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.     

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.     

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.     

Development of a spore-based formulation of microbial pesticides for control of rice sheath blight

An effective formulated biopesticide for controlling sheath blight in rice was developed using three microbial antagonists (Bacillus megaterium, Bacillus subtilis and Aspergillus niger) isolated from the rice sheath. The efficiency of spore-based formulations of the above microbial antagonists was investigated and their effectiveness in controlling sheath blight was demonstrated. Application of talc-based formulations of individual antagonists and mixtures of the three antagonists as spray treatments or soil applications were effective in reducing the incidence by up to 45% at 27 days after inoculation of the pathogen of sheath blight and increased rice yield. The use of spores of a fungal antagonist (A. niger), in comparison to commonly used bacterial antagonists, is a novel feature of the present study. Optimum sporulation conditions of the antagonists for preparation of spore-based formulations and their commercially desirable features such as the ability to maintain spore viability in storage were also determined. Culturing in the synthetic replacement sporulation medium (SRSM-2) for 72 hours was the most effective for sporulation of the two bacterial antagonists while culturing in potato dextrose broth (PDB) for 7 days was the most effective for sporulation of the fungal antagonist. It was demonstrated that talc-based formulations of all antagonists, either in refrigerated storage (4°C) or at room temperature (28±2°C), were able to maintain greater spore viability over a longer period (>6 months) than spore suspensions. In view of the relatively shorter life spans of formulations based on vegetative cells, spore-based formulations have a distinct advantage in achieving longer-lasting control, especially under harsh field conditions.     

Identification of rice sheath blight and blast quantitative trait loci in two different O. sativa/O. nivara advanced backcross populations

Two accessions of Oryza nivara, a wild ancestral species of rice (O. sativa) identified as being moderately resistant to sheath blight and leaf blast disease, were used as donor parents to develop two advanced backcross populations with the US rice cultivar, Bengal, as the recurrent parent. The O. nivara donor parent for Wild-1 (252 BC2F1 lines) was acc. IRGC100898 and for Wild-2 (253 BC2F1 lines) was acc. IRGC104705. Both populations were genotyped with 131 simple sequence repeat markers and the linkage maps covered 1,567.5 cM (Wild-1) and 1,312.2 cM (Wild-2). Sheath blight (ShB) disease was evaluated in both inoculated greenhouse and field conditions. Days to heading (DH), plant height (PH), and plant type (PT), confounding factors for sheath blight disease under field conditions, were recorded. Leaf blast disease was rated under inoculated greenhouse conditions. Multiple interval mapping identified qShB6 with resistance to sheath blight disease attributed to the O. nivara parent in the greenhouse. In the field, qShB6 also was the most significant ShB quantitative trait locus (QTL) in all trials, with resistance attributed to O. nivara. In addition, qShB1 and qShB3 were identified in all trials but were not always attributed to the same parent. The qShB6 QTL is in the same region as the DH-QTL, qDH6, and qShB1 is in the same region as the major PH-QTL, qPH1, suggesting that these ShB-QTL may be confounded by other traits. Although qShB3 did not have as large an effect as other loci, it was not confounded by either DH or PH. For leaf blast, qBLAST8-1 was found in both populations providing resistance to races IB1 and IB49, whereas qBLAST12 providing resistance to both races, was only found in Wild-2. Resistance was attributed to O. nivara for both QTL, and blast resistance genes have been previously reported in these regions.     

Two-helper-strain co-culture system: a novel method for enhancement of 2-keto-l-gulonic acid production

A novel two-helper-strain co-culture system (TSCS) was developed to enhance 2-keto-l-gulonic acid (2-KLG) productivity for vitamin C production. Bacillus megaterium and B. cereus (with a seeding culture ratio of 1:3, v/v), used as helper strains, increased the 2-KLG yield using Ketogulonigenium vulgare compared to the conventional one-helper-strain (either B. cereus or B. megaterium) co-culture system (OSCS). After 45 h cultivation, 2-KLG concentration in the TSCS (69 g l^?1) increased by 8.9 and 7 % over that of the OSCS (B. cereus: 63.4 g l^?1; B. megaterium: 64.5 g l^?1). The fermentation period of TSCS was 4 h shorter than that of OSCS (B. cereus). The increased cell numbers of K. vulgare stimulated by the two helper strains possibly explain the enhanced 2-KLG yield. The results imply that TSCS is a viable method for enhancing industrial production of 2-KLG.     

