水稻内生放线菌OsiRt-1的分离鉴定及对稻瘟病的防治作用

【目的】从水稻中分离、筛选并鉴定出对稻瘟病真菌具有拮抗性能的内生放线菌,对高抗菌株进行稻瘟病生防效果评定,初步探讨其作用机制。【方法】采用4种分离培养基对水稻内生放线菌进行分离,用平板对峙法筛选出对稻瘟病菌拮抗性能最好的菌株,结合其菌落形态、生理生化及16S rRNA基因序列分析进行鉴定;用环境扫描电镜(SEM)观察拮抗菌对稻瘟病菌菌丝形态影响,用菌丝生长速率法对拮抗菌发酵滤液进行抗菌活性评价;大田条件下,喷洒拮抗菌孢子液于水稻,检测其稻瘟病控制效果;同时,对拮抗菌进行产酶和次级代谢产物分析,检测其聚酮合酶基因(PKSⅡ型)和非核糖体多肽合成酶基因(NRPS)。【结果】从1 800个水稻样品中分离出117株内生放线菌,从中筛选出拮抗性能最好的菌株Osi Rt-1,鉴定为米修链霉菌Streptomyces misionensis。该菌株使稻瘟病菌菌丝出现畸形,其无菌滤液对病菌菌丝生长的抑制率为28.06%;大田条件下,Osi Rt-1对水稻苗瘟、穗瘟均有较好防效,其中对苗瘟防效为7.76%,穗瘟防效高达25.65%,损失率降低了17.46%。与之对应,Osi Rt-1处理过的水稻地上部分,Osi Rt-1所占内生放线菌的比例明显高于未处理水稻。该菌株具有可能降解真菌细胞壁的纤维素酶、蛋白酶活性,同时可产生植物生长促进剂铁载体、IAA、ACC脱氨酶。菌株Osi Rt-1呈现PKSⅡ型和NRPS阳性。【结论】菌株Osi Rt-1是一株具生防潜力的内生放线菌,在农业上具有实际研发价值。     

冷风干燥制备高活菌的恶臭假单胞菌菌粉

【目的】获得高活菌恶臭假单胞菌菌粉,提高菌体干燥及保藏存活率。【方法】选用冷风干燥法制备活菌粉,并优化吸附载体与保护剂。【结果】冷风干燥制备恶臭假单胞菌菌粉干燥存活率普遍达到65%以上,显著优于喷雾干燥(24%);对载体与保护剂进行正交试验优化,确定了载体为混合的硅藻土和碱处理玉米芯粉,混合比为1:2,保护剂(质量比)为甘露醇7%、谷氨酸钠5%、甘油1%,制得菌粉活菌数为1.03×1011 CFU/g,室温保藏30 d和4 °C保藏60 d存活率分别达到40.54%和71.67%。【结论】冷风干燥温度相对较低(10?40 °C),对菌体损伤小,碱处理玉米芯粉、甘露醇和谷氨酸钠是提高菌粉保藏存活率的重要因子,此法克服了革兰氏阴性菌菌粉不易制备和不耐保藏的瓶颈。     

Biological control agent of common scab disease by antagonistic strain Bacillus sp. sunhua

AIMS: To identify an antagonistic strain against Streptomyces scabiei and to characterize the antibiotic agent. The efficacy of the isolated strain in controlling common scab disease was also evaluated. METHODS AND RESULTS: A bacterial strain antagonistic against S. scabiei was isolated from the soil of a potato-cultivating area. This bacterium was identified as a Bacillus species by 16S rRNA gene sequence analysis and was designated Bacillus sp. sunhua. Antibiotics produced by this strain were proven to be stable within a broad pH range and at high temperatures. The culture broth was extracted with ethyl acetate, and then the crude extract was applied to HPLC. Two compounds were isolated and identified as iturin A and macrolactin A by 1H-NMR, 13C-NMR, HMBC, HMQC and mass spectrometer. The culture broth of Bacillus sp. sunhua had a suppressive effect on common scab disease in a pot assay, decreasing the infection rate from 75 to 35%. This strain also suppressed Fusarium oxysporum, the pathogen of potato dry rot disease. CONCLUSIONS: Bacillus sp. sunhua was shown to inhibit S. scabiei effectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report demonstrating that macrolactin A and iturin A inhibit S. scabiei. This study demonstrated the possibility of controlling potato scab disease using Bacillus sp. sunhua.     

