Evaluation of the strains of Acinetobacter and Enterobacter as potential biocontrol agents against Ralstonia wilt of tomato

Bacterial wilt (Ralstonia solanacearum) of tomato, Lycopersicon esculentum, causes a considerable amount of damage to tomato in Southern China. Biological control is one of the more promising approaches to reduce the disease incidence and yield losses caused by this disease. Based on antagonistic activity against R. solanacearum and three soil-borne fungal pathogens as well as biocontrol efficacy in the greenhouse, two bacterial strains Xa6 (Acinetobacter sp.) and Xy3 (Enterobacter sp.) were selected out of fourteen candidates as potential biocontrol agents. In order to find a suitable antagonist inoculation method, we compared the methods of root-dipping with soil-drenching in the aspects including rhizocompetence, biocontrol efficacy, and effect of promoting plant growth under greenhouse conditions. The drenching treatment resulted in a higher biocontrol efficacy and plant-yield increase, and this method was also easier to operate in the field on a large scale. Field trials were conducted for further evaluation of these two antagonistic strains. In both greenhouse and field experiments, the strain Xy3 had a better control effect against bacterial wilt than Xa6 did, while Xa6 caused higher biomass or yield increases. As recorded on the 75th day after treatment in two field experiments, biocontrol efficacy of Xy3 was about 65% in both field trials, and the yield increases caused by Xa6 were 32.4 and 40.7%, respectively, in the two trials. This is the first report of an Acinetobacter sp. strain used as a BCA against Ralstonia wilt of tomato.     

Elsinochrome A production by the bindweed biocontrol fungus Stagonospora convolvuli LA39 does not pose a risk to the environment or the consumer of treated crops

Biological control as an alternative to chemical pesticides is of increasing public interest. However, to ensure safe use of biocontrol methods, strategies to assess the possible risks need to be developed. The production of toxic metabolites is an aspect which has so far largely been neglected in the risk assessment and the registration process for biocontrol products. We have evaluated the risks of elsinochrome A (ELA) and leptosphaerodione production by the fungus Stagonospora convolvuli LA39, an effective biocontrol agent used against bindweeds. The toxicity of the two metabolites to bacteria, protozoa, fungi and plants was evaluated in in vitro assays. The most sensitive bacteria and fungi were already affected at 0.01-0.07 microM ELA, whereas plants were far less sensitive. Leptosphaerodione was less toxic than ELA. Subsequently, it was investigated whether ELA is present in the applied biocontrol product or LA39-treated bindweed and crop plants. In plants ELA was never detected and in the biocontrol product the ELA concentration was far too low to have toxic effects even on the most sensitive organisms. We conclude that the production of ELA by biocontrol strain LA39 does not pose a risk to the environment or to the consumer.     

ISSR fingerprinting for the assessment of the bindweed biocontrol agent Stagonospora convolvuli LA39 after field release

AIMS: To develop a molecular identification method based on ISSR fingerprints to monitor the fungal leaf pathogen Stagonospora convolvuli LA39 used to biologically control bindweeds after a field release. METHODS AND RESULTS: The developed method proved to be suitable to clearly distinguish LA39 from resident Stagonospora spp. and was applied in two field experiments. First, the environmental persistence of LA39 was assessed in an overwintering experiment. LA39 could be re-isolated from infected bindweed 1 year after field application, but with very low frequency of occurrence. Secondly, LA39 was applied in an area with natural bindweed infestation and re-isolated from infected bindweed. The dispersal of LA39 during one season was poor (4-5 m). CONCLUSIONS: ISSR fingerprinting has been shown to be a valuable tool to monitor the environmental fate of S. convolvuli in the field. It is concluded that an LA39-based mycoherbicide will have minimal environmental impact caused by the restricted mobility, poor proliferation and poor persistence over seasons of LA39. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies about the dispersal and survival of biocontrol agents after field release as well as the development of methods needed for this purpose are indispensable for a comprehensive risk assessment for biocontrol agents.     

