence analysis and morphological characteristics showed that it was Fusarium solani, the anamorph of Nectria haematococca.The perithecia of N. haematococca were accasionally found in some severely infected stems. This is the first report of the stem canker disease of Sapindus mukorossi infected by N. haematococca in China.

The diseased roots of Glycyrrhiza uralensis with root rot symptom were sampled in Ningxia, China. Seventy eight fungal isolates were finally isolated from the materials and the pathogens were identified according to Koch’s postulate. Fusarium solani and Fusarium oxysporum were found to be pathogenic to G. uralensis plants. Based on the morphological characteristics and sequence analysis of EF-1α, the representative isolates G013 and FLR were identified as F. solani and F. oxysporum, respectively. The host range test showed that the two pathogens could also infect Solanum tuberosum and Glycine max, but not Triticum aestivum Linn, Vigna unguiculata, Medicago sativa, Phaseolus vulgaris, Cucumis sativus Linn, Solanum melongena L., Capsicum frutescens, Solanum lycopersicum, Zea mays L. and Gossypium spp.. This is the first report of root rot caused by F. solani and F. oxysporum on G. uralensis in China.

The pathogenicity of three strains (F-SD-8, F-BJ-2c-2 and F-HN-2a-1) of Valsa mali var. pyri causing pear canker and one strain (F-SX-A6) of V. mali var. mali causing apple canker in China were comparatively tested by wound inoculation on in vitro twigs of pear, apple and some other woody plants, and in vivo twigs of pear. Significant pathogenicity differentiation was detected in V. mali var. pyri. Generally strains F-SD-8 and F-BJ-2c-2 were highly pathogenic on pear although their culturing characteristics differed greatly. The strain F-SX-A6 was more aggressive on apple than on pear, and the strain F-HN-2a-1 showed significant lower pathogenicity on ten pear cultivars and other seven species of woody plants. Our results confirmed that two variants of V. mali had host preference and were also aggressive to crabapple, apricot, and peach besides apple and pear. Meanwhile, strains F-SD-8 and F-BJ-2c-2 could induce the formation of pycnidia on in vivo twigs of pear, which was not observed on in vivo twigs inoculated with F-HN-2a-1 and F-SX-A6.

In order to develop a rapid, sensitive and specific qPCR assay for detection and quantification of Tomato yellow leaf curl virus (TYLCV), a pair of primers and TaqMan probe were designed according to the conserved sequence of known TYLCV isolates. Combining with MNP technique, a novel MNP-qPCR detection method was established and verified based on specificity, sensitivity and reproducibility tests. The results indicated that the Ct value of plotted standard curve showed good linear relationship(R² =0.9994)with the log of copy number of template. The established method showed a high specificity for TYLCV detection without crossing reaction with Tomato severe leaf curl virus and Tomato yellow leaf curl Sadinia virus, and was 10-fold more sensitive than routine PCR. Both coefficients of variation were less than 2%, indicating a good reproducibility. We have provided a novel method for detection of TYLCV in plant samples rapidly and quantitatively.

A new sheath disease of rice was found in Huayuan county of Hunan province in July 2011. One isolate (named as HNHY001) with 4\|septate conidia was identified as Curvularia, which caused black spots on rice sheaths by artificial inoculation both in vitro and in vivo. The other one identified as Nigrospora sp. (named as HNHY002) did not induce any lesion. In seed detection, 27 Curvularia isolates were obtained from the 150 rice seed sets in Hunan province. Among those, four isolates were found to contain 4\|septate conidia, one of which could not produce branchy stroma and cause disease in an artificial inoculation test. However, the rest 3 isolates caused the typical sheath black spot symptoms. The results of BLAST analysis showed that the rDNA\|ITS sequence (JQ360963) of HNHY001 mostly resembled to that of Cochliobolus geniculatus and its mitosporatic 4\|septate conidial Curvularia spp.. Considering the fact that the sexual stage of HNHY001 during confrontation cultures with other 4\|septate conidial Curvularia isolates were not observed, we therefore regarded the isolate HNHY001 as Curvularia fallax, one of C. geniculatus mitosporatic Curvularia spp. based on the asexual stage characteristics, i.e., slightly straight 4\|septate conidium and branchy stromata. This is the first report of rice sheath black spot disease in the world.

To identify the secretion and translocation of the type Ⅲ effectors in Xanthomonas campestris pv. campestris (Xcc) by immunological detection method, the cyaA gene was cloned to pJXG harboring 3×FLAG, yielding the reporter plasmid pJAA carrying 3×FLAG and cyaA simultaneously. The reporter plasmid was verified with the promoter and signal region of XC_1553, a known type Ⅲ effector in Xcc. The plasmid pJAA1553 was transferred into Xcc 8004^* and 8004^*ΔhrcV. The secretion of XC_1553 was investigated by Western blotting. As a result, the secretion of XC_1553 was detected in extracellular protein of 8004^*/pJAA1553, but not in 8004^*ΔhrcV/pJAA1553. For the translocation of XC_1553, the cAMP levels in plants inoculated with Xcc strains were determined. The concentration of cAMP in plants inoculated with 8004^*/pJAA1553 was 15 times higher than that inoculated with 8004^*ΔhrcV/pJAA1553. To sum up, the reporter plasmid pJAA can be applied to identify the type Ⅲ effectors of Xanthomonas campestris pv. campestris.

