A leaf spot disease was found on fresh corn WN2000 in Hebei province and seeding corn in Shandong province.The disease was characterized with round or oval grey-whitish and brown-margined spots on affected tissues, which were surrounded by yellow - brown halos. The spots on WN2000 were larger than those on normal corn, where were(3-10) mm×(3-5 )mm and (1-2) mm×(2-3) mm in size, respectively. This study proves the causal agent of this disease as Bipolaris sorokiniana by pathogen isolation, pathogenicity test, morphological observation, and rDNA-ITS and 3HNR gene sequence analyses. The field investigation showed that the disease incidence varied significantly. Guanyu6 was highly susceptible with disease incidence of 52%, wuyue88 showed strong resistances with disease incidence of 2%. Among the fresh maize, only cultivar WN 2000 was infected with disease incidence of 32%. This is the first report of B. sorokiniana causing leaf spot disease on maize with two sizes of lesions in the world.

Through the conventional tissue isolation approach from the infected tissues of grapevines with anthracnose symptoms, a total of 37 and 31 isolates from Nanning and Hechi, Guangxi was isolated and purified, respectively. Based on the morphology and rDNA ITS sequences analysis, 37 isolates from Nanning was the same strain as well as 31 isolates from Hechi, which were named NN and HC strains, respectively. Even though NN and HC strains both fit the description of Elsinoe ampelina, the two strains showed big differences on morphology and pathogenicity. NN colonies were red brown, suborbicular with smooth edges, white mycelium and transparent sticky droplets on the hummocky raised surface, regular folds on the surrounding edge. HC colonies were light orange, suborbicular with smooth edges, few white mycelium on the hummocky raised surface, but no droplets, irregular folds on the surrounding edge. By inoculation of young grapevine shoots, both strains could cause typical symptom of grape anthracnose disease, while the NN strain showed stronger pathogenicity than HC. PCR amplification using a pair of rDNA ITS primers ITS1F/ITS4 resulted in 1 124 bp and 818 bp fragments from NN and HC, respectively. The 1 124 bp fragment showed 92% coverage and 99% similarity to the sequence of Elsinoe ampelina (GenBank AY826763.1). The 818 bp fragment showed 75% coverage and 99% similarity to the sequence of Elsinoe ampelina (GenBank AY826762.1). Therefore, NN and HC are both pathogens causing grape anthracnose in Guangxi.

Forty-five leaf-samples of stone fruit crops collected in West Liaoning area were detected for Prunus necrotic ringspot virus (PNRSV) by RT-PCR, and nine out of the 45 were positive. Using total RNAs of the positive samples as templates, nearly full-length RNA genome segment 3 (RNA3) of PNRSV were cloned and sequenced. The size of the obtained RNA3 fragments ranged from 1 612 to 1 619 bp and shared 93.8%-100% sequence identity. Another 42 RNA3 sequences of PNRSV were retrieved from GenBank for phylogenetic analysis, and the sequences of cp gene, mp gene and RNA3 were selected, respectively, to construct phylogenic trees, and consistent results were obtained. Phylogenic analysis indicated that the 51 PNRSV isolates could be classified into four groups, including PV32, PV96, PE5 and a possible novel group, the nine isolates obtained in West Liaoning belong to group PV32 and PV96, respectively. Our study presented the genetic diversity of PNRSV in West Liaoning and the suitability of using RNA3 for PNRSV diversity analysis. Meanwhile, a novel PNRSV group was identified, which would be important for further understanding the genetic diversity of PNRSV.

The peach constriction canker, caused by Phomopsis amygdali is one of the most important diseases of peach trees. Because the disease transmission is mainly through infected seedlings and scions, strict quarantine measures are the key to prevent the pathogen into the disease-free area. The accurate, sensitive and fast detection method is an effective tool for quarantine and study on the occurrence of disease. In comparison of histone H3 gene of P. amygdali with that of the closely related species, histone H3 was a suitable target for molecular detection. Based on the histone H3 gene, we designed the specific primers and TaqMan probe, and the TaqMan Fluorescence Quantitative PCR Method (FQ-PCR) was established for the detection of P. amygdali. Results showed that the developed FQ-PCR was specific for P. amygdali detection; and this method was able to detect as little as 4×10¹ copies of recombinant plasmid DNA. Its sensitivity was 1 000 times as high as routine PCR. The detectable limits of P. amygdali were 2 conidia, which were 10 times higher than that of routine PCR; the whole detection process could be finished in one hour. Compared to culture and routine PCR, the positive detection rates of suspicious samples by FQ-PCR were the highest, at 86%. In conclusion, the TaqMan Fluorescence Quantitative PCR developed in this study was simple, fast, sensitive, and specific, and can be used to detect P. amygdali in high-speed from infected peach trees in the field.