Detection of the nematophagous fungus Hirsutella rhossiliensis in soil by real-time PCR and parasitism bioassay

Hirsutella rhossiliensis, a nematophagous fungus, has shown potential in biocontrol of plant-parasitic nematodes. Monitoring the population dynamics of a biocontrol agent in soil requires comprehensive techniques and is essential to understand how it works. Bioassay based on the fungal parasitism on the juveniles of soybean cyst nematode, Heterodera glycines, can be used to evaluate the activity of the fungus but fails to quantify fungal biomass in soil. A real-time polymerase chain reaction (PCR) assay was developed to quantify the fungal population density in soil. The assay detected as little as 100 fg of fungal genomic DNA and 40 conidia g⁻¹ soil, respectively. The parasitism bioassay and the real-time PCR assay were carried out to investigate the presence, abundance and activity of H. rhossiliensis in soil after application of different inoculum levels. Both of the percentage of assay nematodes parasitized by H. rhossiliensis based on the parasitism bioassay and the DNA yield of the fungus quantified by real-time PCR increased significantly with the increase of the inoculum levels. The DNA yield of the fungus was positively correlated with the percentage of assay nematodes parasitized by H. rhossiliensis. The combination of the two is useful for monitoring fungal biomass and activity in soil.     

撕裂蜡孔菌(Emmia lacerata)SR5抑菌特性及生防潜力评价

【背景】撕裂蜡孔菌(Emmia lacerata)是一种在世界范围内广泛分布的白腐真菌,对植物病原真菌有较好的抑制作用,可作为生防真菌进行开发和利用。【目的】对撕裂蜡孔菌SR5的抑菌能力和胞外产铁载体能力进行测定,挖掘其生防潜力。【方法】采用平板对峙法检测SR5对9种植物病原真菌的抑菌能力,并通过不同浓度的发酵原液测定真菌胞外代谢物的抑菌效果;结合铬天青S(chrome azurol S, CAS)检测法测定真菌产铁载体能力,明确SR5抑菌特性。【结果】SR5以过度生长的方式快速竞争营养和生存空间,拮抗9种植物病原真菌,抑菌率为23.7%–62.7%,对可可毛色二孢(Lasiodiplodia theobromae)的拮抗等级为Ⅳ级,而对其余8种病原真菌的拮抗等级为Ⅲ级,其中对香港丽赤壳(Calonectria hongkongensis)和间座壳(Diaporthe sp.)抑菌效果最佳;CAS检测法表明SR5能产生分泌型铁载体,产铁载体能力中等,最高铁载体活性单位(siderophore unit, SU)为44.1%。【结论】SR5以过度生长方式快速竞争营养和生存空间,而且以分泌...     

Effects of temperature and water activity on <Emphasis Type="Italic">Lecanicillium</Emphasis> spp. conidia germination and growth,and mycosis of <Emphasis Type="Italic">Pissodes strobi</Emphasis>

Selecting entomopathogenic fungal isolates for use as biocontrol agents requires an assessment of their growth and virulence characteristics as affected by environmental conditions. Here we demonstrate a wide temperature and moisture range for colony growth, effective conidial germination and virulence against Pissodes strobi Peck (white pine weevil) of several isolates of Lecanicillium Gams and Zare, an entomopathogenic fungus distributed worldwide and indigenous to forests on Vancouver Island, British Columbia, Canada. In order to examine the potential Lecanicillium as a biological control agent, the pathogenicity of isolates collected from different geographical locations on P. strobi cadavers was assessed, and colony growth at different temperatures was evaluated. Colony growth was evident between 5 and 30°C, with optimal growth occurring at 25°C. Various combinations of water activity (0.55, 0.76, 0.85 and 0.99 a _w) and temperature (10, 15, 20, and 25°C) were also used to evaluate environmental impacts on conidial germination and cumulative mycosis of adult P. strobi. Certain Lecanicillium isolates displayed xerophilic (0.85 a _w) or psychrophilic (10°C) growth optima. Ultimately, identifying the abiotic limits of this entomopathogenic fungus will be used to determine which isolates have potential for future in situ biocontrol trials.     

