Efficacy of Bacillus subtilis MBI 600 Against Sheath Blight Caused by Rhizoctonia solani and on Growth and Yield of Rice

Rice sheath blight disease （ShB）, caused by Rhizoctonia solani, gives rise to significant grain yield losses. The present study evaluated the efficacy of Integral, the commercial liquid formulation of Bacillus subtilis strain MBI 600, against rice ShB and for plant growth promotion. In greenhouse studies, four log concentrations of Integral （from 2.2×10^6 to 2.2×10^9 cfu/mL） were used as seed treatment （ST）. After 25 d, seedlings were dipped （SD） into Integral prior to transplanting. At 30 d after transplanting （DAT）, leaf sheaths were inoculated with immature sclerotia of the pathogen. At 45 DAT, a foliar spray （FS） with Integral was applied to some treatments. The fungicide control was 50% carbendazim at 1.0 g/L, and a nontreated control was also included. Overall, there were 10 treatments, each with five replications. ShB severity was rated at 52 DAT, and seedling height and number of tillers per plant were rated at 60 DAT. In 2009, two field trials evaluated Integral at 2.2×10^8 and 2.2×10^9 cfu/mL. Integral was applied as ST, and seedlings were produced in a nursery bed. After 32 d, seedlings were treated with Integral as SD and transplanted into 10 m^2 blocks. Foliar sprays were given at 45 and 60 DAT. There were seven treatments, each with eight replications arranged as a factorial randomized complete block design. At 20 DAT, the plots were broadcast inoculated with R. solani produced on rice grains. Seedling height before transplanting, ShB severity at 90 DAT, and grain yield at harvest were recorded. Integral at 2.2×10^9 cfu/mL provided significant increase of seedling heights over other treatments under greenhouse conditions. The Integral treatments of ST ＋ SD ＋ FS at 2.2×10^9 cfu/mL significantly suppressed ShB over other treatments. In field studies, Integral provided significant increase of seedling height in nursery, and number of tillers per plant, compared with the control. ShB severity was significantly suppressed with higher concentrations of Integral compared to lower concentrations. Grain yield were the highest at an Integral concentration of 2.2×10^9 cfu/mL. Overall, Integral significantly reduced ShB severity, enhanced seedling growth, number of tillers per plant and grain yield as ST ＋ SD ＋ FS at the concentration of 2.2×10^9 cfu/mL under the conditions evaluated.

Advances in Mapping Loci Conferring Resistance to Rice Sheath Blight and Mining Rhizoctonia solani Resistant Resources

Sheath blight （SB） caused by Rhizoctonia solani is one of the three major diseases of rice, and now has become the most severe disease causing rice yield loss in China. Breeding and use of varieties resistant to SB is crucial in controlling the disease, but the advances achieved have been limited due to the tack of highly SB-resistant rice germplasm. Genetic analysis revealed that the SB resistance in rice was a typical quantitative trait controlled by multi-genes. Although many QTLs conferring resistance to SB have been identified in recent years, most of the QTLs only showed small effects and few of them have been evaluated for utilization potential. Many R. solani-resistant resources have been found in wild rice species, microorganisms and other plant species. It is already known that the SB-resistance could be improved in transgenic rice plants by genetic transformation. This paper reviewed the genetic mapping of loci associated with resistance to rice SB, the evaluation of the potential of resistance QTLs, and the resistant resources found in various organisms besides rice. To develop SB-resistant rice varieties, it is important to develop and explore new resistant rice germplasms, fine map and evaluate resistance QTLs, and also to pay attention to various bio-resources showing resistance to R. solani.

