Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, the rice sheath blight pathogen

Fourteen strains of Pseudomonas fluorescens isolated from rhizosphere soil of rice were tested for their antagonistic effect towards Rhizoctonia solani, the rice sheath blight fungus. Among them, PfMDU2 was the most effective in inhibiting mycelial growth of R. solani in vitro. Production of chitinase, beta-1,3-glucanase, siderophores, salicylic acid (SA) and hydrogen cyanide (HCN) by P. fluorescens strains was evaluated. The highest beta-1,3-glucanase activity, siderophore production, SA production and HCN production were recorded with PfMDU2. A significant relationship between the antagonistic potential of P. fluorescens against R. solani and its level of beta-1,3-glucanase, SA and HCN was observed.     

Characterization of quantitative trait loci (QTLs) in cultivated rice contributing to field resistance to sheath blight (Rhizoctonia solani)

Sheath blight, caused by Rhizoctonia solani, is one of the most important diseases of rice. Despite extensive searches of the rice germ plasm, the major gene(s) which give complete resistance to the fungus have not been identified. However, there is much variation in quantitatively inherited resistance to R. solani, and this type of resistance can offer adequate protection against the pathogen under field conditions. Using 255 F₄ bulked populations from a cross between the susceptible variety Lemont and the resistant variety Teqing, 2 years of field disease evaluation and 113 well-distributed RFLP markers, we identified six quantitative trait loci (QTLs) contributing to resistance to R. solani. These QTLs are located on 6 of the 12 rice chromosomes and collectively explain approximately 60% of the genotypic variation or 47% of the phenotypic variation in the LemontxTeqing cross. One of these resistance QTLs (QSbr4a), which accounted for 6% of the genotypic variation in resistance to R. solani, appeared to be independent of associated morphological traits. The remaining five putative resistance loci (QSbr2a, QSbr3a, QSbr8a, QSbr9a and QSbr12a) all mapped to chromosomal regions also associated with increased plant height, three of which were also associated with QTLs causing later heading. This was consistent with the observation that heading date and plant height accounted for 47% of the genotypic variation in resistance to R. solani in this population. There were also weak associations between resistance to R. solani and leaf width, which were likely due to linkage with a QTL for this trait rather than to a physiological relationship.     

Bacterization of peanut withPseudomonas fluorescens for biological control ofRhizoctonia solani and for enhanced yield

Severity of stem-rot disease of peanut caused byRhizoctonia solani was reduced by 54.9 and 68% in plants of two cultivars treated in the greenhouse with antagonistic strains ofPseudomonas fluorescens. These strains were selected based on theirin vitro toxicity to mycelial growth and sclerotial germination ofR. solani. In field experiments, bacterization of peanuts withP. fluorescens resulted in taller plants (by 25.7%) and increased yields (by 59.0%).     

Potential of Serratia marcescens strain B2 for biological control of rice sheath blight

Serratia marcescens strain B2 inhibited mycelial growth of the rice sheath blight pathogen Rhizoctonia solani AG-1 IA. Rice plants were treated with bacterial suspension and then challenge inoculated with the pathogen. Application of S. marcescens effectively reduced the incidence of sheath blight. S. marcescens survived in soil under glasshouse conditions at ca. 10⁸ colony forming units g^-1 of soil for 4 weeks after application. These results suggest that S. marcescens has potential as an effective and persistent biological control agent for rice sheath blight.     

Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24

Pseudomonas fluorescens 2P24 is a biocontrol agent isolated from a wheat take-all decline soil in China. This strain produces several antifungal compounds, such as 2,4-diacetylphloroglucinol (2,4-DAPG), hydrogen cyanide and siderophore(s). Our recent work revealed that strain 2P24 employs a quorum-sensing system to regulate its biocontrol activity. In this study, we identified a quorum-sensing system consisting of PcoR and PcoI of the LuxR–LuxI family from strain 2P24. Deletion of pcoI from 2P24 abolishes the production of the quorum-sensing signals, but does not detectably affect the production of antifungal metabolites. However, the mutant is significantly defective in biofilm formation, colonization on wheat rhizosphere and biocontrol ability against wheat take-all, whilst complementation of pcoI restores the biocontrol activity to the wild-type level. Our data indicate that quorum sensing is involved in regulation of biocontrol activity in P. fluorescens 2P24.     