Pseudomonads: major antagonistic endophytic bacteria to suppress bacterial wilt pathogen, <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in the eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

Endophytic bacteria of eggplant, cucumber and groundnut were isolated from different locations of Goa, India. Based on in vitro screening, 28 bacterial isolates which effectively inhibited Ralstonia solanacearum, a bacterial wilt pathogen of the eggplant were characterized and identified. More than 50% of these isolates were Pseudomonas fluorescens in which a vast degree of variability was found to exist when biochemical characteristics were compared. In greenhouse experiments, the plants treated with Pseudomonas isolates (EB9, EB67), Enterobacter isolates (EB44, EB89) and Bacillus isolates (EC4, EC13) reduced the wilt incidence by more than 70%. All the selected isolates reduced damping off by more than 50% and improved the growth of seedlings in the nursery stage. Most of the selected antagonists produced an antibiotic, DAPG, which inhibited R. solanacearum under in vitro conditions and might have been responsible for reduced wilt incidence under in vivo conditions. Also production of siderophores and IAA in the culture medium by the antagonists was recorded, which could be involved in biocontrol and growth promotion in crop plants. From our study we conclude that Pseudomonas is the major antagonistic endophytic bacteria from eggplants which have the potential to be used as a biocontrol agent as well as plant growth-promoting rhizobacteria. Large scale field evaluation and detailed knowledge on antagonistic mechanism could provide an effective biocontrol solution for bacterial wilt of solanaceous crops.     

Biological control of Chenopodium album L. in Europe

Ascochyta caulina (P. Karst) v.d. Aa and v. Kest is aplant pathogenic fungus which is specific to Chenopodium albumL. It has been suggested as a potential mycoherbicide to this weed,which is important and wide spread in arable crops throughout Europe. Toinvestigate its potential as a biocontrol agent, the fungus has beentested in glasshouse and field experiments. Formulations containingdifferent combinations of A. caulina conidia, the phytotoxinsfrom the fungus and low doses of herbicides have been tested.Significant improvement in the efficacy of the fungus was achieved inglasshouse trials with an aqueous formulation containing PVA(0.1% v/v), Psyllium (0.4% w/v), Sylgard 309(0.1% v/v), nutrients and conidia (5 ×10⁶/ml). The extracellular, hydrophilic phytotoxinsproduced by A. caulina were purified and their structuresdetermined. The main toxin, named ascaulitoxin, was characterised as theN²--D-glucopyranoside of the unusual bis-aminoacid2,4,7-triamino-5-hydroxyoctandioic acid. Two other toxins proved to betrans-4-amino-D-proline and the aglycone of ascaulitoxin. Thesetoxins have shown promising herbicidal properties. Field trials haveinvestigated the performance of A. caulina conidia applied atdifferent developmental stages of C. album either as a singletreatment or combined with sub-lethal doses of herbicides or with thefungal phytotoxins. With the available formulation, favourable weatherconditions are needed to obtain infection in the field. The efficacy ofthe strain of A. caulina used so far has proved to beinadequate to justify its development as a bioherbicide. This isprobably due to its low virulence.     

The potential of Ulocladium botrytis for biological control of Orobanche spp.

A strain of Ulocladium botrytis isolated from diseased Orobanche crenata shoots caused disease on the parasitic weed in pathogenicity tests. The potential of the fungus to be developed as a mycoherbicide for Orobanche spp. was further investigated. Although the fungus significantly decreased O. crenata germination in vitro by 80%, it did not generally lead to a decreased number of O. crenata shoots or tubercles in inoculated root chambers or pots. However, the number of diseased or dead tubercles and underground shoots was significantly increased compared to the noninoculated treatments. Postemergence inoculation of O. crenata shoots with a conidial suspension resulted in the death of almost all inoculated plants 14 days after application under greenhouse conditions. In preliminary host-range studies, the pathogen caused disease on Orobanche cumana on sunflower whereas on Orobanche aegyptiaca shoots parasitizing tomato only minimal disease symptoms could be detected after postemergence inoculation. Based on the results of our investigations, we conclude that Ulocladium botrytis has only a limited potential to be used as a biocontrol agent against Orobanche spp.     