Synthesis and characterization of hydroxyapatite-ciprofloxacin delivery systems by precipitation and spray drying technique

This investigation synthesized and characterized hydroxyapatite (HAP) microspheres, agglomerated microspheres, and implants containing ciprofloxacin. This delivery system is to be used as an implantable drug delivery system for the treatment of bone infections. The HAP microspheres were made by chemical precipitation followed by a spray-drying technique. Agglomerated microspheres were prepared by a wet granulation process using a granulator. Implants were prepared by direct compression of the granules on a Carver press. Ciprofloxacin was analyzed by high-performance liquid chromatography. Characterization of the HAP microspheres include particle size, size distribution, physical state of the drug in the microsphere, and microstructure of the drug delivery system before and after in vitro release. The particle size, porosity, and morphology of the microspheres were dependent on viscosity and concentration of the slurry as well as the atomization pressure used during spray drying. Even at the highest drug load (2% wt/wt), the drug was present in a noncrystalline state. The drug release from the agglomerated microspheres was quick and almost complete within 1 hour. However, compressing the same amount of agglomerated microspheres into an implant greatly reduced the rate of ciprofloxacin release. Only 12% (wt/wt) of the drug was released from the implant within 1 hour. The in vitro release of ciprofloxacin from these implants follows a diffusion-controlled mechanism. This method provides a unique way of producing various shapes and drug loads of HAP microspheres that can be easily manufactured on a commercial scale. Published: January 28, 2002.     

Enhanced resistance to the rice blast fungus Magnaporthe grisea conferred by expression of a cecropin A gene in transgenic rice

Cecropins are a family of antimicrobial peptides, which constitute an important key component of the immune response in insects. Here, we demonstrate that transgenic rice (Oryza sativa L.) plants expressing the cecropin A gene from the giant silk moth Hyalophora cecropia show enhanced resistance to Magnaporthe grisea, the causal agent of the rice blast disease. Two plant codon-optimized synthetic cecropin A genes, which were designed either to retain the cecropin A peptide in the endoplasmic reticulum, the ER-CecA gene, or to secrete cecropin A to the extracellular space, the Ap-CecA gene, were prepared. Both cecropin A genes were efficiently expressed in transgenic rice. The inhibitory activity of protein extracts prepared from leaves of cecropin A-expressing plants on the in vitro growth of M. grisea indicated that the cecropin A protein produced by the transgenic rice plants was biologically active. Whereas no effect on plant phenotype was observed in ER-CecA plants, most of the rice lines expressing the Ap-CecA gene were non-fertile. Cecropin A rice plants exhibited resistance to rice blast at various levels. Transgene expression of cecropin A genes was not accompanied by an induction of pathogenesis-related (PR) gene expression supporting that the transgene product itself is directly active against the pathogen. Taken together, the results presented in this study suggest that the cecropin A gene, when designed for retention of cecropin A into the endoplasmic reticulum, could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.     

QTL analysis and mapping of pi21, a recessive gene for field resistance to rice blast in Japanese upland rice