Secondary metabolites of rice sheath blight pathogen Rhizoctonia solani Kühn and their biological activities

Eight compounds were isolated from the fermentation cultures of rice sheath blight pathogen Rhizoctonia solani Kuhn. They were identified as ergosterol （1）, 6β-hydroxysitostenone （2）, sitostenone （3）, m-hydroxyphenylacetic acid （4）, methyl m-hydroxyphenylacetate （5）, m-hydroxymethylphenyl pentanoate （6）, （Z）-3-methylpent-2-en-1,5-dioic acid （7） and 3-methoxyfuran-2-carboxylic acid （8） by means of physicochemical and spectroscopic analysis. Among them, 2, 3, 5-8 were isolated from R. solani for the first time. All the compounds were evaluated for their biological activities. 4-6 and 8 showed their inhibitory activities on the radical and germ elongation of rice seeds. 1,4 and 7 showed moderate antibacterial activity to some bacteria. 4, 7 and 8 exhibited weak inhibitory activities on spore germination of Magnaporthe oryzae. 8 showed moderate antioxidant activity with the 1,1-diphenyl-2-picryhydrazyl （DPPH） andβ-carotene-linoleic acid assays. This is the first time to reveal compounds 5, 6 and 8 from rice sheath blight pathogen R. solani to have in vitro phytotoxic activity.

Establishment of Agrobacterium tumefaciens-Mediated Transformation System for Rice Sheath Blight Pathogen Rhizoctonia solani AG-1 IA

To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1 IA,the virulent isolate GD118 of this pathogen was selected as an initial isolate for transformation.The conditions for transformation of isolate GD118 were optimized in five aspects,i.e.pre-induction time,co-culture time,acetosyringone(AS) concentration at the co-culture phase,co-culture temperature and pH value of induction solid medium(ISM) at the co-culture phase.Finally,a system of Agrobacterium tumefaciens-mediated transformation(ATMT) for R.solani AG-1 IA was established successfully.The optimal conditions for this ATMT system were as follows:the concentration of hygromycin B at 30 μg/mL for transformant screening,8 h of pre-induction,20 h of co-culture,200 μmol/L of AS in ISM,co-culture at 25 ℃ and pH 5.6 to 5.8 of ISM at the co-culture phase.The transformants still displayed high resistance to hygromycin B after subculture for five generations.A total of 10 randomly selected transformants were used for PCR verification using the specific primers designed for the hph gene,and the results revealed that an expected band of 500 bp was amplified from all of the 10 transformants.Moreover,PCR amplification for these 10 transformants was carried out using specific primers designed for the Vir gene of A.tumefaciens,with four strains of A.tumefaciens as positive controls for eliminating the false-positive caused by the contamination of A.tumefaciens.An expected band of 730 bp was amplified from the four strains of A.tumefaciens,whereas no corresponding DNA band could be amplified from the 10 transformants.The results of the two PCR amplifications clearly showed that T-DNA was indeed inserted into the genome of target isolate GD118.

Diversity of ds RNA Viruses Infecting Rice Sheath Blight Fungus Rhizoctonia solani AG-1 IA

Rice sheath blight,caused by Rhizoctonia solani(Kühn),is a notorious soil-borne disease prevalent in many rice-growing regions.Although several sporadic studies of mycoviruses in R.solani AG-1 IA have been reported for single strain of R.solani AG-1 IA,there have been no reports describing the distribution and diversity of mycoviruses in natural populations.In this study,43 R.solani AG-1 IA strains collected from different locations in China were examined for the presence of ds RNA elements to confirm the presence of viral infections.Electrophoretypes showed that 16 of the 43 fungal strains(37.2%)contained ds RNAs that can be characterized as viruses.Furthermore,the species-specific reverse transcription PCR(RT-PCR)showed ds RNA bands with similar sizes do not always contain the same virus but exist as mixed mycoviral infections.Thus,our findings indicate mycoviruses infecting R.solani AG-1 IA in China are diverse,widespread and universal.

Influence of different inoculation methods on the evaluation of the resistance to rice sheath blight(Rhizoctonia solani Kuhn)

To establish a standard system for geneticstudies on sheath blight resistance, a field testwas conducted at the experimental farm ofYangzhou University to compare several pro-cedures for inoculating rice plants with R.solani Kuhn (RH9). The varieties used wereJasmine 85, Teqing (resistant or moderatelyresistant), and Lemont (susceptible). They

Three Genes Related to Trehalose Metabolism Affect Sclerotial Development of Rhizoctonia solani AG-1 IA,Causal Agent of Rice Sheath Blight