Biological control of snow mould (Microdochium nivale) in winter cereals by Pseudomonas brassicacearum, MA250

Seed lots of winter wheat and rye, naturally infested with Microdochium nivale and Fusarium spp., were treated with an isolate of Pseudomonas, which was recovered from roots of Brassica napus. Seeds were treated with bacterial fermentate and dried before sowing or they were directly sprayed in the furrow-opener at the moment of sowing. Besides field experiments, parallel climate chamber bioassays were performed to assess the effect of bacterial treatment on snow mould caused by seed-borne M. nivale and Fusarium spp. The biocontrol effect was assessed by plant density counts and by measuring yield. Significant biocontrol activity, measured by plant density counts, was detected both in field and climate chamber experiments sown with wheat. Biocontrol effect after spray application at sowing was less pronounced, although a slight increase in plant density was observed. The cell concentration required to obtain adequate biocontrol effect was 10⁹ CFU per ml for the dose used. The bacterial isolate was identified by 16S rDNA sequencing and biochemical tests as a Pseudomonas brassicacearum strain.     

Biological control of root rot-root knot disease complex of tomato

Efficacy of Pseudomonas aeruginosa alone or in combination with Paecilomyces lilacinus was evaluated in the control of root-knot nematode and root-infecting fungi under laboratory and field conditions. Ethyl acetate extract (1 mg/ml) of P. lilacinus and P. aeruginosa,respectively, caused 100 and 64% mortality of Meloidogyne javanica larvae after 24 h. Ethyl acetate fractions of biocontrol agents were more effective than hexane extracts in the suppression of M. javanica larvae, indicating that active nematicidal compounds are intermediary in polarity. In field experiments, biocontrol fungus and bacterium significantly suppressed soilborne root-infecting fungi including Macrophomina phaseolina, Fusarium oxysporum, Fusarium solani, Rhizoctonia solani and Meloidogyne javanica, the root-knot nematode. P. lilacinus parasitized eggs and female of M. javanica and this parasitism was not significantly influenced in the presence of P. aeruginosa. P. aeruginosa was reisolated from the inner root tissues of tomato, whereas P. lilacinusdid not colonize tomato roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Interactions of Fusarium moniliforme,its metabolites and bacteria with corn

Fusarium moniliforme Sheldon is an economically important pathogen of corn (Zea mays L.) which causes stalk, root and ear rot. Several mycotoxins have also been isolated, identified and implicated in both animal and human toxicoses. The fungus can be disseminated in symptomless corn seed and can also survive in crop residues in the soil. Asymptomatic infection may be related to different corn cultivars, fungal strains, and environmental factors. Symptomatic expression of pathogenicity may vary, but usually the result of such infections is death of the plant. The greatest concern is the asymptomatic infection, since it is in this form that fungal toxins may surreptitiously enter animal and human food chains. F. moniliforme produces both fusaric acid, which is phytotoxic to corn and interferes with seed germination, and plant growth regulators that may affect pathogenicity of the fungus or be associated with the production of mycotoxins. Other metabolites, including fusarin C, moniliformin, and the fumonisins, may or may not be phytotoxic, but are associated with animal and human toxicoses. The control of F. moniliforme in corn is therefore quite important. One potential means to accomplish this reduction is biocontrol by the application of antagonistic rhizobacteria to corn kernels at planting. To be effective the bacteria must be able to colonize the corn root system and be able to prevent root infection by successful competing with F. moniliforme which may be accomplished by siderophore and or antibiotic activity.     

甲基营养型芽孢杆菌拮抗玉蜀黍尾孢菌、链格菌和灰葡萄孢菌及环脂肽分析

[目的]探究甲基营养型芽孢杆菌(Bacillus methylotrophicus)对植物病原菌玉蜀黍尾孢菌(Cercospora zeae-maydis Tehon et Daniels)、链格菌(Alternaria alternate)和灰葡萄孢菌(Botrytis cinerea)的拮抗作用并鉴定抗菌物质,为其...     