Fusarium graminearum and F. verticillioides, the two main pathogens causing maize ear rot, were set as targets for the quick and quantitative analyses. In this study the duplex PCR was established using the specific primer pairs for F. graminearum and F. verticillioides. The reaction was optimized from the aspects of DNA concentration, primer ratio and annealing temperature. The results showed that the annealing temperature of 54℃ and 58℃ displayed efficient amplification, the optimum primer ratio of F. graminearum to F. verticillioides was 0.2 μmol·L^-1/0.4μmol·L^-1, and the sensitivity for the reaction reached as low as 6.25 ng·μL^-1. The duplex PCR developed in the study provides an accurate and sensitive approach to simultaneously detect F. graminearum and F. verticillioides.

Chalcone syhthase is a key enzyme in the process of plant disease resistance. Based on the Expressed Sequence Tag (EST) of chalcone synthase gene obtained from wheat, an approximately 2 543 bp sequence was isolated from the DNA of wheat by TAIL-PCR technique, named TaCHS. Sequence analysis showed that this DNA fragment had an open reading frame of 1 185 bp and a promoter region of 1 264 bp, and the DNA had two extons interrupted by a 94 bp intron. The sequence was over 88% homologous to the other reported CHS sequences from Gramineous plants. The plant CARE analysis showed that 5′-upstream sequence from ATG had the feature of promoter: TATA-box, CAAT-box, ABRE, MBS, light responsive element, and so on. The RT-PCR result showed that TaCHS was highly expressed at 4 d post-infection by Greumannomyces graminis var. tritici.

Rice ragged stunt virus (RRSV) is a member of the genus Oryzavirus, family Reoviridae. Nonstructural protein Pns7 encoded by RRSV induces the formation of filament structures protruding from the surface of insect cells. In this study, based on a rice protoplast culture system, the expression dynamics of Pns7 protein in rice protoplasts were studied. Pns7 gene was cloned into the Gateway prokaryotic expression vector, and large amount of protein was obtained after IPTG induction. Polyclonal antiserum against Pns7 protein was prepared by immunizing the rabbit. The specificity of the antiserum was confirmed by Western blot analysis. The titer of the antiserum was measured by indirect ELISA and found to be 1∶2 500. Real-time quantitative RT-PCR was used to analyze the amount of Pns7 RNA in rice protoplasts after virus infection. Results showed that, accumulation of Pns7 RNA arised at 8 h after virus inoculation, and reached a maximum at about 24 h. The level of Pns7 RNA declined to a platform stage after 32 h. Meanwhile, using the prepared antiserum as the probe, Western blot analysis indicated that Pns7 protein started to express at 16 h after the virus inoculation, and reached a maximum level at about 32 h. The expression of Pns7 protein began to decline after 60 h, but still held a high level.

Rice stripe disease, induced by Rice stripe virus (RSV), is a destructive disease in japonica rice-grown regions of China.RSV is mainly transmitted by small brown planthopper (Laodelphax striatellus,SBPH)in a circulative, persistent and propagative manner.The spread and replication of RSV in the insect body shortens the period of 5^th instar and the total nymph, resulting decreased number of adults that can finally finish eclosion. To investigate molecular basis for the in fluence of RSV on SBPH, the transcriptional level of some developmental genes in SBPH after acquiring RSV were analyzed by RT-qPCR. Initially, 10 development-related genes in SBPH were identified through blasting corresponding mRNA sequences of Drosophila melanogaster against a SBPH local database derived from transcriptome assembly. Next,the relatively transcriptional level of the 10 genes between the viruliferous and healthy SBPH were evaluated by the RT-qPCR. Results showed that the transcription of 8 genes showed significant difference for the case of RSV-infected SBPH (2 upregulated and 6 downregualted). The differentially of dynein heavy chain and sodium/potassium-transporting ATPase subunit alpha may associate with developmental defect of nervous system and abnormal behavior in SBPH. The decreased accumulation of paxillin may affect wing development,and five cytoskeleton proteins were also downregulated, which may interfere with the eclosion process. Differential transcription between the viruliferous and healthy SBPH presented in our study would aid understanding of the interaction between RSV and SBPH.

Rice sheath blight is the serious fungual diseases in rice. The traditional method to test the pathogenicity of Rhizoctonia solani based on the relative height of lesion on rice needs to inoculate rice at late tillering or heading stage which is time-consuming and difficult to control the conditions. In this study, compared with the traditional way, the evaluation of Rhizoctonia virulence and bioactivity of crude toxin was conducted on the way of detached rice leaf and rice leaf sheath. The results showed that, although all the three approaches of inoculation were significantly positive to evaluate the pathogenicity and crude toxin activity, the detached ways seem to be more accurate with the advantage of easy handling and condition controlling. Meanwhile, we noticed the rice sheath are more sensitivity to crude toxin of R. solani than rice leaf,which might be the variation of sensitivity of the different rice tissues to the toxin. Therefore, the detached rice leaf and leaf sheath methods may take place of the traditional way of height of lesion to determine virulence of R. solani. The combination of the three approaches can be used as a new method to determine toxin activity of R. solani. The lesions difference between rice leaf and leaf sheath suggested that the susceptibility of different rice tissues to the fungus is varied. In addition, the significant differences with the severity of disease index caused by crude toxin and isolate inoculation imply that the other virulence factors should be taken into concern besides the toxin. During the quantitative analyzing the interactions of Rhizoctonia solani AG-1 IA and host rice, the different rice tissues should be taken consideration together for more accurate exploring of the interaction mechanism.