Trehalose metabolism is related to the sclerotial development of Rhizoctonia solani AG-1 IA,the causal agent of rice sheath blight(RSB).Here,we further elucidated the functions of three genes Rstre,Rstps1 and Rstpp that encode three key enzymes trehalase(TRE),alpha,alpha-trehalosephosphate synthase(TPS1)and trehalose 6-phosphate phosphatase(TPP)in the sclerotial development of R.solani AG-1 IA.Due to the lack of a stable genetic transformation system for R.solani,the heterologous expression of these three genes in Pichia pastoris GS115 was performed.The results showed that reactive oxygen species(ROS)contents and enzyme activities in R.solani decreased significantly in the treatments of the fermentation broths of Rstps1 and Rstpp transformants,and that in the treatment of the fermentation broth of Rstre transformant visibly increased.Furthermore,the fermentation broths of the transformants of all the three genes were added to potato dextrose agar(PDA)medium for the cultivation of R.solani,as a result,the dry weight of sclerotia in each PDA plate containing the fermentation broths of Rstps1 and Rstpp transformants significantly increased compared with the control,and that of Rstre transformant obviously decreased.Finally,178 proteins were found to interact with RSTPS1,and 16 of them were associated with ROS.Taken together,the findings suggest that all these three genes related to trehalose metabolism play important roles in the sclerotial development of R.solani AG-1 IA,and can be used as new targets for the development of novel high-efficiency fungicides for the controlling of RSB.

iTRAQ-based quantitative proteomics analysis of defense responses triggered by the pathogen Rhizoctonia solani infection in rice

The soil-borne necrotrophic fungus Rhizoctonia solani is one of destructive fungi causing severe yield losses in various important crops. However, the host defense mechanisms against the invasion of this pathogen are poorly understood. In this study, we employed an i TRAQ-based quantitative proteomic approach to investigate host proteins responsive to R. solani using the resistant rice cultivar YSBR1. As a whole, we identified 319 differentially accumulated proteins(DAPs) after inoculation of rice plants with R. solani. Functional categorization analysis indicates that these DAPs cover a broad range of functions. Notably, a substantial portion of the DAPs are involved in cell redox homeostasis, carbohydrate metabolism, and phenylpropanoid biosynthesis, or belong to pathogenesis-related proteins, indicating that these processes/proteins play important roles in host defense against R. solani. Interestingly, all of the DAPs involved in photosynthesis and chlorophyll biosynthetic processes, and part of the DAPs involved in phenylpropanoid biosynthesis, show reduced accumulation after R. solani infection, suggesting that R. solani probably inhibits host photosynthetic system and phenylpropanoid biosynthesis to facilitate infection and colonization. In conclusion, our results provide both valuable resources and new insights into the molecular mechanisms underlying rice and R. solani interaction.

武夷菌素对玉米纹枯病菌Rhizoctonia solani生长发育的影响

玉米纹枯病近年来已成为我国很多玉米主产区最重要的病害,而武夷菌素(wuyiencin)是来源于不吸水链霉Streptomyces ahygroscopicus var.wuyiensis的一种微生物源类杀菌剂.本研究测试了武夷菌素对玉米纹枯病菌生长发育的影响,结果表明在含有武夷菌素的PDA培养基上,玉米纹枯病菌生长缓慢,菌丝分支致密且部分菌丝尖端出现原生质体渗透;菌丝致病力下降.随着武夷菌素质量浓度的增高,菌丝受抑制程度加重,在培养后期,菌株形成的菌核数量和质量均显著下降.当武夷菌素质量浓度为50 mg/L时,玉米纹枯病菌菌落直径减少75%以上,菌丝致病力降低达99%,形成的菌核数量和质量分别降低67%和61%.武夷菌素可显著抑制玉米纹枯病菌的生长发育,在玉米纹枯病的控制中具有重要的应用潜力.武夷菌素对田间玉米纹枯病的有效防治有待进一步研究.

