Biocontrol of Rhizoctonia solani Damping-Off of Tomato with Bacillus subtilis RB14

Bacillus subtilis RB14, which showed antibiotic activities against several phytopathogens in vitro by producing the antibiotics iturin A and surfactin, was subjected to a pot test to investigate its ability to suppress damping-off of tomato seedlings caused by Rhizoctonia solani. To facilitate recovery from soil, B. subtilis RB14-C, a spontaneous streptomycin-resistant mutant of RB14, was used. Damping-off was suppressed when the culture broth, cell suspension, or cell-free culture broth of RB14-C was inoculated into soil. Iturin A and surfactin were recovered from the soils inoculated with the cell suspension of RB14-C, confirming that RB14-C produced them in soil. The gene lpa-14, which was cloned from RB14 and required for the production of both antibiotics, was mutated in RB14-C, and a mutant, R(Delta)1, was constructed. The level of disease suppressibility of R(Delta)1 was low, but R(Delta)1(pC115), a transformant of R(Delta)1 with the plasmid pC115 carrying lpa-14, was restored in suppressibility. These results show that the antibiotics iturin A and surfactin produced by RB14 play a major role in the suppression of damping-off caused by R. solani. RB14-C, R(Delta)1, and R(Delta)1(pC115) persisted in soil during the experimental period and were recovered from the soil, mostly as spores.     

Effect of Validamycin on Production of Some Enzymes in Rhizoctonia solani

A remarkable effect of validamycin on the morphology of Rhizoctonia solani was seen after 2 days culture when the fungus was cultivated in a Roux flask with standing. In accordance with the morphological change, the production of laminarinase and glucan synthetase by the fungus was affected by validamycin.

The production of laminarinase was increased in the culture filtrate, and significantly decreased in the mycelium in the presence of validamycin. While the intracellular production of glucan synthetase in the culture with validamycin (10~50μg/ml) increased by 40~60% compared with that in the control culture.     

Mycolytic effect of fluorescent Pseudomonas in biocontrolling of fungal phytopathogenic Curvularia lunata,Fusarium oxysporum,Alternaria padwickii and Rhizoctonia solani

About 50 bacterial strains, each of Pseudomonas fluorescens, from different rhizospheric soil of different plants were screened for antagonistic activity against Curvularia lunata, Fusarium oxysporum, Alternaria padwickii, Rhizoctonia solani causing black kernel, kernel spotting, root rots, stackburn and sheath blight diseases of rice (Oryza sativa L.). Out of the 50 isolates, 15 isolates were found to be effective in lysing the cell wall of the above-mentioned putative pathogens tested in vitro. These Pseudomonas isolates produced mycolytic enzymes, viz. β-1,3-glucanases, β-1,4-glucanases and lipases. P. fluorescens PAK1 and PAK12 among the strains were more effective for the production of these enzymes while PAK12 produce good level of β-1,3-glucanases, β-1,4-glucanases and lipases against tested fungal pathogens. These findings demonstrate a mechanism of antagonism by P. fluorescens against different fungal plant pathogens.     

Characterization of Genes Involved in Biosynthesis of a Novel Antibiotic from Burkholderia cepacia BC11 and Their Role in Biological Control of Rhizoctonia solani

Genetic manipulation of fluorescent pseudomonads has provided major insight into their production of antifungal molecules and their role in biological control of plant disease. Burkholderia cepacia also produces antifungal activities, but its biological control activity is much less well characterized, in part due to difficulties in applying genetic tools. Here we report genetic and biochemical characterization of a soil isolate of B. cepacia relating to its production of an unusual antibiotic that is very active against a variety of soil fungi. Purification and preliminary structural analyses suggest that this antibiotic (called AFC-BC11) is a novel lipopeptide associated largely with the cell membrane. Analysis of conditions for optimal production of AFC-BC11 indicated stringent environmental regulation of its synthesis. Furthermore, we show that production of AFC-BC11 is largely responsible for the ability of B. cepacia BC11 to effectively control the damping-off of cotton caused by the fungal pathogen Rhizoctonia solani in a gnotobiotic system. Using Tn5 mutagenesis, we identified, cloned, and characterized a region of the genome of strain BC11 that is required for production of this antifungal metabolite. DNA sequence analysis suggested that this region encodes proteins directly involved in the production of a nonribosomally synthesized lipopeptide.     

