Pathogenicity and repulsion for toxin‐producing bacteria of dominant bacteria on the surface of American pine wood nematodes

Bacteria were isolated from the surface of two samples of American pine wood nematodes to identify methods of controlling pine wilt disease. The dominant bacterial strains were identified, and their toxicity and pathogenicity, in addition to their competitiveness with other pathogenic bacteria, were measured to ascertain how bacteria on the surface of American pine wood nematodes might be used to prevent and control pine wilt disease. The bacterial isolates show that the dominant bacteria carried by the two samples of pine wood nematodes are US4, US5, Smal‐007 and Rrad‐006. Based on routine staining, morphological observation and 16S rDNA sequence analysis, the four strains were identified as Delftia lacustris, Pseudomonas putida, Stenotrophomonas maltophilia and Rhizobium nepotum. The incubation of four dominant bacterial strains and Chinese dominant bacterial strains on the surface of aseptic nematodes and in nutrient broth showed that Smal‐007 and Rrad‐006 have strong competitiveness on the surface of pine wood nematodes. Using a bacterial culture medium to measure the propensity of pine seedlings to wilt, all the American dominant bacterial strains were shown to be less toxic than the Chinese dominant strains. If pine seedlings are inoculated with both bacterial and aseptic pine wood nematodes, American dominant bacterial strains present less pathogenicity than the Chinese dominant bacterial strains. In particular, Smal‐007 and Rrad‐006 show the lowest pathogenicity. If pine seedlings are inoculated with both bacterial and wild pine wood nematodes, American dominant bacterial strains significantly reduce the pathogenicity of wild pine wood nematodes isolated from Zhejiang Province, China. The effects of Smal‐007 and Rrad‐006 are confirmed as the most prominent. The American dominant strains Smal‐007 and Rrad‐006 satisfy two main requirements: excellent repulsion performance and low pathogenicity. Therefore, they can be used as candidate strains for biocontrol bacteria.     

Molecular characterization of Alternaria alternata population isolated from Upper Egyptian tomato fruits

Alternaria alternata is the most common fungal pathogen of tomatoes in Upper Egypt. Morphological identification of this fungus is challenging; therefore, this study searched for new classification tools based on molecular techniques. Using a dilution plating method, 67 strains of A. alternata were isolated from 34 samples of rotten tomato fruits representing the Giza 80 and Edkawy cultivars. The collected strains were identified using the amplification products of the internal transcribed spacer (ITS) region, glyceraldehyde 3‐phosphate dehydrogenase (Gpd) and Alt a1, which is a gene involved in the production of most of the allergens produced by A. alternata. The screening revealed that A. alternata constituted more than half of the total fungi recovered from rotten tomatoes in this study. According to the phylogenetic analysis using these three loci, the collected strains clustered in accordance with the host cultivar type from which they had been isolated. Specific gene random primer polymerase chain reaction (SGRP‐PCR) techniques indicated that the A. alternata population in the tested region has a high genetic diversity. The pathogenicity test showed that most of the A. alternata isolates (67.2%) were highly pathogenic, and no correlation was found between the phylogenetic analysis and pathogenicity. In addition, the influence of the fungicide Disan 80% on the collected strains showed significant differences that were attributed to the source of isolation.     

Susceptibility of Dendroctonus simplex to Hypocreales fungi: towards the development of a biological control strategy