PevD1, a fungal protein elicitor secreted from Verticillium dahliae can induce necrosis and systemic acquired resistances (SAR) in tobacco plants. In our previous study, we identified a PevD1 interaction protein Nbnrp1 in Nicotiana benthmiana. Here, we investigate the role of Nbnrp1 in PevD1-induced disease resistance. Nbnrp1 expresses in all tobacco tissues and is located around cytoplasm and nucleus in tobacco cells. Nbnrp1-silence plants displayed reduced necrosis and disease resistance,and transcription levels of defense-related gene PR1-a,PR1-b were also reduced compared to those of the wild type plants after PevD1 infiltration, suggesting that Nbnrp1 is involved in PevD1-induced defense response and disease resistance. The results provide a foundation for further understanding the mechanism of PevD1-induced resistance and Verticillium dahliae-tobacco interaction.

Our study analysis the diversity of endophytic bacteria in red globe grape leaves sampled from four different regions (i.e. Beijing, Chengdu, Hefei and Wuhan), three different growth stages (i.e. flowering, maturing and senescence), by traditional plating isolation and denaturing gradient gel electrophoresis (DGGE) method. Results show that the population of endophytic bacteria in samples from different regions ranged from 8.0×10³ -2.8×10⁴ CFU·g^-1, between which the highest was from Wuhan. Population of endophytic bacteria in samples from different growth stages were between 0.8×10³ -1.5×10⁴ CFU·g^-1, and the largest was in mature period. All the strains isolated from leaves collected from different regions and different growth period were identified as Bacillus, Pantoea, Curtobacterium, Pseudomonas and Staphylococcus, based on 16S rRNA gene sequence aligment. Bacillus sp. was the dominant genus. DGGE profiles showed that samples from the same area or the same growth stage were of highly similarities, and easy to gather as a class. The species diversity was mainly expressed in samples from Chengdu and samples from mature stage. Uncultured bacteria and unrecognized marine planktonic bacteria were abounded in grape leaves as a result of cloning and sequencing for the characteristic bands.

In 2015, symptoms of leaf spot were observed on water spinach (Ipomoea aquatica) in fields in Jianshui County,Hani-Yi autonomous prefecture of Honghe, Yunnan Province, China. The causal pathogen was identified as Myrothecium roridum based on the morphological characteristics, pathogenicity test, and molecular identication. Host range of this pathogen was evaluated, and its pathogenicity to 10 plants was significantly different with higher virulence to eggplant and water spinach. To our knowledge, this is the first report of M. roridum causing leaf spot on I. aquatica in China.

To detect whether pathogenic group of Puccinia striiformis f. sp. tritici (Pst) are virulent to Guinong 22 in Shaanxi, ninety four of single uredium-derived Pst isolates collected randomly from wheat fields in Chencang district, Baoji in western Central Shaanxi, were established and identified pathogenically based on Chinese differentials consisting of 19 wheat cultivars. The results showed that 23 out of them belongs to pathogenic group virulent to Guinong 22 (G22), accounting for an occurrence frequency of 24.47%. Eight of the 23 isolates were divided into four G22 pathotypes, including G22-40, G22-60, G22-68, and G22-72 with an occurrence frequency of 1.06 %, 1.06%, 5.33%, and 1.06%, respectively, and 15 of the rest G22 isolates were unknown races (pathotypes) and were categorized into 14 pathotypes. The pathogenic groups virulent to Suwon 11 (Su11) and Hybrid 46 (Hy46) had a respective occurrence frequency of 41.48% and 22.34%. Races (pathotypes) of CYR33, CYR32, and G22-68 had high occurrence frequency of 17.02%, 6.38%, and 5.33%, respectively. This study reported the dynamics of G22 pathogenic group of Pst in western Central Shaanxi. The G22 pathogenic group was highly virulent to resistance genes Yr24 (=Yr26) and Yr10, and had a huge potential threat to wheat production. Therefore, strengthening surveillance and forecast of G22 pathogenic group is necessary and will provide a basis for controlling wheat stripe rust and for breeding wheat resistant to the disease in Shaanxi.

Yunnan Province, the Southwest border of China, is the key initial inoculum source region for the spread and epidemic outbreaks of wheat stem rust. Revealing genes conferring stem rust resistance in wheat cultivars is an important theoretical base for the prevention and control of the disease. Six races of Puccinia graminis f. sp. tritici and three molecular markers, specific for detection of stem rust resistance genes Sr31, Sr32 or Sr38, respectively were used for analyzing the resistance at wheat seedling stage and for detecting resistance genes in 116 wheat cultivars (lines) from Yunnan Province. The result indicated that eighty cultivars showed different resistance to all the tested races, accounting for 69.0% of the tested cultivars (lines). Molecular detection result showed 43 cultivars carrying Sr31, 10 cultivars carrying Sr38, accounted for 37.1% and 8.6% of the tested cultivars or lines, respectively. Only wheat cultivar 91E001 was detected to contain Sr32 that is resistant to Ug99. These results indicate that the resistance of cultivars in Yunnan to wheat stem rust is in a moderate level. Sr31 is present in relatively more cultivars, while Sr38 with excellent resistance to Chinese Pgt gene is relatively fewer. Sr32 conferring resistance to Ug99 is rarely present in Yunnan cultivars.