Factors affecting antifungal activity of Streptomyces philanthi RM-1-138 against Rhizoctonia solani

Sheath blight disease of rice caused by Rhizoctonia solani Kühn is economically important disease in most of the world’s rice growing areas. The disease causes severe yield losses of >20 % of rice in Thailand. Our previous investigation reported the antifungal activity of Streptomyces philanthi RM-1-138 against R. solani PTRRC-9. In this study, glucose yeast-malt extract medium, initial pH of 7.5 and a temperature of 30 °C were found to be optimum for both cell growth and antifungal activity of S. philanthi RM-1-138. The inhibition of 94 and 100 % on the growth of R. solani PTRRC-9 were achieved from the antifungal metabolites of the 6 and 9-days-old culture filtrates of S. philanthi RM-1-138, respectively. Heat treatment on the culture filtrate had slight effect on its antifungal activity. The culture broth demonstrated higher antifungal activity on growth of R. solani PTRRC-9 (90.4 %) than the culture filtrate (31.5 %) and its effective dose was at 0.1 % (v/v). The present results indicated the possibilities of using either the culture broth or culture filtrate of S. philanthi RM-1-138 to inhibit growth of R. solani PTRRC-9.     

三种药剂对早稻二化螟的防效及施药适期探讨

试验结果表明,氯虫苯甲酰胺、氯虫·噻虫嗪有对二化螟Chilo suppressalis(Walker)有较好的防效和较长的持效,一次施药可兼治多个峰次。20%氯虫苯甲酰胺150mL/hm2、40%氯虫·噻虫嗪120g/hm2和2.15%甲氨基阿维菌素苯甲酸盐750mL/hm2在二化螟2龄幼虫高峰期施药,药后8d对幼虫防效分别达94.7%、91.2%和91.0%;药后18d的防效依次为99.0%、98.0%、73.2%,氯虫苯甲酰胺、氯虫·噻虫嗪处理防效仍高于药后8d,但甲氨基阿维菌素苯甲酸盐处理防效明显下降;药后18d对主峰保苗效果(枯心)氯虫苯甲酰胺和氯虫·噻虫嗪分别为99.0%、98.0%,甲氨基阿维菌素苯甲酸盐处理偏低,为73.2%。对后峰保苗效果(枯鞘)氯虫苯甲酰胺为84.9%好于氯虫·噻虫嗪处理的69.3%,甲氨基阿维菌素苯甲酸盐的防效仅为7.1%。氯虫·噻虫嗪不同时期施药处理后,主峰卵孵高峰后14d考查,防治效果和保苗效果以卵孵高峰施药最好,其次为2龄幼虫高峰期施药;主峰卵孵高峰后24d考查,主峰卵孵高峰至3龄幼虫高峰施药对主峰均有较好的防效,但对后峰的防效,随着施药时间推迟而提高,但在螟虫重发地区施药过迟,前期的大量枯鞘会影响稻苗生长与分蘖。因此作者建议在二化螟发生量大、后峰数量多地区,采用主峰卵孵高峰和后峰卵孵高峰2次防治对策,在螟虫发生较轻、后峰数量较少或全代发生较为集中地区,可根据当地虫情发生情况,适当推迟施药,达到一次用药,解决全代螟害。甲氨基阿维菌素苯甲酸盐持效期较短,可根据螟虫发生峰次施用。     

Characterisation,screening and selection of Bacillus subtilis isolates for its biocontrol efficiency against major rice diseases

A Soil bacterium, Bacillus subtilis, isolated from the rhizosphere of rice, showed high biocontrol activity against blast, sheath blight and bacterial blight. In the present study, four B. subtilis strains isolated from paddy soil were studied under laboratory, greenhouse and field conditions. Among the four strains assayed, UASP17 gave maximum inhibition of paddy pathogens and was validated under field trials. B. subtilis (UASP17) under different doses and methods of applications was evaluated for two seasons at Agriculture Research Station (UAS, Raichur). UASP17 was effective in reducing the severity of blast (9.00% and 15.57%), bacterial leaf blight (BLB) (5.00% and 6.11%) and sheath blight (11.93% and 4.17%) diseases for two seasons. The application of bioagent also increased the paddy grain yield (61.00 and 64.30?Q/ha) in the two seasons, respectively. Taken together, these results indicate that B. subtilis UASP17 as seedling dip for 30?min (10?mL/L of water) prior to transplanting and 2.50?L/ha foliar application was effective in managing the diseases of paddy.     