Field resistance is defined as the resistance that allows effective control of a parasite under natural field conditions and is durable when exposed to new races of that parasite. To identify the genes for field resistance to rice blast, quantitative trait loci (QTLs) conferring field resistance to rice blast in Japanese upland rice were detected and mapped using RFLP and SSR markers. QTL analysis was carried out in F₄ progeny lines from the cross between Nipponbare (moderately susceptible, lowland) and Owarihatamochi (resistant, upland). Two QTLs were detected on chromosome 4 and one QTL was detected on each of chromosomes 9 and 12. The phenotypic variation explained by each QTL ranged from 7.9 to 45.7% and the four QTLs explained 66.3% of the total phenotypic variation. Backcrossed progeny lines were developed to transfer the QTL with largest effect using the susceptible cultivar Aichiasahi as a recurrent parent. Among 82 F₃ lines derived from the backcross, resistance segregated in the expected ratio of resistant 1 : heterozygous 2 : susceptible 1. The average score for blast resistance measured in the field was 4.2 ± 0.67, 7.5 ± 0.51and 8.2 ± 0.66, for resistant, heterozygous and susceptible groups, respectively. The resistance gene, designated pi21, was mapped on chromosome 4 as a single recessive gene between RFLP marker loci G271 and G317 at a distance of 5.0 cM and 8.5 cM, respectively. The relationship to previously reported major genes and QTLs conferring resistance to blasts, and the significance of marker-assisted selection to improve field resistance, are discussed. Received: 8 June 2000 / Accepted: 24 November 2000     

云南地方稻种持久抗瘟性的研究

1996～1999年,在不同纬度、不同海拔和不同稻作生态类型的重病区设立5个持久抗性稻瘟病鉴定圃.试验材料为云南的74份地方稻种资源,其中粳稻56份,籼稻18份(含野生稻3份).通过多个抗性组分进行了系统研究,初步表明大白谷(粳、墨江县)、毫弄早(籼、勐海县)、毫玉浪(籼、勐海县)、疣粒野生稻(野、西双版纳自治州)等具有持久抗瘟性能;其中疣粒野生稻高抗细菌性条斑病,对白叶枯病抗性为0级,接近免疫,中抗稻瘟病.通过对品种多抗性组分分析和品种抗性系统聚类分析,提出在不同生态类型时、空动态的病叶片上的产孢量和病斑表型可作为简易、快速鉴定持久抗瘟性指标.     

Nitrogen form and silicon nutrition effects on resistance to blast disease of rice

The effect of N form and Si nutrition on rice (Oryza sativa L.) susceptibility to blast disease (caused by Pyricularia oryzae Cav.) was assessed in the greenhouse with nutrient solution culture. The N form supplied to the susceptible cultivar IR50 affected the relative infection efficiency (RIE) of P. oryzae measured as lesions/cm² leaf. Plants given NO₃ ^- were more susceptible than plants receiving NH₄ ⁺-N. This result may partially explain why plants grown in nonflooded soil, where NO₃ ^- is the main source of inorganic N, are more susceptible to blast than plants grown in flooded soils, where NH₄ ⁺ is the main inorganic N source. Nitrate-N and Mn concentration were higher in leaf blades of plants grown with NO₃ ^-. Total-N, Si, and Fe concentration were not affected by N form. The addition of Si significantly increased IR50 resistance to blast. With 2.2 mol m^-3 Si in solution, RIE values were lower by more than 90% than the control with no Si added in solution. The effect of Si accumulation in leaves at various positions was further studied in cultivars having differing levels of resistance (IR50, IR36, and IAC165). Silicon addition significantly reduced RIE in the three cultivars. Silicon concentration in the topmost leaves (the only leaves showing typical blast lesions) was not significantly different among the three cultivars when 2.2 mol m^-3 Si was used. Silicon was an important component in the mechanism of resistance to blast and it was effective regardless of the original level of resistance of the cultivar used. Contribution from the Agronomy Unit, Agronomy-Physiology-Agroecology Division, International Rice Research Institute (IRRI), P.O. Box 933, 1099 Manila, Philippines, and Colegio de Postgraduados, Mexico. Part of a thesis submitted by the senior author in partial fulfillment of the requirements for the M.S. degree.     