Cloning and Expression of the Serine Carboxypeptidase Gene in Zea mays and Its Antifungal Activity against Rhizoctonia solani

The authors cloned and identified a new maize serine carboxypeptidase gene named ZmSCP from R15 inbred lines seedlings which were induced by Rhizoctonia solani AGI-IA. ZmSCP encodes a 332 amino acid protein with a predicted molecular mass of 36.5 kDa and pI of 4.75. Phylogenetic analysis revealed that ZmSCP showed closer kinship with Oryza sativa and sorghum, which belong to the same evolutionary branch. Amino acid sequence analysis revealed that there are four types of amino acids in ZmSCP, the percentages of them are 43.1%, 26.9%, 13.9% and 13.1%. The authors subsequently purified the recombinant protein which expressed in Escherichia coli BL21 and analyzed its antimicrobial activities in vitro. Results showed that the recombinant protein inhibited hyphal growth of Rhizoctonia solani. The study suggests that the expression of ZmSCP is closely related to maize sheath blight resistance caused by Rhizoctonia solani. Further, the antifungal activity showed that ZmSCP may play at role in the disease resistance response.

Dissipation of pencycuron in rice plant

Pencycuron is a non-systemic protective fungicide for controlling sheath blight of rice. However, information on the fate of pencycuron in rice plant is lacking. The degradation of pencycuron in waterlogged tropic rice field was investigated.Pencycuron was applied at recommended field dose (187.5 g a.i./ha) and double recommended dose to cropped plots for three consecutive years. Pencycuron was rapidly degraded in rice plant at all doses of pencycuron application with first order half-lives of 1.57～2.77 d. The study revealed that pencycuron is safe from the human and environmental contamination point of view.

哈茨木霉T-102菌剂的制备及其对花生纹枯病和土壤生物多样性的影响

由立枯丝核菌(Rhizoctonia solani)引起的纹枯病是花生生产中一种重要的土传病害,严重威胁花生的产量安全和质量安全,是花生产业可持续健康发展中的主要障碍因子。本研究利用微生物深层发酵的方法,将分离获得的具有较好防效的哈茨木霉菌株T-102进行发酵,在150 r/min、20℃及初始接种浓度为7.5%的最佳条件下,获得活菌数可达105 cfu/mL的水悬浮剂。通过在山东临沂和河北唐山开展的田间防控试验结果表明,哈茨木霉菌株T-102水悬浮剂对花生纹枯病的防效明显,与对照相比,病情指数显著降低,同时具有显著的促生增产作用,增产效果分别达到41.2%和31.7%。另外,研究发现,应用生防菌剂能够显著改善花生根际微生物的种群结构和多样性,降低有害微生物的种群丰度。该菌剂的研发成功,有望进一步的商业化开发与应用。

中空介孔二氧化硅负载戊唑醇纳米缓释颗粒的制备及生物活性

本研究以中空介孔纳米二氧化硅(hollow mesoporous nano silica, HMS)为载体负载戊唑醇(tebuconazole, Teb),制备了戊唑醇@中空介孔纳米二氧化硅(Teb@HMS)缓释颗粒。通过扫描电镜(SEM)、透射电镜(TEM)、傅立叶变换红外光谱仪(FTIR)、比表面积分析仪(BET)及热重分析仪(TGA)等仪器对其形貌、结构与性能进行了表征。通过高效液相色谱(HPLC)分析研究了戊唑醇在缓释颗粒中的释放行为。通过菌丝生长速率法、盆栽试验和田间防效试验测定了Teb@HMS缓释颗粒对立枯丝核菌的抑制活性及对水稻纹枯病的防治效果。通过水稻种子发芽试验和斑马鱼试验对Teb@HMS的安全性进行了评价。结果表明:所制备的纳米二氧化硅载药粒子呈规整的中空介孔结构,对戊唑醇的载药率为52.02%,缓释时间长达400 h;Teb@HMS缓释颗粒降低了戊唑醇对非靶标生物水稻种子及斑马鱼的毒性,抑菌活性明显优于戊唑醇原药;盆栽和田间试验结果显示,施药后第18天Teb@HMS针对水稻纹枯病的保护作用防效分别达61.79%和70.42%。该研究可为中空介孔纳米二氧化硅缓释颗粒在农药减量化和植物病害绿色可持续防控方面提供技术支撑和理论指导。