The previous study found that MoCOS1 might function as a transcription factor. Mutant of MoCOS1 produced no conidiophore and less melanin in comparison with wild type strain Y34. RNA-Seq analysis revealed that 442 genes were regulated by MoCOS1. Of them, MoCMR1 was down-regulated. In this study, real-time quantitative PCR analysis further confirmed that mutation of MoCOS1 down-regulated the expression level of MoCMR1. Mutant of MoCMR1 had no obvious difference in quantity and shape of conidium. It was observed that the mutant of MoCMR1 produced slightly less melanin. However, the expression of gene MoCOS1 in MoCMR1Δ remained similar level with that of wild type strain Y34.

The chitinase gene of Magnaporthe oryzae was cloned into the fusion expression vector pET\|32a and expressed in Escherichia coli BL21(DE3) host cells.After the expression strain was induced for 4\|6 hours by 0.5 mmoL IPTG, soluble chitinase were obtained from upper liquid and purifed by Ni²⁺\|NTA affinity chromatography column. This fusion protein was injected into New Zealand rabbits to get polyclonal antibody. ELISA analysis showed that the titer of this polyclonal antibody with good specificity was 1: 204 800. Culture filtrates of 4 Magnaporthe oryzae wild type strains were tested with this antibody by ELISA and in vivo chitinase detection during the pathogen infection was carried by Western blot. The result showed that the expression level of the chitinase appeared to be significantly different among different strains as well as different infection stages of the strain 70\|15. Whether the expression level of the chitinase is related to the biological and physiological characteristics such as pathogenicity among different strains still remains further study.

PevD1, a secrete elicitor from Verticillium dahliae, activated hypersensitive response (HR) and systemic acquired resistance (SAR) in tobacco. In order to investigate the induced resistance and mechanisms of PevD1 in cotton, the PevD1 was expressed in E. coli and assayed the cotton resistance against V. dahliae and immune responses. The results showed that purified recombinant protein PevD1 was not a phytotoxic factor in No.42 Xin-luzao cotton variety. Injection of PevD1 in leaves improved cotton resistance at a concentration as low as 8 μg/mL, the highest disease reduction was 35.04% at the 15^th day post V. dahliae inoculation. This protein was able to systemically induce hydrogen peroxide and nitric oxide generation, vessel reinforcement, lignin and terpenoids deposition. PevD1 enhanced the expression of pathogenesis-related genes and lignin biosynthesis-related genes PAL, C4H1 and 4CL, and also increased enzyme activity of PAL, POD and PPO. The results demonstrated that the PevD1 protein triggered the plant immune responses and improved disease resistance in cotton. The research not only lay a theoretical foundation for cotton Verticillium wilt disease control but also provided a scientific basis for elucidating plant-elicitor interactions.

Rhizoctonia solani Kühn, the causal agent of corn sheath blight, are mostly multinuclear and he\|terokaryotic fungi, and it is difficult to obtain the simplifying nucleus during the perfect stage. In the study, the nucleus of mononuclear R. solani strain JN was purified by protoplast regeneration method and the rDNA\|ITS sequences of the cultures that each was regenerated from single protoplast was compared. Since the rDNA\|ITS sequences among the cultures were still different, the expected nucleus purification effect was not obtained. Therefore, amplification of the rDNA\|ITS sequences of the dicaryon and polykaryon was conducted. How\|ever, the difference among their autologous sequences were also observed. Based on the results, it was consi\|dered that the difference among rDNA\|ITS sequences in R. solani might be caused by the absence of the perfect stage during the evolution but irrelevant to the number of nucleus. In addition, the stable transformants of R. solani strain JN were unable to obtain by conventional vectors suitable for filamentous fungal transformation. A vector pRsA that hph gene expression was regulated by the promoter and terminator of R. solani AG\|3 actin gene was constructed. The pRsA was then transformed into the protoplasts of R. solani strain JN by PEG\|mediated transformation, and three transformants were identified by PCR amplification. However, the transformants lost hygromycin resistance after reculturing for five generations on PDA medium. The results might have a positive on the improvement of the R. solani transformation method.