两株棉花立枯病拮抗菌MH1和MH25的筛选与鉴定

从棉花根际分离了1277个细菌分离物, 以棉花立枯病病原真菌立枯丝核菌(Rhizoctonia solani Kuhn)为靶标菌, 通过平板对峙法获得25个具有拮抗性能的分离物, 其中MH1和MH25具有较强的拮抗性能, 且拮抗性能稳定, 具有较好的生防潜力。经过形态观察、生理生化特征分析及16S rDNA序列分析, MH1为短芽孢杆菌(Brevibacillus brevis), MH25为枯草芽孢杆菌(Bacillus subtilis)。MH1和MH25的16S rDNA序列在GenBank中注册号分别为: EF488102, EF488103。     

Characterization of field isolates of Trichoderma antagonistic against Rhizoctonia solani

The aim of the present study was to characterize sixteen isolates of Trichoderma originating from a field of sugar beet where disease patches caused by Rhizoctonia solani were observed. Use of both molecular and morphological characteristics gave consistent identification of the isolates. Production of water-soluble and volatile inhibitors, mycoparasitism and induced systemic resistance in plant host were investigated using in vitro and in vivo tests in both sterilized and natural soils. This functional approach revealed the intra-specific diversity as well as biocontrol potential of the different isolates. Different antagonistic mechanisms were evident for different strains. The most antagonistic strain, T30 was identified as Trichoderma gamsii. This is the first report of an efficient antagonistic strain of T. gamsii being able to reduce the disease in different conditions. The ability to produce water-soluble inhibitors or coil around the hyphae of the pathogen in vitro was not related to the disease reduction in vivo. Additionally, the strains collected from the high disease areas in the field were better antagonists. The antagonistic activity was not characteristic of a species but that of a population.     

一株抗水稻纹枯病菌的解淀粉芽胞杆菌分离与鉴定

【目的】筛选对水稻纹枯病菌(Rhizoctonia solani)具有强拮抗作用的细菌菌株。【方法】用指示菌法筛选拮抗菌株;通过形态观察、生理生化实验、Biolog及16S rDNA序列分析鉴定目标菌株;利用平板双向培养法和滤纸片扩散法测定抑菌谱及拮抗性质。【结果】分离到一株高活力的水稻纹枯病菌拮抗菌株YB-3,该菌株属于解淀粉芽胞杆菌(Bacillus amyloliquefaciens);菌株YB-3对常见的14株病原真菌和7株细菌具有较强的拮抗作用,并发现其对亲缘关系较近的芽孢菌属有较强的拮抗作用;该菌株的抑制活性具有温度稳定、耐酸、但对蛋白酶敏感的特点。【结论】通过指示菌法筛选到一株对水稻纹枯病菌有强拮抗作用的解淀粉芽胞杆菌(B.amyloliquefaciens)YB-3,它具有广谱、高效的植物病原菌拮抗活性。     

Molecular Characterization of the Rice Pathogenesis-related Protein, OsPR-4b, and Its Antifungal Activity Against Rhizoctonia solani

We cloned and identified a new rice pathogenesis‐related (PR)‐4 gene, OsPR‐4b. OsPR‐4b encodes a 151 amino acid protein with a predicted molecular mass of 16.47 kDa and pI of 4.42. The putative OsPR‐4b shows high similarity to PR‐4 type proteins from various plant species and belongs to the Barwin family. Like other PR‐4s from monocot plants, OsPR‐4b contains a conserved Barwin domain and has a signal peptide at its N‐terminus. Recombinant OsPR‐4b protein expressed in Escherichia coli showed antifungal activity in vitro against the sheath blight fungus, Rhizoctonia solani. The results suggest that the OsPR‐4b may play a role in the disease resistance responses of rice against pathogen attacks through its antifungal activity.     

两株棉花立枯病拮抗菌MH1和MH25的筛选与鉴定

从棉花根际分离了1277个细菌分离物,以棉花立枯病病原真菌立枯丝核菌(Rhizoctonia solani Kuhn)为靶标菌,通过平板对峙法获得25个具有拮抗性能的分离物,其中MH1和MH25具有较强的拮抗性能,且拮抗性能稳定,具有较好的生防潜力.经过形态观察、生理生化特征分析及16S rDNA序列分析,MH1为短芽孢杆菌(Brevibacillus brevis),MH25为枯草芽孢杆菌(Bacillus subtilis).MH1和MH25的16S rDNA序列在GenBank中注册号分别为:EF488102,EF488103.     