玉米纹枯病菌菌核形成相关基因CYP450表达特性分析

为了从立枯丝核菌菌核形成探寻纹枯病防治方法,试验从田间采集玉米纹枯病发病植株,分离纯化立枯丝核菌菌株。使用生物信息学分析软件对CYP450基因的结构与功能进行分析,可知CYP450可能是一种亲水性蛋白且不存在跨膜序列,由397个氨基酸共同编码,含有信号肽。利用实时荧光定量PCR技术,对立枯丝核菌CYP450基因菌核形成过程中基因表达量变化进行研究,探寻CYP450基因在菌核形成过程中的作用。分析结果显示CYP450基因在菌核形成过程中表达量有显著差异,其中在菌丝时期表达量低,菌核形成后表达量有逐渐增高趋势,即在立枯丝核菌菌核形成过程中,CYP450可能参与了玉米纹枯病菌菌核的形成,该结果为玉米纹枯病的防治及抗病育种提供理论依据。

Enrichment of bacteria involved in the nitrogen cycle and plant growth promotion in soil by sclerotia of rice sheath blight fungus

Rice sheath blight pathogen,Rhizoctonia solani,produces numerous sclerotia to overwinter.As a rich source of nutrients in the soil,sclerotia may lead to the change of soil microbiota.For this purpose,we amended the sclerotia of R.solani in soil and analyzed the changes in bacterial microbiota within the soil at different time points.At the phyla level,Proteobacteria,Acidobacteria,Bacteroidetes,Actinobacteria,Chloroflexi and Firmicutes showed varied abundance in the amended soil samples compared to those in the control.An increased abundance of ammonia-oxidizing bacterium(AOB)Nitrosospira and Nitrite oxidizing bacteria(NOB)i.e.,Nitrospira was observed,where the latter is reportedly involved in the nitrifier denitrification.Moreover,Thiobacillus,Gemmatimonas,Anaeromyxobacter and Geobacter,the vital players in denitrification,N2O reduction and reductive nitrogen transformation,respectively,depicted enhanced abundance in R.solani sclerotia-amended samples.Furthermore,asymbiotic nitrogen-fixing bacteria,notably,Azotobacter as well as Microvirga and Phenylobacterium with nitrogen-fixing potential also enriched in the amended samples compared to the control.Plant growth promoting bacteria,such as Kribbella,Chitinophaga and Flavisolibacter also enriched in the sclerotia-amended soil.As per our knowledge,this study is of its kind where pathogenic fungal sclerotia activated microbes with a potential role in N transformation and provided clues about the ecological functions of R.solani sclerotia on the stimulation of bacterial genera involved in different processes of N-cycle within the soil in the absence of host plants.

水稻纹枯病菌拮抗菌的筛选、鉴定及培养条件探索

从不同土壤、植物和水稻纹枯病菌Rhizoctonia solani菌核样品上分离到细菌菌株325株和放线菌菌株86株。通过琼脂平板对峙法及发酵滤液介质筛选法,获得了对水稻纹枯病菌具有较强拮抗活性的细菌和放线菌菌株各1株,它们对水稻纹枯病菌菌丝生长的抑制率分别为75.56%和84.07%。采用形态学和生理生化学以及分子生物学方法,将细菌菌株NB12鉴定为枯草芽胞杆菌Bacillus subtilis、放线菌菌株NA1鉴定为Streptomyces triostinicus。对它们产生抑菌物质的发酵条件进行了探索,明确了菌株NB12的最佳发酵条件为:初始pH 7.0的LB或BPY培养液、装液量40 mL/250 mL、培养温度30℃、摇床转速180 r.min 1、培养时间48 h;菌株NA1的最佳发酵条件为:初始pH 6.0～9.0的大豆粉培养液或大豆粉–玉米粉培养液、装液量130 mL/250 mL、培养温度35℃、摇床转速140 r.min 1、培养时间≥72 h。

水稻抗纹枯病突变体的离体筛选

以水稻成熟胚培养产生的愈伤组织和悬浮细胞为筛选材料,以广东省水稻纹枯病菌Rhizoctonia solaniKühn强致病力菌株GD-11和GD-118产生的粗毒素为选择压力,从水稻品种的选择、粗毒素致病力的鉴定、突变体的筛选方法、突变体的再生以及突变体再生植株对粗毒素的抗病性鉴定等方面展开研究,建立了水稻抗纹枯病突变体的筛选体系并获得了抗病突变体.筛选体系为:两步筛选法,即以粤香占的愈伤组织和悬浮细胞为筛选材料,以φ(粗毒素)为3.5%和4.0%分别作为第1、2步的处理浓度,每一步处理的时间为20 d;筛选出的突变体再生苗的病情指数为37.55,而原始型再生苗的病情指数为87.50,抗病效果达57.08%.