The use of Hypocreales fungi against bark beetles is an attractive alternative strategy for biological control in forestry. However, fungal inoculation methods remain a major challenge due to the cryptic behaviour of these beetles. Experiments were conducted to evaluate the susceptibility of adult eastern larch beetles, Dendroctonus simplex, to different isolates of Hypocreales fungi and to assess the efficiency of direct inoculation of fungal conidia using an assisted autodissemination device. Among the nine fungal isolates tested, Beauveria bassiana INRS‐242 was selected as the utmost isolate causing a mortality rate of 64.4% of D. simplex adults within 10 days and having a high conidial viability of 81.7%. Using an assisted autodissemination device, a fungal‐coated pouch was a promising contamination chamber. Once it had fallen onto the fungal‐coated pouch, each D. simplex adult loaded an average lethal dose of 2.1 × 10⁹ conidia, which induced a mortality rate of 98.0%. Our results demonstrated the fungal pathogenicity of B. bassiana INRS‐242 against the eastern larch beetles and pointed out the potential of the assisted autodissemination device as an effective method of conidia inoculation. These findings represent the first step to develop a biological control strategy against D. simplex populations.     

Identification of Fungal Species Associated with Cladode Spot of Prickly Pear and Their Sensitivity to Chitosan

México is the most important producer of prickly pear (Opuntia ficus‐indica) in the world. There are several fungal diseases that can have a negative impact on their yields. In this study, there was a widespread fungal richness on cladodes spot of prickly pears from México. A total of 41 fungi isolates were obtained from cladodes spot; 11 of them were morphologically different. According to the pathogenicity test, seven isolates caused lesions on cladodes. The morphological and molecular identification evidenced the isolation of Colletotrichum gloeosporioides, Alternaria alternata, Fusarium lunatum, Curvularia lunata. All these species caused similar symptoms of circular cladodes spot. However, it is noticeable that some lesions showed perforation and detachment of affected tissues by Fusarium lunatum. To our knowledge, this is the first report of the Fusarium lunatum as phytopathogenic fungus of cladodes of prickly pear. The chitosan inhibited the mycelium growth in the seven isolates of phytopathogenic fungi. Chitosan applications diminished the disease incidence caused by C. gloeosporioies and F. lunatum in 40 and 100%, respectively. Likewise, the lesion severity index in cladodes decreased. There are no previous reports about the application of chitosan on cladodes of prickly pears for the control of phytopathogenic fungi. Therefore, this research could contribute to improve the strategies for the management of diseases in prickly pear.     

Plant host and soil origin influence fungal and bacterial assemblages in the roots of woody plants

Microbial communities in plant roots provide critical links between above‐ and belowground processes in terrestrial ecosystems. Variation in root communities has been attributed to plant host effects and microbial host preferences, as well as to factors pertaining to soil conditions, microbial biogeography and the presence of viable microbial propagules. To address hypotheses regarding the influence of plant host and soil biogeography on root fungal and bacterial communities, we designed a trap‐plant bioassay experiment. Replicate Populus, Quercus and Pinus plants were grown in three soils originating from alternate field sites. Fungal and bacterial community profiles in the root of each replicate were assessed through multiplex 454 amplicon sequencing of four loci (i.e., 16S, SSU, ITS, LSU rDNA). Soil origin had a larger effect on fungal community composition than did host species, but the opposite was true for bacterial communities. Populus hosted the highest diversity of rhizospheric fungi and bacteria. Root communities on Quercus and Pinus were more similar to each other than to Populus. Overall, fungal root symbionts appear to be more constrained by dispersal and biogeography than by host availability.     

Introduction of Beauveria bassiana as a biological control agent for Tribolium castaneum in Kerman province

Barley, Hordeum vulgare, one of the important crops in the word, is used in malting, feed and food industries. The red flour beetle, Tribolium castaneum, was found wherever grains or other dried foods are stored. Disinfestations of barley using chemical methods to kill insects, in this research, for the first time we isolated the pathogenic KB512 of entomopathogenic fungi Beauveria bassiana from soil and insects, which produced aerial and submerged conidia and blastospores in laboratory conditions. We investigated the best conditions for the production and utilisation of spore suspension to spray the larvae of T. castaneum, which is one of the important pests in Kerman province (Iran). One hundred and eighty isolates that naturally infected by T. castaneum were reared during spring and summer seasons 2010–2011. The pathogenicity test was carried out with direct spray. To bioassay the isolates, three concentrations of the spore suspension were prepared as follows: 1?×?10⁶ and 1?×?10⁸ conidia/ml. The pests were sprayed by aerial conidial suspension, which was prepared by 0.01% Tween 80 in distilled water, and the controls were sprayed by 0.01% Tween 80 in distilled water. After spraying the pests, the plates were incubated at 25?±?1?°C and 80% of relative humidity. Then, the treated pests were monitored every day for the fungal growth and mortality.     