Characterization of a novel plant growth-promoting bacteria strain Delftia tsuruhatensis HR4 both as a diazotroph and a potential biocontrol agent against various plant pathogens

A novel, plant growth-promoting bacterium Delftia tsuruhatensis, strain HR4, was isolated from the rhizoplane of rice (Oryza sativa L., cv. Yueguang) in North China. In vitro antagonistic assay showed this strain could suppress the growth of various plant pathogens effectively, especially the three main rice pathogens (Xanthomonas oryzae pv. oryzae, Rhizoctonia solani and Pyricularia oryzae Cavara). Treated with strain HR4 culture, rice blast, rice bacterial blight and rice sheath blight for cv. Yuefu and cv. Nonghu 6 were evidently controlled in the greenhouse. Strain HR4 also showed a high nitrogen-fixing activity in N-free Döbereiner culture medium. The acetylene reduction activity and ¹⁵N₂-fixing activity (N₂FA) were 13.06 C₂H₄ nmol ml⁻¹ h⁻¹ and 2.052 ¹⁵Na.e.%, respectively. The nif gene was located in the chromosome of this strain. Based on phenotypic, physiological, biochemical and phylogenetic studies, strain HR4 could be classified as a member of D. tsuruhatensis. However, comparisons of characteristics with other known species of the genus Delftia suggested that strain HR4 was a novel dizotrophic PGPB strain.     

Identification and characterization of rhizosphere fungal strain MF‐91 antagonistic to rice blast and sheath blight pathogens

Aim

To examine the inhibition effects of rhizosphere fungal strain MF‐91 on the rice blast pathogen Magnaporthe grisea and sheath blight pathogen Rhizoctonia solani.

Methods and Results

Rhizosphere fungal strain MF‐91 and its metabolites suppressed the in vitro mycelial growth of R. solani. The inhibitory effect of the metabolites was affected by incubation temperature, lighting time, initial pH and incubation time of rhizosphere fungal strain MF‐91. The in vitro mycelial growth of M. grisea was insignificantly inhibited by rhizosphere fungal strain MF‐91 and its metabolites. The metabolites of rhizosphere fungal strain MF‐91 significantly inhibited the conidial germination and appressorium formation of M. grisea. Moreover, the metabolites reduced the disease index of rice sheath blight by 35·02% in a greenhouse and 57·81% in a field as well as reduced the disease index of rice blast by 66·07% in a field. Rhizosphere fungal strain MF‐91 was identified as Chaetomium aureum based on the morphological observation, the analysis of 18S ribosomal DNA internal transcribed spacer sequence and its physiological characteristics, such as the optimal medium, temperature and initial pH for mycelial growth and sporulation production.

Conclusions

Rhizosphere fungus C. aureum is effective in the biocontrolling of rice blast pathogen M. grisea and sheath blight pathogen R. solani both in in vitro and in vivo conditions.

Significance and Impact of the Study

This study is the first to show that rhizosphere fungus C. aureum is a potential fungicide against rice blast and sheath blight pathogens.     