To investigate the distribution and population of Plasmodiophora brassicae and resistance levels of cruciferous vegetables to the pathogen in Jiangxi province, the physiological races of P. brassicae collected from counties of Jiangxi province were identified by ‘Williams Identifials’ in this paper. A total of 39 samples were collected and tested. Three races including race No. 8, 9 and 16 were identified, among them race No.16 was reported for the first time in China, and the occurrence frequency of race No. 9 was as high as 94.87%. Based on the field investigation and glasshouse inoculation, race No. 9 was non-pathogenic on head cabbage and cauliflower, but it could infect Chinese cabbage, bok-choy and purple pakchoi with disease incidence ranging from 49.06% to 91.49% for different cultivars.

In order to detect the sensitivity of Rhizoctonia cerealis in Henan Province to triadimefon, two-hundred R. cerealis isolates were collected from 32 counties in 16 cities in 2013 and 2015, and their sensitivity to triadimefon was determined in vitro by mycelial growth rate method. The isolates collected from different regions showed different sensitivities. However, most isolates collected from different years in the same region had the same sensitivity. Though there had appeared the isolates which showed decreased sensitivity to triadimefon in the field, the frequency was very low, which meant that triadimefon could still be used to control wheat sharp eyespot and the sensitivity to triadimefon should be monitored continuiously.

Brown rot is a devastating fungal disease on stone and pome fruits worldwide. Remarkable progresses have been made to understand this disease and its causal agents based on researches for more than one hundred years. In this article, it has been reviewed on various aspects including pathogen populations, life cycle and symptoms, distributions and host ranges, biological characteristics (colony morphology, conidial size and shape, conidial germination profile and virulence), molecular detection methods, fungicide resistance and mana-gement strategies. Prospects were also provided for further researches on brown rot of fruits. Such information are crucial for better understanding of both brown rot disease and its causal agents and improving the outcomes of brown rot management, thus to keep safe production of stone and pome fruits.

Bipolaris zeicola is the pathogen of northern corn leaf spot. The species-specific PCR primers Y-EF-F and Y-EF-R for B.zeicola were designed after multiple sequences alignment of elongation factor 1α of B. zeicola with the other allied species of Bipolaris. A 137 bp single DNA fragment from the isolates of B. zeicola was amplified by PCR approach and no product was amplified from other fungi. The detection sensitivity of the pathogen by the species-specific primers is 1 pg·μL^-1. The identical 137 bp bands were amplified using total DNA extracted from the diseased tissue including maize leaves, maize bracts and maize kernels inoculated with B. zeicola. The maize leaves were inoculated in the field with spore suspension, and the obvious disease symptoms could be seen in the fifth days post inoculation and the pathogen could be detected from the symptom-free leaves inoculated in the third days post inoculation. The PCR protocol provides a rapid and reliable tool for routine detection and identification of B. zeicola. In addition, it also provides an early detection approach for the latent pathogen in symptom-free maize, which is conductive for the disease control timely.

Lotus (Nelumbo nucifera) was the unique aquatic one ranked in the top ten famous flowers and plants in China, and was cultivated extensively in the pearl river delta wetlands. In recent years, rhizome rot of lotus took place in large area, affecting the production and tourism industry of lotus. In order to effectively control this disease, it is necessary to clarify its causal agent. In this study, the pathogen causing rhizome rot of lotus was identified through pathogenicity tests, morphological studies, specific PCR diagnosis, and phylogene-tic analysis based on combined sequences of translation elongation factor 1-α (EF-1α), the mitochondrial small subunit (mtSSU) ribosomal DNA, and the nuclear ribosomal intergenic spacer region (IGS). The results show that: (i) both the morphological and molecular identification confirmed that the pathogen was Fusarium commune, which is a sister taxon of F. oxysporum and was firstly reported on lotus. (ii) F. commune was highly similar to F. oxysporum on morphology, whereas production of longer monophialides (>25 μm) and polyphialides by F. commune can distinguish it from F. oxysporum. Moreover, DNA sequences of the mtSSU、EF-1α and IGS regions were also useful to distinguish F. commune from F. oxysporum. (iii) Genetic differentiation existed among the pathogens of lotus rot from different areas, and the pathogens of both lotus rhizome rot and Chinese water chestnut (Eleocharis dulcis) wilt belong to the same species.

Seleng wormwood (Artemisia selengensis Turcz. ex Bess.) is a fragrant edible plant that is widely cultivated in China, especially in Nanjing. In the Baguazhou area of Nanjing, the seleng wormwood industry is estimated to be worth hundreds of millions RMB annually. Since 2012, a bacterial soft rot has been observed annually on seleng wormwood (cv. Dayebai) during the months of August and September. Black, necrotic areas with a foul odor appeared on the stems in soil and resulted in wilt and death of the plants. Disease incidences ranged from 10% to 20% in the affected fields and some seriously damaged fields could be no yield. From 2012, more than 30 bacterial strains with consistent appearance were isolated from symptomatic seleng wormwood samples and 12 of them from different fields were selected for characterization. Based on morphological observation,physiological,biochemical and Biolog test, as well as multilocus sequence analysis, the causal pathogen was identified as Pectobacterium carotovorum subsp. carotovorum.