Biocontrol of strawberry fruit infected by Botrytis cinerea: Effects on the microbial communities on fruit assessed by next‐generation sequencing

Fruit grey mould, caused by the fungus Botrytis cinerea, is known to be a harmful disease of strawberry at postharvest stage. However, effects of an application of biological control agents (BCAs) on strawberry fruit in terms of shift in the microbial community are still unknown. The present research aimed to investigate the effects of an application of BCAs on postharvest microbial populations present on strawberry fruits. Strawberry plants were sprayed with three kinds of BCA, RhizoVital 42 fl. (Bacillus amyloliquefaciens FZB42), Trianum‐P (Trichoderma harzianum T22) and Naturalis (Beauveria bassiana ATCC 74040), targeting Botrytis cinerea fungus. Control plots were composed of water and fungicide treatments. Microbial communities (bacteria and fungi) were analysed via next‐generation sequencing on an Illumina MiSeq. Analysis of 16S RNA and ITS rRNA sequences indicated that the BCAs application modified both bacterial and fungal community compositions and diversity. An application of two BCAs together had more effects on microbial community composition than a single application. These results suggest that BCAs can modify bacterial and fungal community composition and diversity on strawberry fruits, which may consequently improve the efficiency and establishment of these products on control of postharvest diseases of fruits, such as grey mould.     

Isolates of Microdochium nivale and M. majus Differentiated by Pathogenicity on Perennial Ryegrass (Lolium perenne L.) and in vitro Growth at Low Temperature

Pink snow mould is a serious disease on grasses and winter cereals in cold and temperate zones during winter. To better understand the basis for the variation in pathogenicity between different isolates of Microdochium nivale and M. majus and to simplify selection of highly pathogenic isolates to use when screening for resistance to pink snow mould in perennial ryegrass, we sought traits correlated with pathogenicity. Isolates of M. nivale were more pathogenic on perennial ryegrass than isolates of M. majus, as measured by survival and regrowth of perennial ryegrass after infection and incubation under simulated snow cover. Pathogenicity as measured by relative regrowth was highly correlated with fungal growth rate on potato dextrose agar (PDA) at 2°C. Measuring fungal growth on PDA therefore seems to be a relatively simple method of screening for potentially highly pathogenic isolates. In a study of a limited number of isolates, highly pathogenic isolates showed an earlier increase and a higher total specific activity of β‐glucosidase, a cell wall‐degrading enzyme, compared with less pathogenic isolates. None of the M. majus isolates was highly pathogenic on perennial ryegrass. Our results indicate biological differences between M. nivale and M. majus and thus strengthen the recently published sequence‐based evidence for the elevation of these former varieties to species status.     

Bioprospecting for fungi with potential pathogenic activity on leaves of Agave tequilana Weber var. Azul