Virus infection disrupts the normal metabolism and biological processes of the host. At the molecular level, virus infection triggers a global change in host gene expressions, in particular, the expression changes of host genes that are associated with the stress and defense against pathogen attacks. Thus, studying the alteration of plant gene expression upon virus infection may provide detailed insights into the molecular interaction between virus and host. The real\|time fluorescence quantitative PCR (qRT\|PCR) has been widely used to investigate the changes in the cellular gene expression. For the accuracy of qRT\|PCR analysis, normalization using the appropriate internal control genes is necessary. However, sometimes the expression of housekeeping genes that are commonly used as internal controls is affected by virus infection. In this study, the effect of Rice black\|streaked dwarf virus (RBSDV) or Rice stripe virus (RSV) infection on the transcript expression levels of 10 rice (Oryza sativa) housekeeping genes including 18S, 25S, ACT, β\|TUB, eEF\|1α, eIF\|4a, GAPDH, UBC, UBQ\|5 and UBQ\|10 were assessed by qRT\|PCR and then further analyzed by three commonly used algorithms: geNorm, NormFinder and BestKeeper. The qRT\|PCR results, combined with three algorithms analyses, revealed that half of the transcript levels of internal control rice genes were affected by RBSDV infection while most of the genes were stable in RSV\|infected rice plants. Among those genes, UBC and β\|TUB were the reference genes with the most unaltered transcript levels by infection with RBSDV and RSV. Taken together, our results showed that RBSDV and RSV infections differently affected the transcriptional expression of housekeeping genes that were commonly used for internal control in qRT\|PCR. Thus, determining the suitable internal reference genes is crucial for profiling the alteration of gene expression pattern by different virus infections.

Cotton wilt disease caused by the soil\|borne vascular wilt fungus is one of the most widespread and damaging diseases and difficult to control. It is beneficial to understand the pathogenesis and utilize the host resistance resource through the research on infection process. The green fluorescent protein\|tagged isolate of Verticillium dahliae was used for studying the infection process in different cotton species. The resistant cotton group containing Gossypium barbadense cv. Hai7124 and G. trilobum and susceptible one containing G. hirsutum cv. Junmian1 and G. davidsonii were used in the study. The results showed that there was no obvious difference in the initial invasion of V. dahliae when the conidia adsorbed on the root surface of all detected cotton 5 hours after inoculation. However, the infection process showed significant difference between susceptible and resistant cotton. In susceptible cotton, V. dahliae infiltrated into the cortex 3 to 5 days after inoculation, and colonized in the vascular 5 to 7 days after inoculation, then the pathogen was spread and reproduced quickly. The systemic infection achieved 14 days after inoculation and formed the symptom of verticillium wilt. The infection of V. dahliae to resistant cotton is a relatively slow process that the pathogen reached to cortex 5 to 7 days and to vascular tissue 7 to 10 days after inoculation. V. dahliae cannot spread 14 days after inoculation and difficult to finish the systemic infection. These results suggested that the structure, physiology and biochemistry of resistant cotton delayed the infection process compared with susceptible cotton. Therefore, the research of V. dahliae infection process to cotton is meaningful to the research of pathogenesis and the utilization of resistance resource.

Citrus tristeza virus (CTV), Citrus tatter leaf virus (CTLV), Citrus exocortis viroid (CEVd), and Candidatus Liberibacter asiaticus (Ca. L. asiaticus) associated with Huanglongbing (HLB) are important graft\|transmissible pathogens of citrus. A rapid multiplex PCR protocol with an internal control was developed for the detection of these citrus pathogens using a timesaving two\|temperature cycling for PCR amplification. The assay was applied for rapid detection of those pathogens in field\|grown trees. The results indicated that 89.3%, 10.7%, 17.9% and 28.6% of the 28 field\|grown citrus samples were infected with CTV, CTLV, CEVd and Ca. L. asiaticus, respectively, and approximately half of the samples were co\|infected with more than one pathogen. In addition, the absence of multiple pathogens in shoot\|tip grafted budlings was evaluated with the multiplex PCR assay.

In order to determine the dynamics of resistant populations of Plasmopara viticola to metalaxyl in the field, metalaxyl sensitivity of P. viticola collected from 11 vineyards of Hebei, Shanxi and Henan provinces was measured with the leaf disc floating method before and after application of fungicides. It was found that the resistance of P. viticola to metalaxyl was stably inherited. The dynamics of resistant populations in different areas varied with the control programs used. When fungicides with different mechanisms were applied to control P. viticol, we need to rationalize the work processes of pesticide application and constantly refine it according to the actual control effects in the field.

Strain 13657 was isolated from Buddhist pine (Podocarpus macrophyllus) imported from Japan in Ningbo port by traditional PDA method. PDA colonies consisted of flat mycelium with pale rose coloured aerial hyphae. Macroconidia with five to nine septa, 58.7 to 86.3 μm long, 5.3 to 8.8 μm wide were observed. Microconidia with zero to one septa, 2.8 to 9.3 μm long, 2.4 to 5.6 μm wide, oval to cylindrical, and produced on monophialides were also observed. Partial sequences of ITS region, 28S rRNA, EF and TUB genes were obtained by PCR amplification of genomic DNA of strain 13657 with universal primers. Analysis of these sequences with the related Albonectria rigidiuscula sequences deposited in the NCBI database showed they had 99% identity. The EF and TUB sequences of strain 13657 were clustered into A. rigidiuscula clade based on phylogenetic results. Pathogenicity tests showed that the isolate infected twigs of Buddhist pine and caused dry leaves and complete twig mortality. A. rigidiuscula were isolated repeatedly from the infected petioles leaves and twigs. Based on morphological characteristics, molecular and pathogenicity test results, the isolate 13657 was identified as A. rigidiuscula. To our knowledge, this is the first report of interception of A. rigidiuscula in China. This is also the first report of A. rigidiuscula being pathogenic on Buddhist pine in Japan and throughout the world.