Genetic Variability in Pectic Enzymes of Rhizoctonia solani Isolates Causing Bare-patch Disease of Cereals

Field isolates of Rhizoctonia solani obtained from three discrete bare patches in a wheat field in Western Australia were characterized by pectic zymogram grouping. The genetic background of pectic enzymes was analysed by comparing the zymograms of asexual homokaryons and sexual progenies derived from field isolates. The 170 field isolates obtained from the field site produced indistinguishable pectic zymograms. However, variations among field isolates of the same zymogram group were detected, on the basis of zymograms of their resultant protoplast-regenerated cultures. Asexual sibling homokaryons derived from each of the field isolates were heterogeneous for their pectic enzymes. Homokaryons with a common heterokary on incompatibility factor, obtained from a field isolates were homogeneous for pectic enzymes. Basidiospore progenies of a field isolate segregated widely in pectic zymograms. It appeared that the expression of pectic enzymes by field isolates involved multiple genetic factors. The variation of zymograms among homokaryotic strains suggests that each field isolate of R. solani contains two types of nuclei, although cells of vegetative hyphae are multinucleate.     

Biocontrol of Rhizoctonia solani damping-off disease in cucumber with Bacillus pumilus SQR-N43

Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Micrographs were used to investigate the ability of Bacillus pumilus (B. pumilus) SQR-N43 to control Rhizoctonia solani (R. solani) Q1 in cucumbers. The root colonization ability of B. pumilus SQR-N43 was analyzed in vivo with a green fluorescent protein (GFP) tag. A pot experiment was performed to assess the in vivo disease-control efficiency of B. pumilus SQR-N43 and its bio-organic fertilizer. Results indicate that B. pumilus SQR-N43 induced hyphal deformation, enlargement of cytoplasmic vacuoles and cytoplasmic leakage in R. solani Q1 mycelia. A biofilm on the root surface was formed when the roots were inoculated with 10(7)-10(8)cells g(-1) of soil of GFP-tagged B. pumilus SQR-N43. In the pot experiment, the biocontrol reduced the concentration of R. solani. In contrast to applications of only B. pumilus SQR-N43 (N treatment), which produced control efficiencies of 23%, control efficiencies of 68% were obtained with applications of a fermented organic fertilizer inoculated with B. pumilus SQR-N43 (BIO treatment). After twenty days of incubation, significant differences in the number of CFUs and the percentage of spores of B. pumilus SQR-N43 were recorded between the N treatment (2.20×10(7)CFU g(-1) of soil and 79%, respectively) and the BIO treatment (1.67×10(8)CFU g(-1) of soil and 52%, respectively). The results indicate that B. pumilus SQR-N43 is a potent antagonist against R. solani Q1. The BIO treatment was more effective than the N treatment because it stabilized the population and increased the active form of the antagonist.     

Methylation and aging in Rhizoctonia solani

Our earlier studies had shown that as fungi age, many of their vital functions decrease; in Rhizoctonia solani, protein synthesis is one of the functions so affected. We now find that the ability to methylate tRNA, a vital component of the protein synthesizing system, also decreases with age. This methylation of Escherichia coli tRNA by R. solani methylase preparations increased with the concentration of enzyme and with time of incubation; in both cases the rate of increase was considerably higher for preparations from young cells than for those from old cells. The methylation reaction also increased with the concentration of substrate tRNA, with temperature, at least to 45° C, and with pH to 9.0. Methylase preparations from R. solani methylated both exogenous E. coli tRNA and yeast tRNA, but were only weakly active on isolated R. solani tRNA. However, acid-precipitated methylases from R. solani were very effective in methylating the homologous exogenous tRNA. Regardless of the source of the tRNA used as substrate, the methylases from older cells were always less active than those from young cells from the same mycelium. No methylase inhibitor was detected in the fungus.     

Use of DAPI for Anastomosis Group Typing of Strains of the Fungus Rhizoctonia solani

Strains of Rhizoctonia solani, a common soil-borne, pathogenic fungus of plants, are assigned to one of 11 anastomosis groups (AGs) based on the occurrence of imperfect fusions (anastomoses) between hyphae of a non-typed strain and a tester strain of one of the 11 AG's. Imperfect fusion is characterized by the death of one or more cells in each of the hyphae involved in the fusion. Although hyphae from branches of the same strain of JR. solani may fuse with each other (self-fusion), cell death does not occur. Cell death is accompanied by nuclear degradation and granulation, or plasmolysis of the cytoplasm, which often is not visible using bright-field microscopy. When the DNA-binding fluorochrome DAPI (4', 6-diamidino-2-phenylindole) is used and the hyphal fusions viewed under fluorescence microscopy, no nuclei are observed in fused hyphal cells from two strains of the same AG of R. solani Because DAPI reacts only with living nuclei, lack of staining is presumptive evidence that the fused cells are dead as a result of imperfect fusion. The use of DAPI reduces the time required for making AG determinations compared to standard methods because it eliminates the need to assess cell wall dissolution and cytoplasmic fusion. Also, it is not necessary to trace the hyphae involved in the fusion to their respective origins to ensure that self-fusion has not occurred.     