In 2015, tomato pith necrosis disease was observed in tomato-growing area in Guangzhou of Guangdong province. Bacteria were consistently isolated from the diseased tomato samples. Pathogenicity test of 5 randomly selected bacterial isolates showed that the inoculated tomato plants exhibited the same symptoms as those observed in the field. 16S rDNA sequencing results indicated that the 5 isolates shared 99.9%～100% sequence identities with each other, and had a 99% sequence match to Pseudomonas cichorii strains MAFF 211996, TIs01 and IP1-05. Using the species-specific primer pair Hrp1a/Hrp2a of P. cichorii, an 897-bp hrcR gene-specific fragment could be amplified from the 5 isolates. Sequencing analysis suggested the amplified hrcR genes shared 99%～99.9% identities with each other, and had a 99.9% sequence identity with the hrcR of P. cichori 83-1. The physiological and biochemical tests further indicated these bacterial isolates were P. cichorii. All above results further support that tomato pith necrosis occured in Guangdong is caused by P. cichorii.

The disease caused by Banana streak virus (BSV) led to great severe loss in banana production and affected the exchange of banana germplasm resources. A TaqMan real-time PCR assay was developed using the primer pair and probe based on the conserved ORF III sequence of BSV. The assay was reproducible and could specifically detect BSV without cross reaction with Cucumber mosaic virus (CMV) and Banana bunchy top virus (BBTV),and showed higher sensitivity than that of Endpoint PCR in detecting either DNA standard plasmid samples or field samples, which had 100 times higher than that by Endpoint PCR when detecting DNA standard plasmid samples, and 10 times higher than that by Endpoint PCR when detecting field samples. The transmission feature of BSV in the micropropagated banana samples was different when they were quantitatively detected by TaqMan real-time PCR. When comparing the differentiated banana shoots of the suckers, the concentrations of BSV in those of ‘Dajiao’ of the 3^th and 9^th generations significantly reduced; while the concentrations of BSV in those of ‘Fenjiao’ firstly increased and then decreased in subsequent generations. However, the concentrations of BSV in those of ‘Yueyoukang 1’ increased gradually in the subsequent generation. Moreover, the concentrations of BSV in ‘Dajiao’,‘Fenjiao’ and ‘Yueyoukang 1’ of the 10^th, 11^th and 12^th generations were higher in the first or (and) second upper leaves than those in the other leaves. Direct binding PCR analysis preliminarily indicated that there is no integration of BSV into the banana genomes in this study. This research elucidated the mechanisms of transmission and distribution of BSV in micropropagated banana by detecting the BSV concentrations using TaqMan real-time PCR, which would be very useful for studying the interaction between BSV and banana and detecting the BSV in the micropropagated banana.

The objectives of this study are to investigate a sample of Netherlands lily showing disease symptoms like that of virus infection. This sample was grown in the quarantined glasshouse. Partial genomic fragments was amplified by RT-PCR using two pairs of degenerate primers (Carla-pCar1I / M4T and Potex 5/Potex 1RC) designed for detection of the genera Calavirus and Potexvirus, respectively. Sequence analysis was performed and phylogenetic tree was reconstructed. RT-PCR amplifications of the positive sample both generated expected fragments (1 257 and 737 nts). Sequence analysis showed that the two fragments shared 79.9%-99.5% and 80.5%-99.3% nucleotide sequence identities with that of the Plantago asiatica mosaic virus (PlAMV) 3′terminal (including TGBp2, TGBp1, CP, 3’-NTS genes) and RNA polymerase gene sequences, respectively. The existence of PlAMV in the Netherlands lily was further confirmed by RT-PCR using the coat protein-specific primers (PlAMV-cpup/PlAMV-cpdw) of this virus. Phylogenetic analysis indicated that this isolate was clustered together with viral isolates from Netherlands lily, suggesting a high homology to the Netherlands lily viral isolates. To our knowledge, this is the first report of capturing PIAMV at the port of entry in China.