Greenhouse and field trials of the bacterial antagonists in pelletformulations to suppress sheath blight of rice caused by Rhizoctoniasolani

Bacterial formulations, produced using both Bacillus megaterium andB. pumilus individually with pharmaceutical technology, were testedunder both greenhouse and field conditions. In the greenhouse testing,some bacterial formulations, for instance For 7 minus Lac and For 16 minusLac, performed as well as freshly prepared bacterial antagonists insuppress sheath blight disease. In the field testing, For 16 minus Lac wasnot effective in suppressing sheath blight development. Failure of the For16 minus Lac to suppress sheath blight disease in the field trial may be dueto the dilution and inactivation of antibiotics produced by B.megaterium in the aquatic environment in the rice field and climaticconditions during the formulation application.     

Water-soluble granules containing <Emphasis Type="Italic">Bacillus megaterium</Emphasis> for biological control of rice sheath blight: formulation,bacterial viability and efficacy testing

Endospores of B. megaterium were formulated in granule formulations with sodium alginate, lactose and polyvinylpyrrolidone (PVP K-30) by the wet granulation technique. The granule formulation exhibited good physical characteristics, such as high-water solubility and optimal viscosity, that would be suitable for spray application. The bacteria remained viable in the dry granule formulation at 10⁹ c.f.u./g after 24 months storage at room temperature. Under laboratory conditions, aqueous solutions of the formulation showed high activity against mycelial growth of R. solani (99.64 ± 0.14% mycelial inhibition). High viability of the bacterial antagonist on leaf sheath and leaf blade at day 7 after spraying with the formulation was observed (approximately 10⁶ c.f.u./g of plant). Application of an equivalent number of un-formulated endospores resulted in much loss of the bacterial endospores even 1 day after application. In a small pilot field study, an aqueous solution of the formulation (3%w/v) applied by spraying at days 1, 5 and 10 after pathogen inoculation of the rice plants was more effective in suppressing rice sheath blight disease than one application of a fungicide (Iprodione) at day 1. Additionally, rice plants sprayed with the aqueous solution of the granule formulation had higher panicle and whole kernel weights than those of fungicide-treated and control (untreated) plants.     

Dispersal of rice sheath blight fungus,Rhizoctonia solani AG-1(IA), and subsequent disease development in paddy fields, from survey of vegetative compatibility groups

The rice sheath blight fungus,Rhizoctonia solani AG-1(IA), was isolated from surface soil and stubble in the pretrans-planting season (early March) and from sheath blight lesions on plants at the maturing stage (late September) in two paddy fields (TW and TM:8 a). Isolates were classified into vegetative compatibility groups (VCG) based on the formation of a barrage zone (or interaction zone) at the colony interface between isolates on paired cultures. In field TW surveyed in 1991 and 1992, 128–142 isolates obtained in the pretransplanting season and 82–85 isolates from lesions on mature plants were classified into 27–29 and 28–30 VCGs, respectively. Seven to 10 VCGs (26–35%) were common between both samples, and of 30 VCGs from mature plants 12 (40%) survived the winter 1991/1992 and recovered from stubble and/or soil. In field TM in 1992, 111 isolates in the pretransplanting season and 99 from mature plants were divided into 18 and 14 VCGs, respectively. Seven (39%) out of 18 VCGs in the pretransplanting season were recovered again from mature plants. Occurence of common VCGs throughout the year indicated that ca. one third of VCGs found in the pretransplanting season caused sheath blight on rice plants about 6 mo later in the same field. Further, it was shown that about the same rate of VCGs which had caused disease overwintered on stubble and/or surface soil until the following pretransplanting season.     

Potential for the integration of biological and chemical control of sheath blight disease caused by Rhizoctonia solani on rice

Biological control using antagonistic microbes to minimize the use of chemical pesticides has recently become more prevalent. In an attempt to find an integrated control system for sheath blight, caused by Rhizoctonia solani in rice, Streptomyces philanthi RM-1-138, commercial formulations of Bacillus subtilis as Larminar^® and B. subtilis strain NSRS 89-24+MK-007 as Biobest^® and chemical fungicides including carbendazim^®, validamycin^®, propiconazole^® and mancozeb^® were applied alone and in combination with S. philanthi RM-1-138. In vitro experiments showed that all treatments tested did provide some control against mycelial growth and sclerotia production by R. solani PTRRS-9. In addition, the four chemical fungicides had no detrimental effects on S. philanthi RM-1-138 even at high concentrations (up to 100 μg/ml). The efficacy of S. philanthi RM-1-138, the commercial formulations of B. subtilis, chemical fungicides alone or in combination with S. philanthi RM-1-138 was also tested in a greenhouse experiment against sheath blight disease on rice plants. All treatments showed some protection of rice for sheath blight by 47–60 % when carbendazim^® was applied alone and up to 74 % when combined with S. philanthi RM-1-138.     