Thirty different fungal strains were isolated from A. tequilana leaves showing disease symptoms such as wilt and curled leaves, black, red and chlorotic spots. Ten genera were identified and confirmed by using the LSU D1/D2 rDNA and ITS1‐5.8S‐ITS2 regions, mainly of the Ascomycota phylum, where the Lasiodiploidia and Neoscytalidium genera were the more (46.6%) abundant. The other genera identified were Cladosporium, Cytospora, Epicoccum, Flavodon, Lasiodiplodia, Myrmaecium, Neoscytalidium, Penicillium, Peniophora, Purpureocillium, Trametes and Fusarium. Five strains of Lasiodiplodia and one of Fusarium were selected based on their representativeness and pathogenic potential on Agaves. Pathogenic potential was analysed by both, an infection assay, evidenced as necrosis, and by pectinolytic activity. Specifically, necrosis infection assay was conducted by puncture (wounded) infection and by direct mycelium contact. In general, Lasiodiplodia strains exhibited different pathogenic profiles according to their necrosis percentages, regardless of the infection method used. Fusarium strain analysed also showed a high necrosis infection (> 99%). Pectinolytic activity used as an indirect measurement of pathogenesis presented a high Fusarium extract activity (peaking at 23.9 U). Lasiodiplodia strains exhibited up 6 times more enzymatic activity (peaking at 143.5) than Fusarium strain analysed. In addition, Agave leaf extracts used totally or partially as carbon source during fungal induction culture may induce different pathogenic activities in these strains. In general, the two pathogenicity assays implemented evidenced differences in the pathogenicity profile of these analysed strains.     

Identification and Molecular Characterizations of Neoscytalidium dimidiatum Causing Stem Canker of Red‐fleshed Dragon Fruit (Hylocereus polyrhizus) in Malaysia

During the year 2008 to 2009, a new disease of stem canker was noticed in most red‐fleshed dragon fruit (Hylocereus polyrhizus) plantations in Malaysia. The symptoms observed were small circular sunken orange spot, black pycnidia and rotted stem. This study was conducted to determine the occurrence of the stem canker on H. polyrhizus in Malaysia, subsequently to isolate, identify and characterize the fungal pathogen based on morphology and molecular characteristics and pathogenicity test. From the surveyed 20 plantations in Malaysia, stem canker was detected in all the plantations. A total of 40 isolates of Scytalidium‐like fungus were isolated and identified as Neoscytalidium dimidiatum based on morphological characteristics and ITS region sequences, which showed 99% similarity to N. dimidiatum (FJ648577). From the phylogenetic analysis using maximum‐likelihood tree, isolates of N. dimidiatum from stem canker of H. polyrhizus were grouped together and did not show any sequence variation. From pathogenicity test, all 40 isolates of N. dimidiatum were pathogenic causing stem canker on H. polyrhizus. To our knowledge, this is the first report of stem canker of H. polyrhizus caused by N. dimidiatum in Malaysia.     

Properties of capsaicinoids for the control of fungi and oomycetes pathogenic to pepper

Capsaicinoids are pungent compounds found in pepper (Capsicum spp.) fruits. Capsaicin showed antimicrobial activity in plate assays against seven isolates of five species of fungi and nine isolates of two species of oomycetes. The general trend was that oomycetes were more inhibited than fungi. Assays of capsaicin biosynthetic precursors suggest that the lateral chain of capsaicinoids has more inhibitory activity than the phenolic part. In planta tests of capsaicinoids (capsaicin and N‐vanillylnonanamide) applied to the roots demonstrated that these compounds conferred protection against the pathogenic fungus Verticillium dahliae and induced both chitinase activity and expression of several defence‐related genes, such as CASC1, CACHI2 and CABGLU. N‐Vanillylnonanamide infiltrated into cotyledons confers systemic protection to the upper leaves of pepper against the fungal pathogen Botrytis cinerea. In wild‐type tomato plants such cotyledon infiltration has no protective effect, but is effective in the Never‐ripe tomato mutant impaired in ethylene response. A similar effect was observed in tomato after salicylic acid infiltration.     