Wheat crown rot disease caused by Fusarium spp. was one of worldwide-spread and devastating disease for wheat production. F. asiaticum was one of the dominant pathogen of wheat crown rot disease in major winter wheat production area in China. It’s beneficial to the pathogenesis analysis and resistance breeding through histological observation of infection process. F. asiaticum labeled by green fluorescent protein (GFP) for studying infection process in wheat was a direct method for in vivo observation of interaction between pathogen and host. Gene gfp was transferred into F. asiaticum strain CF0915 via PEG-CaCl₂ mediated transformation method. Based on analysis of fluorescence expression, PCR verification, genetic stability, growth parameters and pathogenicity analysis, transformants CF0915-GFP resembling the wild-type strain was chosen to investigate infection process on wheat cultivars of different resistance. The results showed that transformants CF0915-GFP showed bright green fluorescence, and gfp gene was integrated into the genome of F. asiaticum by PCR amplification verification and inherited normally and stably, the growth characters and pathogenicity were comparable to wild strain. Numerous conidiospores attached root hairs and root epidermis of wheat and germinated at 1 day after inoculation in susceptible cultivar ‘Yangmai 158’, until 2 dpi germination of conidiospores was observed in moderately-resistant cultivars ‘CI12633’ and ‘Sumai 3’. The hyphae infected root hairs and grooves along junctions of adjacent epidermal cells and then spread into cortex of roots at 3 dpi. The hyphae spread fast onto leaf shealth from roots at 8 dpi, while at 10 dpi massive hyphae invaded into root cortex cells and epidermal cells of leaf shealth and meanwhile abundant macroconidiaspores were observed, brown disease spots appeared on basal stems of susceptible cultivar. Massive hyphae were filled in xylems vessels of roots and leaf shealths at 14 dpi, subsequently numerous chlamydospores were observed in the epidermal cells of leaf shealth. Until 25 dpi most seedlings appeared wilt and dead. Compared to susceptible cultivar, infection process was retarded later in moderately-resistant cultivars. In this study the histopathological observation of infection process of F. asiaticum in wheat provided an important theoretical basis for disease control and resistance resource utilization.

Alternaria leaf spot caused by Alternaria alternata is one of the key causes of cotton premature senescence and often leads to severe loss in cotton production. Scopoletin biosynthesis pathway, a branch of phytoalexin metabolic pathway for synthesis of the scopoletin, acts the roles of growth suppression of phytopathogenic fungi and enhancement of plant disease resistance. In order to clarify the resistant roles of cotton scopoletin biosynthesis against A. alternata, cotton leaf crumple virus-induced gene silencing (VIGS) techniques was performed to silence three key enzymes encoding genes of the scopoletin biosynthesis pathway, Ghpal for phenylalanine ammonia lyase, Ghc4h for cinnamate 4-hydroxylase, and Gh4cl for 4-coumarate-CoA ligase. The results showed that the expression levels of Ghpal, Ghc4h, and Gh4cl in cotton leaves were induced by A. alternata, suggesting these genes play important roles in the early defense response against A. alternata in cotton. The results of VIGS experiments showed that disease index of the silenced Ghpal, Ghc4h, or Gh4cl plants were 43.36, 38.27, 44.78, respectively, which were significantly higher than the mock one of the wild type and the empty vector transgenic plants. These results showed that Ghpal, Ghc4h, Gh4cl in the scopoletin biosynthesis played an important role in resistance to A. alternata. Moreover, exogenous feeding of scopoletin can recover the resistance of VIGS cotton plants to A. alternata. Taken together, our study revealed that the cotton scopoletin biosynthesis played an important role in the resistance to A. alternata.

Using the Illumina HiSeq^TM2000 platform, the transcriptomes of healthy and infected mango buds were sequenced. Through Blastx comparison to Nr、Swiss-Prot、KEGG and COG databases, functional annotations of the differentially expressed genes (DEGs) were obtained. The DEGs were then carried out GO and KEGG pathway analysis. The results showed that: a total of 119 815 unigenes was obtained from the two samples, with 880 bp in the average length and 1 546 bp in N50 value. Through the transcriptomes analysis with the RPKM (reads per kb per million reads method), 29 878 DEGs were acquired. Additionally, 11 DEGs were chosen for qRT-PCR verification and the results were consistent to the RPKM analysis. Taking corrected P-value ≤ 0.05 as a threshold, DEGs were enriched in 22 pathways, most of which were closely related to stress responses. A total of 153 DEGs were involved in metabolism of starch and sucrose, most of which encode racemase and epimerase, hydrolase and intramolecular transferase and take part in the glucose catabolic process, carbohydrate metabolic process, pyruvate metabolic process, nucleotide metabolic process, and so on. In the antioxidant activity process, twenty DEGs that encoded enzymes involved in reactive oxygen metabolism were acquired with ratio larger than ten and 14 DEGs were up-regulated. It demonstrated that reactive oxygen metabolism played an important role in the mango-Fusarium mangiferae interaction. With ratio larger than ten, fifty-three DEGs related to phenolic metabolism were acquired, forty of which were up-regulated. It indicated that mango may protect itself from the F. mangiferae infection by accelerating the synthesis of secondary substances, such as lignin and phenolic compounds. Calcium signal transduction pathways, salicylic acid (SA) pathway and mitogen-activated protein kinase (MAPK) signal pathways were down-regulated in the mango-F. mangiferae interaction. RPM1, an important gene related to plant disease resistance, was also down-regulated, which was probably the main reason to the susceptibility of mango to F. mangiferae.