印度梨形孢(Piriformospora indica)增强植物抗逆境胁迫能力的研究进展

印度梨形孢是一种可在多种植物根部定殖的内生真菌,能与多种植物形成共生体,提高植物对外界营养的吸收能力,促进次生代谢产物的积累,提高植物对生物及非生物胁迫的抵抗能力,同时可增加植物的生物产量,对宿主植物产生许多有益影响。因此,印度梨形孢作为优良的生物防治和土壤改良因子,在农业生产方面显示出巨大的应用前景。本文结合本课题组近年研究结果及近10年间相关科学工作者的研究,系统总结了印度梨形孢在增强植物抗生物胁迫与非生物胁迫方面的研究进展,旨在为更好地发挥其潜在价值提供参考。     

Biological control of root-rot of Coleus forskohlii Briq. using microbial inoculants

The root-rot or wilt of Coleus forskohlii is a very serious soil-borne disease caused by Fusarium chlamydosporum. A field study was undertaken to study the possibility of controlling the disease using three biocontrol agents viz., Glomus mosseae, Pseudomonas fluorescens, Trichoderma viride, singly and in combination. Planting of coleus cutting was done in wilt sick soil. Inoculation with Trichoderma viride + Glomus mosseae gave the best result in controlling the disease. The same treatment also resulted in maximum growth, yield and root forskolin concentration of coleus. Plants treated with T. viride + G. mosseae showed a disease severity index of 33.28% compared to uninoculated control plants, which had a maximum disease severity index of 85.5%. The fungicide Emisan (0.2%) was not as effective as the biocontrol agents in controlling the pathogen.     

The susceptibility of immature stages of <Emphasis Type="Italic">Bemisia tabaci</Emphasis> to the entomopathogenic fungus <Emphasis Type="Italic">Lecanicillium muscarium</Emphasis> on tomato and verbena foliage

Lecanicillium muscarium is a widely occurring entomopathogenic fungus. Laboratory studies were conducted to determine the efficacy of L. muscarium against different instars of Bemisia tabaci on tomato and verbena foliage after two incubation times (3 and 7 days). Significant reduction in B. tabaci numbers were recorded on fungus treated plants (p < 0.001). Second instar B. tabaci proved most susceptible to L. muscarium infection. There was no significant difference in mortality of B. tabaci second instars after either 3 or 7 days exposure to L. muscarium on either host plant. The importance of the speed of pest mortality following treatment and the potential of L. muscarium to be incorporated into an integrated pest management strategy for the biocontrol of B. tabaci on tomato and verbena plants are discussed.     

Methods for studying the nematophagous fungus Verticillium chlamydosporium in the root environment

In order to exploit fully the biocontrol potential of the nematophagous fungus Verticillium chlamydosporium, it is important to understand the ecology of the fungus in soil, and interactions with both plant and nematode hosts. Several approaches for studying the fungus in soil and the root environment are compared. These include a semi-selective medium for V. chlamydosporium, PCR primers specific for the fungal -tubulin gene, and monoclonal antibodies. In addition to providing a target for species-specific primers, the -tubulin gene is implicated in resistance to the fungicides used in the semi-selective medium, and the genetic basis for this is investigated. Culture and PCR-based methods were used to screen for the presence of the fungus in field soils known to have been suppressive to cereal cyst nematode and that contained V. chlamydosporium populations. Monoclonal antibodies specific for either hyphae or conidia of the fungus were obtained, and their application as a tool for visualising the infection process on the root was explored.     