Bulb and Root Rot in Lily (Lilium longiflorum) and Onion (Allium cepa) in Israel

In the past 10 years, there has been a substantial increase in reports, from growers and extension personnel, on bulb and root rots in lily (Lilium longiflorum) in Israel. Rot in these plants, when grown as cut flowers, caused serious economic damage expressed in reduction in yield and quality. In lily, the fungal pathogens involved in the rot were characterized as binucleate Rhizoctonia AG‐A, Rhizoctonia solani, Pythium oligandrum, Fusarium proliferatum (white and purple isolates) and F. oxysporum, using morphological and molecular criteria. These fungi were the prevalent pathogens in diseased plants collected from commercial greenhouses. Pathogenicity trials were conducted on lily bulbs and onion seedlings under controlled conditions in a greenhouse to complete Koch's postulates. Disease symptoms on lily were most severe in treatments inoculated with binucleate Rhizoctonia AG‐A, P. oligandrum and F. proliferatum. Plant height was lower in the above treatments compared with the control plants. The least aggressive fungus was R. solani. In artificial inoculations of onion, seedling survival was significantly affected by all fungi. The most pathogenic fungus was F. proliferatum w and the least were isolates of F. oxysporum (II and III). All fungi were successfully re‐isolated from the inoculated plants.     

Potential of Cactoblastis cactorum as a Vector for Fungi Pathogenic to Pricklypear, Opuntia inermis

In Australia, fungi associated with larvae of the biological control agent Cactoblastis cactorum may contribute to the control of the exotic weed pricklypear (Opuntia inermis). C. cactorum larvae were assessed for their ability to vector pathogenic fungi into O. inermis by the infestation of larvae with fungal suspensions. Six fungal isolates caused disease after being carried into the host on external surfaces of larvae, and propagules of one isolate (UQ5109) initiated disease after being transferred from the cladode epidermis into the host by larvae feeding on the plant. Scanning electron microscopy revealed extensive hyphal growth on the external surfaces of larvae infested with several of the isolates. Fungi isolated from field-grown O. inermis cladodes were tested for pathogenicity to this plant in an in vivo plant assay. In total, 152 isolates were screened, 22 of which infected the host in pathogenicity tests. Only 1 (UQ5115) infected undamaged host tissue, whereas the remainder required the host to be wounded before infection could proceed. The majority of isolates were only weakly pathogenic, even when inoculated via wounds, suggesting that most were either saprophytes or weak parasites. This study demonstrates that it is possible for larvae of C. cactorum to transmit fungal pathogens into O. inermis tissue and it has provided a sound basis for future field work to determine the contribution that fungi make to the control of O. inermis.     

Collophora hispanica,a New Pathogen and Potential Threat to the Almond Industry in Iran

Trunk diseases are potential threats for almond productivity and longevity worldwide, including Iran. In a recent survey on fungal species associated with trunk diseases of almonds in north‐western Iran, Collophora isolates (tentatively identified as Collophora hispanica) were recovered with high frequency from wood samples with internal necrosis and brown to black vascular streaking of almond trees showing symptoms of decline. However, the pathogenic potential of Collophora isolates on almond trees in Iran remains unproven. In this study, the identity of the isolates was further confirmed as C. hispanica based on a combination of morphological data and sequence data of ITS‐rDNA region, and pathogenicity of C. hispanica isolates on almond was evaluated using excised shoot method and in greenhouse experiments. Collophora hispanica isolates induced lesions statistically different from the control, in both excised shoot method and greenhouse assays. Significant differences were observed among the isolates in the length of the lesion induced on wood. Collophora hispanica should be considered as the main trunk pathogens of almond trees in north‐western region of Iran. The distribution and host range of this new pathogen on almond remains to be studied.     

Pyrene degradation and copper and zinc uptake by Fusarium solani and Hypocrea lixii isolated from petrol station soil