利用纤维二糖差向异构酶制备乳果糖的研究进展

乳果糖是由D-半乳糖和D-果糖两个基团通过β-1,4糖苷键连接而成的还原型二糖;乳果糖口服液具有治疗慢性便秘和肝性脑病的功效,在100多个国家作为常见非处方药(OTC)使用,需求量十分巨大;乳果糖还可以作为益生元改善人体肠道菌群关系。乳果糖的生产依赖化学法,其催化剂对人体有害,下游分离难度大。近年来,纤维二糖差向异构酶被发现能够高效催化乳糖制备乳果糖,该技术绿色环保、步骤简单,具有很强的产业化前景。本文结合自身研究经历对纤维二糖差向异构酶的研发情况进行总结,并综述了乳果糖酶法制备技术的现状。     

Identification of major quantitative trait loci qSBR11-1 for sheath blight resistance in rice

Sheath blight caused by Rhizoctonia solani Kühn is one of the important diseases of rice, resulting in heavy yield loss in rice every year. No rice line resistant to sheath blight has been identified till date. However, in some rice lines a high degree of resistance to R. solani has been observed. An indica rice line, Tetep, is a well documented source of durable and broad spectrum resistance to rice blast as well as quantitative resistance to sheath blight. The present study identified genetic loci for quantitative resistance to sheath blight in rice line Tetep. A mapping population consisting of 127 recombinant inbred lines derived from a cross between rice cultivars HP2216 (susceptible) and Tetep (resistant to sheath blight) was evaluated for sheath blight resistance and other agronomic traits for 4 years across three locations. Based on sheath blight phenotypes and genetic map with 126 evenly distributed molecular markers, a quantitative trait loci (QTLs) contributing to sheath blight resistance was identified on long arm of chromosome 11. Two QTL mapping approaches i.e., single marker analysis and composite interval mapping in multi environments were used to identify QTLs for sheath blight resistance and agronomical traits. The QTL qSBR11-1 for sheath blight resistance was identified between the marker interval RM1233 (26.45 Mb) to sbq33 (28.35 Mb) on chromosome 11. This region was further narrowed down to marker interval K39516 to sbq33 (~0.85 Mb) and a total of 154 genes were predicted including 11 tandem repeats of chitinase genes which may be responsible for sheath blight resistance in rice line Tetep. A set of 96 varieties and a F₂ population were used for validation of markers linked to the QTL region. The results indicate that there is very high genetic variation among varieties at this locus, which can serve as a starting point for allele mining of sheath blight resistance.     

Baseline sensitivity and efficacy of thifluzamide in Rhizoctonia solani

Thifluzamide is a SDHI (succinate dehydrogenase inhibitor) fungicide, which interferes with succinate ubiquinone reductase in the mitochondrial electron transport chain of fungi. Presently, jinggangmycin is the major fungicide extensively used for the control of rice sheath blight caused by Rhizoctonia solani and resistance to jinggangmycin was first reported to occur in China. A total of 128 isolates of R. solani from Anhui Province of China were characterised for the baseline sensitivity to thifluzamide. The isolates were very sensitive to thifluzamide and the baseline sensitivity curve was unimodal with an average EC₅₀ value of 0.058 ± 0.012 µg mL^?1. However, EC₅₀ values of boscalid (another SDHI fungicide) for inhibition of mycelial growth of 22 arbitrarily selected R. solani isolates ranged from 1.89 to 2.68 µg mL^?1. Thifluzamide applied at 110 µg mL^?1 exhibited excellent protective and curative activity against rice sheath blight and provided 81.1–91.0% protective or curative control efficacy. In field trials in 2010 and 2011, control efficacies of thifluzamide at 82 g.a.i ha^?1 15 and 30 days after second application were 84.2% and 86.7%, respectively, suggesting excellent activity against sheath blight. There was a statistically significant difference in the efficacy between thifluzamide and boscalid or jinggangmycin. These results suggested that thifluzamide should be a good alternative fungicide to jinggangmycin for the control of rice sheath blight.     

Suppression of wheat Fusarium head blight by novel amphiphilic aminoglycoside fungicide K20