Suppression of gray leaf spot (blast) of perennial ryegrass turf by Pseudomonas aeruginosa from spent mushroom substrate

Bacteria isolated from spent mushroom substrate (SMS) were evaluated for the suppression of Pyricularia grisea, the causal agent of gray leaf spot of perennial ryegrass (Lolium perenne) turf. Thirty-two of 849 bacterial isolates (3.8%) from SMS significantly inhibited the mycelial growth of P. grisea in vitro. Six bacterial isolates that afforded maximum inhibition of P. grisea were also refractory to Rhizoctonia solani, Rhizoctonia cerealis, Sclerotinia homoeocarpa, and Fusarium culmorum. Each of the six isolates was identified as Pseudomonas aeruginosa by fatty acid profile analysis. The biocontrol activity of the bacterial isolates was not compromised by a prolonged exposure to UV radiation in vitro. In controlled-environment chamber experiments, all 32 bacterial isolates were tested for suppression of gray leaf spot on Pennfine perennial ryegrass when applied as seed treatment or foliar sprays. Foliar applications of the bacteria (10⁸ cfu/ml 0.1% carboxymethylcellulose), but not the seed treatment, significantly reduced disease severity and incidence. The three most efficient isolates from foliar application treatments, which were among the six bacterial isolates identified as P. aeruginosa, were further evaluated for suppression of gray leaf spot as a function of timing of application. The three isolates of P. aeruginosa suppressed gray leaf spot in perennial ryegrass in Cone-tainers when applied at 1, 3, and 7 days prior to inoculation with P. grisea both in controlled-environment chamber experiments, and in potted ryegrass plants maintained in the field. All application intervals, regardless of the bacterial isolate, provided significant reduction of gray leaf spot severity. Suppression of gray leaf spot by isolates of P. aeruginosa under controlled-environment chamber conditions was not different from that observed in potted ryegrass plants maintained in the field. In field experiments, an isolate of P. aeruginosa provided significant suppression of gray leaf spot when applied at 1, 7, and 14 days prior to inoculation with P. grisea. The bacterium proved effective against gray leaf spot of perennial ryegrass maintained as fairway and rough heights. These results indicate that P. aeruginosa may be a potential biocontrol agent for gray leaf spot of perennial ryegrass turf.     

Dual Antagonism of Aldehydes and Epiphytic Bacteria from Strawberry Leaf Surfaces against the Pathogenic Fungus Botrytis cinerea in vitro

Dual culture experiments were conducted in vitro to evaluate the potential combined biological effect of epiphytic bacteria and plant volatiles formed during fatty acids degradation on the pathogenic fungus Botrytis cinerea. The aliphatic aldehydes hexanal, (E)-2-hexenal, (Z)-3-hexenal and (E)-2-nonenal showed an enhancing effect on the antagonistic interaction between the epiphytic bacteria Pseudomonas lurida, Pseudomonas rhizosphaerae, Pseudomonas parafulva, and Bacillus megaterium against the pathogenic fungus. The unsaturated aldehydes were found to be the most potent with the minimum effective concentration being 1 ppm. Increasing volatile concentrations led to the inhibition of Botrytis cinerea growth with concomitant increase of colony diameters of epiphytic bacteria. Especially (E)-2-nonenal showed a stronger inhibitory effect on different strains of the plant pathogenic fungus Botrytis cinerea than on the epiphytic bacteria. These results suggest that co-application of antagonistic bacteria with natural plant volatiles can enhance the effectiveness of the biocontrol agents against B. cinerea.     

Control and possible applications of a novel carrot-spoilage basidiomycete, Fibulorhizoctonia psychrophila

A novel cold-tolerant fungus, Fibulorhizoctonia psychrophila, was isolated from a refrigerated carrot storage facility and identified as an anamorph of Athelia, often classified in Rhizoctonia s.l. Growth of this fungus was observed between 0 and 20°C with an optimum at 9–12°C, while incubation of mycelium grown at 15–32°C resulted in absence of growth even after the fungus was transferred back to 15°C. Growth was inhibited in the presence of the antifungals sorbic acid or natamycin, in particular when the fungus was incubated at 18°C. F. psychrophila produces polysaccharide degrading enzymes that, when compared to enzymes from the ascomycete fungus Aspergillus niger, retain a larger proportion of their activity at lower temperatures. This indicates that F. psychrophila could be used as a source for novel industrial enzymes that are active at 4–15°C.