Aims: This study aimed to isolate and identify potential polycyclic aromatic hydrocarbon (PAH)‐degrading and/or metal‐tolerant fungi from PAH‐contaminated and metal‐contaminated soils. Methods and Results: Pyrene‐degrading fungi were isolated from contaminated soil and tested for metal (Cu, Zn and Pb) compound solubilization and metal accumulation. Three strains of Fusarium solani and one of Hypocrea lixii were able to degrade more than 60% of initial supplied pyrene (100 mg l^?1) after 2 weeks. The isolates were grown on toxic metal (Cu, Pb and Zn)‐containing media: all isolates accumulated Cu in their mycelia to values ranging from c. 5·9 to 10·4 mmol per kg dry weight biomass. The isolates were also able to accumulate Zn (c. 3·7–7·2 mmol per kg dry weight biomass) from zinc phosphate‐amended media. None of the isolates accumulated Pb. Conclusions: These fungal isolates appear to show promise for use in bioremediation of pyrene or related xenobiotics and removal of copper and zinc from wastes contaminated singly or in combination with these substances. Significance and Impact of the Study: Microbial responses to mixed organic and inorganic pollution are seldom considered: this research highlights the abilities of certain fungal strains to interact with both xenobiotics and toxic metals and is relevant to other studies on natural attenuation and bioremediation of polluted sites.     

Genetic relationships of Edwardsiella strains isolated in China aquaculture revealed by rep-PCR genomic fingerprinting and investigation of Edwardsiella virulence genes

Aims: The aim of this study was to classify Edwardsiella strains isolated from China aquaculture based on biochemical and molecular methods. Methods and Results: In this study, biochemical characterization of 19 Edwardsiella tarda isolates and two Edwardsiella ictaluri isolates was performed with API 20E system. Other pathogenicity‐related phenotypes such as haemagglutination, haemolytic activities and lethality to fish were also examined in these strains. As it was difficult to categorize the subgroups of Edw. tarda according to their origins or phenotypic properties, three PCR‐based methods, i.e. PCR amplification of virulence genes, Enterobacterial repetitive intergenic consensus‐PCR and BOX‐PCR, were carried out to further resolve the relatedness of the Edw. tarda isolates. As a result, all Edw. tarda isolates could be generally grouped into pathogenic and nonpathogenic branches before being classified into strain‐specific or origin‐specific clades. Conclusions: Biochemical characterization was sensitive for interspecific typing, while PCR‐based approaches permitted a more accurate discrimination for intraspecific typing resulting in pathogenic and nonpathogenic clusters and further more delicate clades for Edwardsiella. Significance and Impact of the Study: PCR‐based genomic fingerprinting to study the relatedness and trace the pathogenicity of the Edwardsiella strains will be helpful in investigating the virulence factors of Edwardsiella and in the development of vaccines and diagnostics for edwardsiellosis.     

VdSho1 Regulates Growth,Oxidant Adaptation and Virulence in Verticillium dahliae

Verticillium dahliae infection leads to Verticillium wilt in cotton and other dicotyledon crops. To reduce the loss of economic crops, more attention has been focused on the key genes involved in pathogenicity of this soil‐borne plant fungal pathogen. Sho1 encodes a conserved tetraspan transmembrane protein which is a key element of the two upstream branches of the HOG‐MAPK pathway in fungi. Sho1 is required for full virulence in a wide variety of pathogenic fungi. In this study, sho1 mutant in V. dahliae (designated ΔVdsho1) was generated by Agrobacterium tumefaciens‐mediated transformation. ΔVdsho1 strain was highly sensitive to menadione (at concentration of 120 μm ) and hydrogen peroxide (at concentration of 250 μm ), displayed delayed spore germination and reduced spore production compared with the wild type and the complemented strains. During infection of host cotton plants, ΔVdsho1 exhibited impaired ability of root attachment and invasive growth. Results from the present work suggest that VdSho1 controls external sensing, virulence and multiple growth‐related traits in V. dahliae and might serve as a potential target for control of Verticillium wilt.     