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.     

水稻纹枯病菌多聚半乳糖醛酸酶基因RsPG5的克隆与功能分析

【背景】纹枯病是水稻最重要的病害之一,目前对其病菌致病机制的研究还较少。【目的】鉴定更多水稻纹枯病菌致病基因,为纹枯病的防治提供理论依据。【方法】采用3''-RACE方法获得RsPG5基因全长,并使用ExPASy等在线软件对其编码产物的结构及生物学特性进行分析,测定其编码产物的致病功能。【结果】RsPG5具有7个外显子和6个内含子,编码区全长1 263 bp,可编码420个氨基酸。编码产物为糖苷水解酶GH28家族成员,具有真菌多聚半乳糖醛酸酶特有的保守序列NTD、DD、GHG和RF(I)K,并且有一个含15个氨基酸的信号肽;二级结构由α-螺旋、β-折叠和随机卷曲螺旋构成,并且可形成4个二硫键;三级结构为由α-螺旋、β-折叠和随机卷曲螺旋按右手螺旋规则形成的具有裂隙区的特定空间结构,裂隙区可能负责着其酶活功能。生物学性质预测表明,RsPG5为稳定、易溶于水的外泌性蛋白,主要定位于细胞壁、液泡和线粒体。RsPG5具有明显的多聚半乳糖醛酸酶活性,可分解果胶,破坏水稻叶鞘细胞;针刺接种分蘖末期水稻叶鞘,72 h后可形成明显的褐色坏死斑;将纹枯病菌接种至水稻叶鞘,在病菌致病过程中RsPG5可上调表达。【结论】RsPG5是一个典型的多聚半乳糖醛酸酶蛋白,为水稻纹枯病菌的重要致病因子。     

Grouping of isolates in AG 2 ofRhizoctonia solani by total cellular fatty acid analysis

Total-cellular fatty acid compositions of 34 isolates ofRhizoctonia solani belonging to intraspecific groups (ISGs) of anastomosis group (AG) 2, i.e., AG 2-1, AG 2-2 IIIB (mat rush), AG 2-2 IV (sugar beet), AG 2-2 LP (turfgrass), and AG 2–3 (soybean), were compared. The major fatty acids identified were palmitic, stearic, and oleic acids. Principal component analysis based on the percentage composition of total cellular fatty acids revealed consistently low variability among isolates of a single ISG of AG 2. Average linkage cluster analysis showed that isolates obtained from turfgrass representing a newly proposed group, AG 2-2 LP, were differentiated from other AG 2 ISGs. Isolates of another newly proposed group AG 2–3, from diseased soybean were also closely related to AG 2-1 and AG 2-2 IIIB but distinguishable from the AG 2-1 and AG 2-2 LP isolates by the average linkage cluster analysis. These results suggested that the percentage composition of total-cellular fatty acids is a distinct characteristic for the five ISGs belonging to AG 2, and fatty acid analysis is useful for the differentiation and characterization of these ISGs of AG 2 inR. solani.     

Effect of peat-bran inoculum ofTrichoderma species on biological control ofRhizoctonia solani in lettuce

A range of known biocontrol or plant growth-stimulating species ofTrichoderma orGliocladium were grown on peat-bran substrate to yield between 5×10⁷–3×10¹⁰ colony forming units (cfu's)g^–1 substrate after 14 days growth. Inocula were incorporated into peat:sand potting compost infested withRhizoctonia solani to give 7–8 × 10⁴ cfu's of antagonist g^–1 compost and assessed for biological control activity using lettuce seedlings. Six of the eight antagonists decreased daming-off and three of these consistently increased yield in comparison withR. solani treatment alone.Subsequently, peat-bran inoculum ofT. harzianum isolate TH1 was incorporated at 0.5% w/v intoR. solani infested potting compost. Both autoclaved and nonautoclaved inoculum ofT. harzianum TH1 decreased disease and increased yield. Incorporation of ethyl acetate-extracted autoclaved inoculum ofT. harzianum TH1 resulted in similar levels of biocontrol and improved plant growth as did incorporation of nonautoclaved and autoclavedT. harzianum TH1 inoculum. The need to standardize inocula and controls is emphasized.     