Co-utilization of Bacillus subtilis and Flutolanil in controlling damping-off of tomato caused by Rhizoctonia solani

Damping-off of tomato caused by Rhizoctonia solani was controlled in a pot test using the biological agent, Bacillus subtilis RB14-C, and the chemical pesticide, Flutolanil. The co-utilization of B. subtilis RB14-C, and Flutolanil decreased the amount of Flutolanil used from 375 g/pot when Flutolanil was used alone to 94 g/pot, while exerting the same effect of reducing disease occurrence.     

牡丹根腐病原菌拮抗细菌抑菌活性物质分析

前期研究发现解淀粉芽胞杆菌(Bacillus amyloliquefaciens)md8和md9对牡丹根腐病原菌具有较好的抑制作用,但其抑菌物质的组成尚不清楚。本文首先明确了2个菌株3种脂肽类物质合成的基因片段,利用酸沉淀和葡聚糖凝胶层析法进行抑菌物质的分离纯化,牛津杯对峙法检测脂肽类粗提物和凝胶层析分离组分的抑菌活性;进一步利用实时荧光定量PCR(RT-qPCR)检测菌株在拮抗根腐病原菌过程中脂肽类物质合成基因相对表达量的变化,基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)分析抑菌物质的种类。结果表明,2个菌株的脂肽类粗提物和凝胶层析分离组分对根腐病原菌均具有良好的平板抑菌效果,RT-qPCR结果表明2个菌株合成伊枯草菌素(iturin)的基因ituD在对峙培养过程中相对表达量显著增加,芬荠素(fengycin)合成基因fenA也呈现上调表达。MALDI-TOF-MS分析表明2个菌株中具有抑菌作用的分离组分主要为伊枯草菌素类物质Iturin B和Bacillomycins D,结合实时荧光定量PCR结果,推测菌株md8和md9在拮抗根腐病原菌过程中发挥主要生防作用的物质为伊...     

The Effect of Mode of Application of Bacillus subtilis RB14-C on its Efficacy as a Biocontrol Agent Against Rhizoctonia solani

Bacillus subtilis RB14‐C, which produces the antibiotic iturin A, was investigated for its effectiveness as a biocontrol agent against Rhizoctonia solani infecting tomato using seed coating and/or direct introduction of the bacteria to the soil. The ability of RB14‐C to colonize plant roots and produce iturin A in soil, depending on the method of bacterial application, was also determined. Seed coating and the combined treatment (soil and seed bacterization) did not protect seedlings against damping‐off caused by R. solani. By contrast, RB14 introduced only to the soil controlled the disease. The total number of RB14‐C bacteria on the roots of plants grown from coated seeds was significantly lower than on the roots of plants grown in soil mixed with the bacteria. In the combined treatment, application of B. subtilis with seeds to soil preinoculated with this bacterium, at first suppressed the population of RB14‐C in the soil. Then the colonization was generally uniform. The concentration of iturin A in non‐planted soil was highest at the beginning of the experiment (i.e. after application of the bacterial suspension) but then decreased, and was undetectable 3 days after incubation. However, after seed planting the antibiotic was produced again around young roots. Bacteria introduced to the soil as a seed coating also released the antibiotic around the seeds.     

Rhizoctonia solani Incited Damping-off in Radish Suppressed by Plasmid and Non-plasmid Carrying Strains of R. solani that Enhance Host Chitinase Activity

Thirteen Rhizoctonia solani isolates belonging to anastomosis group four were examined. All of the isolates carry plasmids except isolates CHU344 and CHU345. Plasmids were analysed by agarose gel electrophoresis and observed by transmission electron microscope. The plasmid DNA is linear, having a size of 2.6 kbp. The termini of the plasmid have a hairpin structure. The replication of plasmid DNA of R. solani CHU341 (pCHU341) was demonstrated by electron microscopy.
Virulence of the fungi was tested on radish seedlings and expressed according to percentage of radish seedlings that damped-off. Strains CHU341, CHU344, CHU345, CHU346, CHU347, CHU348, CHU350 and CHU353, suppressed damping-off of radish seedlings by the virulent isolates, CHU342, CHU349, CHU351 or CHU352 and was expressed according to a suppressiveness index. The chitinase activities of radish seedlings increased from 10 to 20 times after inoculation with suppressive isolates, CHU341, CHU344 and CHU345.
The results indicate that damping-off of radish seedlings caused by R. solani can be protected by related strains with or without plasmids. The protection of the seedlings may result from the enhancement of chitinase activities induced by the suppressive strains of R. solani.