Antimicrobial Activity and Biodiversity of Endophytic Fungi in <Emphasis Type="Italic">Dendrobium</Emphasis><Emphasis Type="Italic">devonianum</Emphasis> and <Emphasis Type="Italic">Dendrobium thyrsiflorum</Emphasis> from Vietman

Endophytic fungi are rich in orchids and have great impacts on their host plants. 53 endophytes (30 isolates from Dendrobium devonianum and 23 endophytic fungi from D. thyrsiflorum) were isolated, respectively, from roots and stems of Dendrobium species. All the fungi were identified by way of morphological and/or molecular biological methods. 30 endophytic fungi in D. devonianum were categorized into 11 taxa and 23 fungal endophytes in D. thyrsiflorum were grouped into 11 genera, respectively. Fusarium was the dominant species of the two Dendrobium species in common. Antimicrobial activity of ethanol extract of fermentation broth of these fungi was explored using agar diffusion test. 10 endophytic fungi in D. devonianum and 11 in D. thyrsiflorum exhibited antimicrobial activity against at least one pathogenic bacterium or fungus among 6 pathogenic microbes (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus). Out of the fungal endophytes isolated from D. devonianum and D. thyrsiflorum, Phoma displayed strong inhibitory activity (inhibition zones in diameter >20 mm) against pathogens. Epicoccum nigrum from D. thyrsiflorum exhibited antibacterial activity even stronger than ampicillin sodium. Fusarium isolated from the two Dendrobium species was effective against the pathogenic bacterial as well as fungal pathogens. The study reinforced the assumption that endophytic fungi isolated from different Dendrobium species could be of potential antibacterial or antifungal resource.     

Identification and characterization of Cucurbita gummy stem blight fungi in Northeast China

Cucurbita maxima (pumpkin) is a typical cucurbit that is susceptible to gummy stem blight. In recent years, this major fungal disease has decimated pumpkin yields in Northeast China (Heilongjiang Province) in increasingly numerous outbreaks with more rapid spread in recent years. After culturing fungi on potato dextrose agar medium, we conducted a systematic study of the growth and morphological characteristics of various purified strains of Cucurbita gummy stem blight (GSB) fungus from across Northeast China. Subsequently, DNA samples of 30 isolates with distinct hyphal variations were subjected to internal transcribed spacer (ITS) sequencing. Sequence analysis resulted in identification of the isolates as Stagonosporopsis cucurbitacearum and demonstrated that this is a dominant and widely distributed fungal species in this region. Subsequently, multi‐site phylogenetic analysis assigned the 30 aforementioned strains to two genotypes that aligned to seven phenotypic types of Cucurbita GSB fungi. By analysing ITS conserved sequences of these phenotypically diverse groups, we found that Cucurbita GSB pathogens broadly shared two motifs that contained sequence variations unique to two groups in addition to common identical motifs. Ultimately, this study provided useful data for rapid and accurate identification of S. cucurbitacearum and diagnosis in early symptoms of Cucurbita GSB. This work also provides tools to explore the distribution and regularity of GSB outbreaks spatially and temporally across Northeast China.     

Application of terminal restriction fragment length polymorphism (T-RFLP) analysis to monitor effect of biocontrol agents on rhizosphere microbial community of hot pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Microbial communities in hot pepper (Capsicum annuum L.) cultivation fields under different cultivation methods were investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis. Rhizosphere soil and leaf samples were collected from control, conventional and nature-friendly cultivation fields between May and July, 2009. Two Bacillus subtilis strains were applied to nature-friendly cultivation fields as biocontrol agents during the sampling period. Relative abundances of bacteria and plant pathogenic fungi related T-RFs were also measured to monitor the effect of biocontrol agents on potential plant pathogenic fungi. In the principal component analysis (PCA) based on T-RFLP profiles, the microbial communities from rhizosphere soil samples in July, including bacteria and fungi, showed distinct difference between nature-friendly cultivation fields and other cultivation fields. However, there was no correlation between cultivation methods and leaf microbial communities at any sampling period. Changes in the abundance of bacteria related T-RF in the rhizosphere of nature-friendly cultivation fields were observed clearly two months after application of biocontrol agent, while the abundance of plant pathogenic fungi related T-RFs significantly decreased.