Control of blast and sheath blight diseases of rice using antifungal metabolites produced by Streptomyces sp. PM5

Two antifungal aliphatic compounds, SPM5C-1 and SPM5C-2 with a lactone and ketone carbonyl unit, respectively obtained from Streptomyces sp. PM5 were evaluated under in vitro and in vivo conditions against major rice pathogens, Pyricularia oryzae and Rhizoctonia solani. These compounds were dissolved in distilled water/medium to get the required concentrations. The well diffusion bioassay indicated that the of SPM5C-1 remarkably inhibited the mycelial growth of P. oryzae and R. solani in comparison to SPM5C-2. Though SPM5C-2 showed low antifungal activity against P. oryzae, it was not active against R. solani. Further, SPM5C-1 completely inhibited the growth of P. oryzae and R. solani at concentrations of 25, 50, 75 and 100 μg/ml. Greenhouse experiments revealed that spraying of SPM5C-1 at 500 μg/ml on rice significantly decreased blast and sheath blight development by 76.1% and 82.3%, respectively, as compared to the control with a corresponding increase in rice grain yield.     

Induction of enzymatic scavengers of active oxygen species in rice in response to infection by <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Changes in the activities of peroxidase, ascorbate peroxidase, catalase and superoxide dismutase in rice in response to infection by Rhizoctonia solani were studied. A significant increase in peroxidase activity was observed in R. solani-inoculated rice leaf sheaths 1 day after inoculation and the maximum enzyme activity was recorded 3 days after inoculation at which period a 3-fold increase in peroxidase activity was observed compared to the untreated control. Three peroxidase isozymes viz., PO-4, PO-5 and PO-6 were induced in rice upon infection by R. solani. Ascorbate peroxidase and catalase activities significantly increased 1–2 days after inoculation and the maximum enzyme activities were recorded 5 days after inoculation. Superoxide dismutase activity increased significantly 2 days after inoculation and increased progressively, reaching four times the control value at 7 days after inoculation.     

A new bio-formulation containing plant growth promoting rhizobacterial mixture for the management of sheath blight and enhanced grain yield in rice

Three plant growth promoting rhizobacterial (PGPR) strains, PF1,FP7 and PB2, were tested alone and in combinations for suppression ofrice sheath blight disease and promotion of plant growth underglasshouse and field conditions. The mixture of PGPR strainssignificantly reduced the sheath blight incidence when applied as eitherbacterial suspension through seed, root, foliar and soil application inglasshouse conditions, or as talc-based formulation under fieldconditions, compared to the respective individual strains. The averagemean of disease reduction was 29.2% for single strains and45.1% for mixtures. In addition to disease suppression, treatmentwith mixture of PGPR strains promoted plant growth in terms of increasedplant height and number of tillers, and ultimately grain yield. Theaverage increases in yield for single strains were 17.7%, and25.9% in case of mixture. Mixture of three PGPR strains reduceddisease and promoted growth to a level equivalent to two strainmixtures. Though seed treatment of either single strain or strainmixtures alone could reduce the disease, subsequent application to root,leaves or soil further reduced the disease and enhanced the plantgrowth. The mixture consisting of PF1 plus FP7 was the most effective inreducing the disease and in promoting plant growth and grainyield.     