K20 is a novel amphiphilic aminoglycoside capable of inhibiting many fungal species. K20's capabilities to inhibit Fusarium graminearum the causal agent wheat Fusarium head blight (FHB) and to this disease were examined. K20 inhibited the growth of F. graminearum (minimum inhibitory concentrations, 7.8–15.6 mg L^?1) and exhibited synergistic activity when combined with triazole and strobilurin fungicides. Application of K20 up to 720 mg L^?1 to wheat heads in the greenhouse showed no phytotoxic effects. Spraying wheat heads in the greenhouse with K20 alone at 360 mg L^?1 lowered FHB severity below controls while combining K20 with half–label rates of Headline (pyraclostrobin) improved its disease control efficacy. In field trials, spraying K20 at 180 mg L^?1 and 360 mg L^?1 combined with half-label rates of Headline, Proline 480 SC (prothioconazole), Prosaro 421 SC (prothioconazole + tebuconazole), and Caramba (metconazole) reduced FHB indices synergistically. In addition, the K20 plus Proline 480 SC combination reduced levels of the mycotoxin deoxinivalenol by 75 % compared to the control. These data suggest that K20 may be useful as a fungicide against plant diseases such as FHB particularly when combined with commercial fungicides applied at below recommended rates.     

Antagonistic potential of native strain Streptomyces aurantiogriseus VSMGT1014 against sheath blight of rice disease

A total of 132 actinomycetes was isolated from different rice rhizosphere soils of Tamil Nadu, India, among which 57 showed antagonistic activity towards Rhizoctonia solani, which is sheath blight (ShB) pathogen of rice and other fungal pathogens such as Macrophomina phaseolina, Fusarium oxysporum, Fusarium udum and Alternaria alternata with a variable zone of inhibition. Potential actinomycete strain VSMGT1014 was identified as Streptomyces aurantiogriseus VSMGT1014 based on the morphological, physiological, biochemical and 16S rRNA sequence analysis. The strain VSMGT1014 produced lytic enzymes, secondary metabolites, siderophore, volatile substance and indole acetic acid. Crude metabolites of VSMGT1014 showed activity against R. solani at 5 µg ml^?1; however, the prominent inhibition zone was observed from 40 to 100 µg ml^?1. Reduced lesion heights observed in culture, cells-free filtrate, crude metabolites and carbendazim on challenge with pathogen in the detached leaf assay. The high content screening test clearly indicated denucleation of R. solani at 5 µg ml^?1 treatment of crude metabolite and carbendazim respectively. The results conclude that strain VSMGT1014 was found to be a potential candidate for the control of ShB of rice as a bio fungicide.     

水稻稻瘟病和纹枯病抗性鉴定方法

水稻(Oryza sativa)是世界上最重要的粮食作物, 但稻瘟病和纹枯病等病害严重危害水稻的产量和品质, 给我国乃至全球粮食安全带来巨大威胁。鉴定水稻抗病资源、克隆抗病基因、揭示抗性机理并在育种中加以利用, 对抵御水稻病害和保障粮食安全具有十分重要的作用。准确评价水稻资源的抗病性, 是开展抗病机理研究和育种生产应用的关键环节。该文详述了水稻幼苗期人工喷雾接种、分蘖期和孕穗期田间注射接种与离体叶片戳伤接种的稻瘟病抗性鉴定方法, 以及水稻分蘖期田间接种、孕穗期温室接种和离体茎秆接种的纹枯病抗性鉴定方法, 以期为同行鉴定水稻资源、开展抗病理论和应用研究提供参考。     

Isolation and Characterization of a Nitrile-Hydrolysing Bacterium Isoptericola variabilis RGT01

A nitrile-hydrolysing bacterium, identified as Isoptericola variabilis RGT01, was isolated from industrial effluent through enrichment culture technique using acrylonitrile as the carbon source. Whole cells of this microorganism exhibited a broad range of nitrile-hydrolysing activity as they hydrolysed five aliphatic nitriles (acetonitrile, acrylonitrile, propionitrile, butyronitrile and valeronitrile), two aromatic nitriles (benzonitrile and m-Tolunitrile) and two arylacetonitriles (4-Methoxyphenyl acetonitrile and phenoxyacetonitrile). The nitrile-hydrolysing activity was inducible in nature and acetonitrile proved to be the most efficient inducer. Minimal salt medium supplemented with 50 mM acetonitrile, an incubation temperature of 30 °C with 2 % v/v inoculum, at 200 rpm and incubation of 48 h were found to be the optimal conditions for maximum production (2.64 ± 0.12 U/mg) of nitrile-hydrolysing activity. This activity was stable at 30 °C as it retained around 86 % activity after 4 h at this temperature, but was thermolabile with a half-life of 120 min and 45 min at 40 °C and 50 °C respectively.