Microbial secondary metabolite induction of abnormal appressoria formation mediates control of rice blast disease caused by Magnaporthe oryzae

Okinawa, the only subtropical area in Japan with numerous island ecosystems, is expected to have diverse microbial resources. Recently, we reported the construction of a culture filtrate library with microbes originally isolated from soils in Okinawa, including the Yaeyama Archipelago, and validated its phylogenetic diversity. In the present study, we investigated the inhibitory effect of the cell extract (CE) from microbial isolate 3–45 against Magnaporthe oryzae in rice (Oryza sativa). Abnormal appressorium formation by M. oryzae was induced in the presence of the CE from isolate 3–45. Additionally, melanization of appressoria was inhibited in the presence of CE from isolate 3–45. Sequence analysis of the 16S rDNA region of isolate 3–45 indicated that it shared similarities with Streptomyces erythrochromogenes. When rice leaves were inoculated with M. oryzae in the presence of CE from isolate 3–45, blast lesion formation was inhibited compared to leaves treated with M. oryzae in the absence of CE from isolate 3–45. In addition, M. oryzae infective activity was significantly inhibited in rice leaf sheaths treated with CE from isolate 3–45. Furthermore, abnormal appressorium formation was observed in the presence of heat‐treated CE from isolate 3–45. These results suggest that CE from isolate 3–45 can protect rice from blast disease caused by M. oryzae. Further studies are required to identify the active compounds present in 3–45‐CE and to clarify its mechanism of inhibition in full detail. The present study on isolate 3–45 might contribute to the development of a new fungicide for controlling rice blast disease caused by M. oryzae.     

Improving blast resistance of maintainer line DRR 9B by transferring broad spectrum resistance gene Pi2 by marker assisted selection in rice

Hybrid rice technology offers great promise to further enhance rice production and productivity for global food security. Improving hybrid rice parental lines is the first step in developing heterotic rice hybrids. To improve resistance against blast disease, a maintainer line DRR 9B was fortified with a major broad-spectrum blast resistance gene Pi2 through marker-assisted selection. The rice blast caused by Magnaporthe oryzae is a major disease and can cause severe yield losses upto 100%. The NILs of Samba Mahsuri namely BA-23-11-89-12-168 possessing Pi2 was utilized as a donor parent. The PCR-based molecular marker tightly linked to Pi2 gene was used for the foreground selection at BC₁F₁ generation. The molecular marker tightly linked to the major fertility restorer gene Rf4 was used for negative selection (i.e., selection of plants possessing non fertility restoring alleles) at BC₁F₁ generation to identify maintainer lines. The positive plants with Rf4 gene were added to the restorer pool for restorer line development. At each stage, MAS for Pi2 coupled with stringent phenotypic selection for agro-morphological and grain quality traits were exercised. At BC₁F₃ generation, one hundred families were screened against blast disease at uniform blast nursery (UBN) and selected resistant lines were advanced to next generations. In the BC₁F₅ generation plants were subjected to agro-morphological evaluation for yield and yield-contributing traits. The selected plants at BC₁F₅ generation were crossed with DRR 9A to assess the maintainer ability of blast resistance lines and for further CMS line conversion for hybrid rice breeding for developing blast resistance rice hybrids.     

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.     

Biological control of oilseed rape Sclerotinia stem rot by Bacillus subtilis strain Em7

In the present study, the endophytic bacterium Bacillus subtilis strain Em7 (GU258545.1) was evaluated as a biological control agent for Sclerotinia sclerotiorum on oilseed rape. In petri dish, strain Em7 not only strongly inhibited pathogen mycelium growth but also germination of sclerotia at concentrations between 10⁹ and 10¹¹ colony forming unit (CFU)·ml^?1. Scanning electron microscopy and transmission electron microscopy studies revealed that in the presence of strain Em7, hyphae of S. sclerotiorum showed leakage and disintegration of hyphal cytoplasm. Furthermore, the strain Em7 showed a broad antifungal spectrum on mycelium growth of numerous important plant pathogenic fungi. Light microscopic observations revealed that strain Em7 caused morphological alterations including increased branching, swelling and collapse of cytoplasm. In the greenhouse, spray treatments of cell suspensions of strain Em7 (1×10⁹ CFU·ml^?1) reduced leaf and stem rot incidence and severity in the seedling and blossom stage. The control efficacy was higher when strain Em7 cell suspension was applied one day prior to inoculation of the pathogen than after inoculation. Three-year field trials showed that two applications of strain Em7 cell suspension at blossom stage significantly reduced disease incidence and severity by 50–70%. There was no significant difference in control efficacy among treatments with strain Em7 cell suspension and the fungicides containing carbendazim or tebuconazole (P = 0.05). Thus, our results strongly suggest that B. subtilis strain Em7 is a promising biological control agent for control of oilseed rape Sclerotinia stem rot.     