The full-length nucleotide sequence and genome organization of a Melon necrotic spot virus isolated from Shandong, China (MNSV-Shandong), were described. The MNSV-Shandong isolate consists of a positive sense single-stranded RNA with 4 267 nt in length. MNSV contains at least five open reading frames (ORFs) encoding p29, p89, p42 and two small p7A and p7B proteins, respectively. The p89 gene shares a common start codon with that of p29 and was generated through read-through of the amber stop codon of p29. Phylogenetic analyses based on complete genomic sequence assigned MNSV-Shandong and MNSV-HM China isolates into a major cluster, MNSV-Shandong isolate and Europe, parts of Asia and North America isolates were in the same branch, but was far from the Japanese isolate. To the best of our knowledge, this is the first report of the genome of a MNSV Shandong isolate in China.

In order to prepare the monoclonal antibodies (MAbs) against Sweet potato latent virus (SPLV) coat protein (CP), spleen cells were isolated from BALB/c mouse immunized by the SPLV CP followed by fusing with mouse myeloma cells (SP2/0) and subcloning. Two hybridoma cell lines (5B₁₁-2 and 5G₈-2) stably secreting MAbs against SPLV CP were obtained and the titres of both MAbs were 1512 000 and 16 400 when using the SPLV CP and diseased leaves, respectively, as coating antigen for the indirect ELISA assay. Isotypes and subclasses analysis of 5B₁₁-2 and 5G₈-2 indicated that both of them belong to IgG1, κ light chain. Western blot analysis further confirmed that both MAbs could specifically react with SPLV CP and sweet potato leaf extracts containing SPLV. ACP-ELISA could successfully detect virus in plant sap diluted by 3 840 fold. The consistency between serological and RT-PCR results showed that the MAbs produced in our study could be used for detecting SPLV in the field samples of sweet potato.

Stripe rust is one of the important epidemical diseases in the wheat-grown areas in China. The change of dominant races in the pathogen population usually results in that resistance varieties ‘loss’ their resistance capability. Developing a set of near isogenic lines (NILs) with a single wheat stripe rust resistance gene is essential for characterization of stripe rust resistance genes in wheat cultivars and monitoring the change of virulence in the pathogen population. In this study, highly susceptible winter variety Mingxian 169 was used as recurrent parent and Jubilejina 2 as the resistance gene donor for developing a set of NILs. Through resistance gene postulation combined with genetic analysis, the resistance gene(s) contained in near isogenic lines N27 and N36 was deduced, and confirmed by PCR with two primers, SSR markers closely linked with YrJu4. The genetic analysis revealed that lines N27 and N36 may contain YrJu3 or YrJu4,or YrJu3 plus YrJu4, and the stripe rust resistance of both lines N27 and N36 to CY17 and CY26, respectively, is controlled by one dominant gene. The PCR results confirmed that the amplicon from Jubilejina 2 with resistance gene YrJu4 was in the same size of those from lines N27 and N36. Therefore, the stripe rust resistance gene YrJu4 from differential variety Jubilejina 2 was successfully transferred into the winter-type recurrent parent, Mingxian 169, and both lines N27 and N36 developed are NILs of Mingxian 169*6/YrJu4, with a single resistance gene YrJu4.

Daily sporec on centration of Blumeria graminis f. sp. tritici were monitored using Burkard volumetric spore samplers in the air of moderately susceptible variety Zhongmai2 and highly susceptible variety Jingshuang16 wheat fields in 2014 and 2015.Daily meteorological factors were recorded by meteorological station in the fields. The correlations were analyzed between spore concentration and meteorological factors, such as air temperature, humidity, rainfall, wind speed and solar radiation. The results showed that spore concentration was mainly positively and significantly correlated to air temperature(r>0.348 3,P<0.05). Then the correlations were calculated between disease index (DI)and four kind of accumulated spore concentrations, which were the accumulated spore concentration before the day of disease measuring, and the accumulated spore concentration before the current week of disease measuring, and the accumulated spore concentration in the previous week of disease measuring, and the accumulated spore concentration in the current week of disease measuring respectively. The data showed that there were exponential relationships between DI and the accumulated spore concentration in mo-derately susceptible variety Zhongmai2 wheat fields in 2014 and 2015, and the best DI estimating model was based on the accumulated spore concentration before the day of disease measuring or on the accumulated spore concentration before the current week of disease measuring; there were mainly logarithmic relationships between DI and the accumulated spore concentration in highly susceptible variety Jingshuang16 wheat fields in 2014 and 2015, and the best DI estimating model was based on the accumulated spore concentration before the current week of disease measuring.