井冈霉素对水稻纹枯病菌生长发育的影响

以水稻纹枯病菌(Rhizoctonia solani Kühn)强致病菌株GD-118为供试菌株,在室内观察了井冈霉素(Jinggangmycin)对其生长发育的影响。结果表明:井冈霉素对水稻纹枯病菌的毒力回归方程为y=3.360 3+1.320 4x,相关系数r=0.962 6,理论抑制菌丝生长的EC50为70.2μg/mL,EC95为6 341.5μg/mL。与不加井冈霉素的空白对照相比,用井冈霉素处理后水稻纹枯病菌的菌落边缘明显凹凸不平,边缘菌丝更密集、颜色加深,并且随着井冈霉素处理浓度的增加,菌丝的干质量逐渐降低,但菌落表面菌丝的密集程度有所增加、颜色更深;空白对照的菌核呈颗粒状、褐色,散生于菌落表面,边缘较多而中间较少;用井冈霉素处理后的菌核多数为粉状、浅褐色,部分菌核会连在一起呈块状,分布在菌落外围呈明显的双环形,具不规则的凹凸型菌落边缘,并且随着井冈霉素处理浓度的增加,菌核的干质量有所增加,菌核出现时间比空白对照提前约24h。另外,随着井冈霉素处理浓度的增加,水稻纹枯病菌的菌丝细胞核平均数目和分布范围均有不正常增多的趋势。

浙江省水稻纹枯病菌的遗传分化与致病力研究

鉴定了从浙江省各地采集的60株水稻纹枯病菌株的菌丝融合群归属情况。该60个菌株均与AG-1融合群的3个亚群AG-1-ⅠA、AG-1-ⅠB、AG-1-ⅠC的标准菌株融合,当属AG-1融合群。其中,有58株均与标准菌株AG-BⅠ融合,所有菌株与其他标准菌株均无融合现象。通过离体接种对菌株致病力进行鉴定,发现各菌株间致病力差异显著。致病力最强的TTZF-1与其中52个菌株差异极为显著。进一步的转录间隔区(ITS)序列分析显示,系统发育树的结果与融合群鉴定的结果一致,与菌株间致病力差异无相关性。

根癌农杆菌介导的水稻纹枯病菌转化系统的建立

为建立水稻纹枯病菌的T-DNA插入诱变转化系统,以水稻纹枯病菌(Rhizoctonia solani AG-1ⅠA)强致病力菌株GD118为转化的初始菌株,从预诱导时间、共培养时间、共培养时乙酰丁香酮的浓度、共培养温度和共培养时固体诱导培养基(SIM)的pH值等5个方面对转化条件进行了优化,成功地建立了适合于水稻纹枯病菌根癌农杆菌介导转化(ATMT)的优化系统。这个优化系统的转化条件如下:以30μg/mL的潮霉素B作为转化子的筛选浓度,预诱导8h,共培养20h,共培养时固体诱导培养基上的乙酰丁香酮浓度为200μmol/L,共培养温度25℃,共培养时固体诱导培养基pH5.6～5.8。采用这个系统筛选到的转化子继代培养5代后,在含30μg/mL潮霉素B的PDA平板上仍表现明显的抗性。从得到的转化子中随机抽取10个,利用根据抗潮霉素hph基因设计的特异性引物进行PCR扩增,转化子均能扩增出500bp左右的预期条带;与此同时,以4个根癌农杆菌作为阳性对照,用根癌农杆菌Vir基因特异引物对转化子进行PCR扩增,以排除转化子受农杆菌污染所致的假阳性,结果表明:4个根癌农杆菌均能扩增出Vir基因条带(730bp),而10个转化子均未能扩增出相应条带。以上两个PCR扩增的实验结果清楚地表明,T-DNA已经插入到目标菌株GD118中。