Perception of the chitin oligosaccharides contributes to disease resistance to blast fungus Magnaporthe oryzae in rice

Chitin is a component of fungal cell walls, and its fragments act as elicitors in many plants. The plasma membrane glycoprotein CEBiP, which possesses LysM domains, is a receptor for the chitin elicitor (CE) in rice. Here, we report that the perception of CE by CEBiP contributes to disease resistance against the rice blast fungus, Magnaporthe oryzae, and that enhanced responses to CE by engineering CEBiP increase disease tolerance. Knockdown of CEBiP expression allowed increased spread of the infection hyphae. To enhance defense responses to CE, we constructed chimeric genes composed of CEBiP and Xa21, which mediate resistance to rice bacterial leaf blight. The expression of either CRXa1 or CRXa3, each of which contains the whole extracellular portion of CEBiP, the whole intracellular domain of XA21, and the transmembrane domain from either CEBiP or XA21, induced cell death accompanied by an increased production of reactive oxygen and nitrogen species after treatment with CE. Rice plants expressing the chimeric receptor exhibited necrotic lesions in response to CE and became more resistant to M. oryzae. Deletion of the first LysM domain in CRXA1 abolished these cellular responses. These results suggest that CEs are produced and recognized through the LysM domain of CEBiP during the interaction between rice and M. oryzae and imply that engineering pattern recognition receptors represents a new strategy for crop protection against fungal diseases.     

Biological control against bacterial wilt and colonization of mulberry by an endophytic Bacillus subtilis strain

Forty-five bacterial isolates were collected from surface-sterilized leaves of mulberry ( Morus alba L.). By screening their antagonistic activities against Ralstonia solanacearum in vitro , four isolates showed a remarkable inhibitory effect. The evaluation of the antagonistic strains against bacterial wilt of mulberry indicated that the strain Lu144 effectively reduced disease incidence. In the greenhouse, Lu144 displayed effective biological control against bacterial wilt of mulberry when it was applied to sterile or nonsterile soil before the infection by the pathogen. Based on bacteriological properties and 16S rRNA gene sequencing, Lu144 was identified as a strain of Bacillus subtilis . The endophytic population and infection process of Lu144 in mulberry seedlings was explored following recovery of the green fluorescent protein (GFP)-labeled Lu144 and examination of the labeled strain by confocal laser scanning microscopy. Interestingly, the infection of GFP-labeled Lu144 cells into the mulberry seedlings occurred through the cracks formed at the lateral root junctions and the zone of differentiation and elongation, and the cells were able to develop and transfer in mulberry and mainly in the intercellular spaces of different tissues. The population of the GFP-labeled Lu144 inoculant was larger and more stable in leaves than that in roots and stems.     

Enhancing blast disease resistance by overexpression of the calcium‐dependent protein kinase OsCPK4 in rice

Rice is the most important staple food for more than half of the human population, and blast disease is the most serious disease affecting global rice production. In this work, the isoform OsCPK4 of the rice calcium‐dependent protein kinase family is reported as a regulator of rice immunity to blast fungal infection. It shows that overexpression of OsCPK4 gene in rice plants enhances resistance to blast disease by preventing fungal penetration. The constitutive accumulation of OsCPK4 protein prepares rice plants for a rapid and potentiated defence response, including the production of reactive oxygen species, callose deposition and defence gene expression. OsCPK4 overexpression leads also to constitutive increased content of the glycosylated salicylic acid hormone in leaves without compromising rice yield. Given that OsCPK4 overexpression was known to confer also salt and drought tolerance in rice, the results reported in this article demonstrate that OsCPK4 acts as a convergence component that positively modulates both biotic and abiotic signalling pathways. Altogether, our findings indicate that OsCPK4 is a potential molecular target to improve not only abiotic stress tolerance, but also blast disease resistance of rice crops.