To investigate the effect of acetic acid on the growth development and nematocidal activity of Purpureocillium lilacinum,the growth rate, spore germination and production, and colony morphology of P. lilacinum were assayed in the presence of acetic acid with different concentrations. The growth development of P. lilacinum was not impaired in the culture medium containing low-dosage acetic acid (200 μg·mL^-1). P. lilac-inum could produce acetic acid with the concentration up to 146 μg·mL^-1 in PDB medium at 3 days post-inoculation, whereas the concentration of it was decreased when the carbon sources were gradually consumed in PDB medium. It was observed that limited carbon sources of culture medium resulted in none of acetic acid produced by P. lilcacinum. In addition, P. lilacinum exhibited good growing status in the medium containing acetate with concentrations ranging from 3 mg·mL^-1 to 10 mg·mL^-1, which suggested that P. lilacinum could make use of acetic acid radical as its carbon source. Furthermore, acetic acid could enhance P. lilacinum nematocidal activity, and the concentration of acetic acid was positive correlation to the inhibition of egg hatch and lethal effect on second-stage juvenile (J2) of root-knot nematode caused by P. lilacinum.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases on wheat in China. Zhong 4, a partial amphiploid (2n=56) carrying resistance from Thinopyrum intermedium and resistant to most of Pst pathotypes identified so far. T4 is one of the very rare Pst pathotypes which are virulent to Zhong 4. With the aim of developing race-specific molecular markers for detection of Pst pathotype T4, 214 random amplified polymorphic DNA (RAPD) primers were used to test polymorphism among pathotype T4 and six predominant pathotypes CYR29, CYR31, CYR32, CYR33, Su11-4 and V26. An unique 821 bp band was identified in T4 with primer S1363. The band was cloned and sequenced. Based on the sequences, sequence characterized amplified region(SCAR) marker T4sp1/sp2 was developed to differentiate T4. The SCAR marker was then validated with DNA samples prepared from leaves of wheat cultivar Mingxian 169 after inoculated with T4, CYR29, CYR31, CYR32, CYR33, Su11-4, V26 and water (mock-inoculated). The specific band was produced only in samples prepared from plants after inoculated with T4 six days. Therefore, the SCAR marker T4sp1/sp2 can specifically detect T4.

Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust, has been known as a heteroecious, macrocyclic rust fungus that requires two unrelated plant species to complete its life cycle since barberry (Berberis spp.) and Mahonia spp. were successively identified as the alternate hosts for the pathogen. There are a great number of species in genera Berberis and Mahonia, however, only 29 species in the genus Berberis and 1 species in Mahonia, M. aquifolium, have been reported to serve as alternate hosts for P. striiformis f. sp. tritici. Most species in these two genera have not been known whether to serve as aecial host for this rust pathogen. In this study, we collected one species, B. germanensis, native to Stuggart, Germany and morphologically differentiated from the known Berberis species that have been confirmed as the alternate hosts of P. striiformis f. sp. tritici. Pycnia and aecia were obtained from infected seedlings of B. germanensis after inoculation with basidiospores of race CYR32 of P. striiformis f. sp. tritici. The resultant aeciospores from the barberry infected susceptible wheat cv. Mingxian 169 to produce sori typical uredia of P. striiformis f. sp. tritici. Therefore, B. germanensis is a new species of alternate host for P. striiformis f. sp. tritici. The result provides useful information for insight into managing wheat stripe rust.

The sensitivity of Rhizoctonia cerealis and Gaeumannomyces graminsis to propiconazol was determined by measuring the mycelial growth on the fungicide-amended media using 98 isolates of R. cerealis and 88 isolates of G. graminsis from Henan Province. The results indicated that 50% effective concentration (EC₅₀) values of R. cerealis and G. graminsis to propiconazol ranged from 0.119 3 to 2.581 3, 0.001 5 to 0.172 4 μg·mL^-1, respectively. The results of the frequency analysis revealed that low sensitivity subcolony to propiconazol had been discovered in R. cerealis and G. graminsis. The mean EC₅₀ values of (0.484 8±0.200 9) and (0.049 3±0.029 0) μg·mL^-1 for most isolates showed a unimodal curve distribution, which were treated as the sensitivity baseline of R. cerealis and G. graminsis to propiconazol, respectively. The isolates collected from different regions showed different sensitivities. The results provided a theoretical basis for the efficient application of propiconazol in the control of wheat sharp eyespot and take-all in the field of Henan Province.

In August 2014, a disease that caused severe losses on sorghum plants in farmland of Inner-Mongolia University for the Nationalities at Tongliao, Inner-Mongolia, China was found. Different pathogenic strains were isolated from symptomatic sorghum leaf tissues. Morphology, biological characteristics, pathogenicity and ITS and EF-1α sequences of these pathogenic strains were analyzed. Based on the morphological features, pathogenicity, sequence similarity of ITS and EF-1α, these pathogenic strains were identified as Alternaria alternata.

Root-knot nematodes (Meloidogyne spp.) are one of the most damaging genera of plant-parasitic nematodes on vegetable crops. In recent years, root-knot nematodes have caused continuous cropping obstacles with the rapid development of protected cultivation in Chifeng city of the Inner Mongolia. In this study, to investigate the species of vegetable root-knot nematodes in Chifeng city, twenty samples of root-knot nematodes collected from the greenhouse in four counties of Chifeng city were identified based on the perineal pattern of mature females and by SCAR-PCR approach. The results showed that the vegetable root-knot nematodes in Chifeng city belong to Meloidogyne incognita.

Site-directed mutagenesis has become an indispensable technique for deciphering protein structure and function. To serve new development for protein structural biology, simultaneous construction for batch of recombinant expression vector with multiple sites mutations is required. Here, we describe a procedure for the unidirectional insertion of site-directed mutagenized DNA into an expression vector via overlap extension and sticky-end PCR. This method takes advantage of two classical PCR-based techniques and has improved the efficiency of the construction of recombinant plasmid with multiple points mutation.