Fusarium head blight biological control with Lysobacter enzymogenes strain C3

Fusarium head blight (FHB), caused by Fusarium graminearum (= Gibberella zeae), is a destructive disease of wheat for which biological controls are needed. Lysobacter enzymogenes strain C3, a bacterial antagonist of fungal pathogens via lytic enzymes and induced resistance, was evaluated in this study for control of FHB. In greenhouse experiments, chitin broth cultures of C3 reduced FHB severity to <10% infected spikelets as compared to >80% severity in the controls in some experiments. C3 broth cultures heated to inactivate cells and lytic enzymes, but retaining the elicitor factor for induced resistance, also were effective in reducing FHB severity, suggesting induced resistance is one mechanism of action. C3 broth cultures also were effective when applied in highly diluted form and when applied 1 week prior to pathogen inoculation. When applied to 8 cultivars of hard red spring wheat in the greenhouse, C3 treatments reduced FHB in 5 cultivars but not in the others. These findings also are consistent with induced resistance. Protection offered by C3 treatments, however, was not systemic and required that C3 be applied uniformly to all susceptible florets. Field tests were conducted in South Dakota and Nebraska to evaluate the efficacy of C3 chitin broth cultures in spring and winter wheat, respectively. In experiments involving two hard red spring wheat cultivars, treatment with C3 reduced FHB severity in ‘Russ’ but not in ‘Ingot’. In three other field experiments comparing C3, the fungicide tebuconazole, and the combination of C3 and tebuconazole, treatments with the bacterial culture alone and the fungicide alone were inconsistent across experiments, each treatment being ineffective in controlling FHB in one experiment. The biocontrol agent–fungicide combination was more consistently effective, reducing FHB incidence or severity in all three experiments. Thus, the potential for using L. enzymogenes C3 as a biological control agent for FHB was demonstrated along with a number of factors that might affect control efficacy in the field.     

Biological control of rice bacterial blight by Lysobacter antibioticus strain 13-1

Bacterial leaf blight (BB) is a worldwide destructive rice disease caused by pathogen Xanthomonas oryzae pv. oryzae (Xoo). A novel strain of Lysobacter antibioticus, which was isolated from the rhizosphere of rice in Yunnan Province of China, can significantly inhibit the growth of various phytopathogenic bacteria and fungi, especially BB pathogen Xoo. In greenhouse experiments, whole bacterial broth culture (WBC) of strain 13-1 was more effective in reducing BB than other components of the culture, with disease suppression efficiency up to 69.7%. However, bacterial cells re-suspended in water, cell-free culture extracts, and heated cultures also significantly reduced BB severity. Suppression efficiencies ranged from 79.0% to 61.8% for undiluted to 100-fold dilution treatments and from 57.6% to 31.7% when the WBC of strain 13-1 (10⁸ CFU/mL) was applied at 3 days and 7 days prior to pathogen inoculation, respectively. In three field trials, strain 13-1 reduced BB incidence by 73.5%, 78.3%, and 59.1%, respectively. Disease suppression by strain 13-1 varied significantly among different rice cultivars, although efficacy was not directly related to the susceptibility level of the cultivars. Efficacy of biocontrol was also affected by different pathogen isolates, with some isolates of Xoo being more sensitive to 13-1 suppression than others. These results suggest that antibiotics and density of colonization on leaves may be involved for biological control of rice BB by strain 13-1. To our knowledge, this is the first report of L. antibioticus being a potential biocontrol agent for rice bacterial blight.     

Generation of selectable marker-free sheath blight resistant transgenic rice plants by efficient co-transformation of a cointegrate vector T-DNA and a binary vector T-DNA in one Agrobacterium tumefaciens strain

Co-transformation of Oryza sativa L. var. Pusa Basmati1 was done using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector and a multi-copy binary vector in the same cell. The T-DNA of the cointegrate vector pGV2260::pSSJ1 carried the hygromycin phosphotransferase (hph) and beta-glucuronidase (gus) genes. The binary vector pCam-chi11, without a plant selectable marker gene, harboured the rice chitinase (chi11) gene under maize ubiquitin promoter. Co-transformation of the gene of interest (chi11) with the selectable marker gene (hph) occurred in 4 out of 20 T(0) plants (20%). Segregation of hph from chi11 was accomplished in two (CoT6 and CoT23) of the four co-transformed plants in the T(1) generation. The selectable marker-free (SMF) lines CoT6 and CoT23 harboured single copies of chi11. Homozygous SMF T(2) plants were established in the lines CoT6 and CoT23. Northern and Western blot analysis of the homozygous SMF lines showed high level of transgene expression. In comparison to untransformed controls, chitinase specific activity was 66- and 22-fold higher in the homozygous SMF T(2) plants of lines CoT6 and CoT23, respectively. The lines CoT6 and CoT23 exhibited 38 and 40% reduction in sheath blight disease, respectively.