Inactivation of phytotoxin produced by the rice sheath blight pathogen Rhizoctonia solani

The rice sheath blight pathogen, Rhizoctonia solani, produces a toxin designated as RS-toxin, a carbohydrate compound containing mainly alpha-glucose and mannose. Different microflora were tested for RS-toxin inactivation. Isolates of Trichoderma viride inactivated this toxin when it was provided as the sole food source, and these isolates reduced the severity of toxin-induced symptoms and electrolyte leakage from rice cells. The best-performing isolate, TvMNT7, produced two extracellular proteins of 110 and 17 kDa. The high molecular mass protein was shown to have alpha-glucosidase activity. The purified 110 kDa protein was able to reduce RS-toxin activity.     

Antifungal effect and mechanism of chitosan against the rice sheath blight pathogen, Rhizoctonia solani

The antifungal properties and mechanism of three types of chitosan against the rice sheath blight pathogen, Rhizoctonia solani, were evaluated. Each chitosan had strong antifungal activity against R. solani and protected rice seedlings from sheath blight, in particular, two types of acid-soluble chitosan caused a 60–91?% inhibition in mycelial growth, 31–84?% inhibition of disease incidence, and 66–91?% inhibition in lesion length. The mechanism of chitosan in protection of rice from R. solani pathogen was attributed to direct destruction of the mycelium, evidenced by scanning and transmission electron microscopic observations and pathogenicity testing; indirect induced resistance was evidenced by the changes in the activities of the defense-related phenylalanine ammonia lyase, peroxidase and polyphenol oxidase in rice seedling. To our knowledge, this is the first report on the antifungal activity of chitosan against rice R. solani.     

Quantification of rice sheath blight progression caused by Rhizoctonia solani

Rhizoctonia solani has a wide host range, including almost all cultivated crops and its subgroup anastomosis group (AG)-1 IA causes sheath blight in rice. An accurate measurement of pathogen’s biomass is a convincing tool for enumeration of this disease. Mycological characteristics and molecular diagnosis simultaneously supported that all six strains in this study were R. solani AG-1 IA. Heterokaryons between strains Rs40104, Rs40105, and Rs45811 were stable and viable, whereas Rs40103 and Rs40106 did not form viable fused cells, except for the combination of Rs40106 and Rs40104. A primer pair was highly specific to RsAROM gene of R. solani strains and the amplified fragment exists as double copies within fungal genome. The relationship between crossing point (CP) values and the amount of fungal DNA was reliable (R ² >0.99). Based on these results, we determined R. solani’s proliferation within infected stems through real time PCR using a primer pair and a Taqman probe specific to the RsAROM gene. The amount of fungal DNA within the 250 ng of tissue DNA from rice cv. Dongjin infected with Rs40104, Rs40105, and Rs45811 were 7.436, 5.830, and 5.085 ng, respectively. In contrast, the fungal DNAs within the stems inoculated with Rs40103 and Rs40106 were 0.091 and 0.842 ng. The sheath blight symptom progression approximately coincided with the amount of fungal DNA within the symptoms. In summary, our quantitative evaluation method provided reliable and objective results reflecting the amount of fungal biomass within the infected tissues and would be useful for evaluation of resistance germplasm or fungicides and estimation of inoculum potential.     

Efficacy ofTrichoderma chitinases againstRhizoctonia solani, the rice sheath blight pathogen

Thirty-five strains ofTrichoderma viride andT. harzianum were screened for their antagonistic ability against the rice sheath blight pathogen,Rhizoctonia solani. The strains that inhibited/overgrew the phytopathogenic fungus were considered effective. Light microscopic studies showed the antagonism of the hyphae of effectiveTrichoderma strains towards their host hyphae. Chitinase activity ofTrichoderma culture filtrates was enhanced, when colloidal chitin was used as the sole carbon source, instead of glucose. Chitinase pattern differed among the four select strains. The chitinase isoforms are induced differentially by carbon sources. The chitin affinity column fraction ofTrichoderma culture filtrate inhibited,in vitro, the growth ofR. solani.     

Functional validation of pathogenicity genes in rice sheath blight pathogen Rhizoctonia solani by a novel host-induced gene silencing system

Rice sheath blight, caused by the soilborne fungus Rhizoctonia solani, causes severe yield losses worldwide. Elucidation of the pathogenic mechanism of R. solani is highly desired. However, the lack of a stable genetic transformation system has made it challenging to examine genes' functions in this fungus. Here, we present functional validation of pathogenicity genes in the rice sheath blight pathogen R. solani by a newly established tobacco rattle virus (TRV)–host-induced gene silencing (HIGS) system using the virulent R. solani AG-1 IA strain GD-118. RNA interference constructs of 33 candidate pathogenicity genes were infiltrated into Nicotiana benthamiana leaves with the TRV-HIGS system. Of these constructs, 29 resulted in a significant reduction in necrosis caused by GD-118 infection. For further validation of one of the positive genes, trehalose-6-phosphate phosphatase (Rstps2), stable rice transformants harbouring the double-stranded RNA (dsRNA) construct for Rstps2 were created. The transformants exhibited reduced gene expression of Rstps2, virulence, and trehalose accumulation in GD-118. We showed that the dsRNA for Rstps2 was taken up by GD-118 mycelia and sclerotial differentiation of GD-118 was inhibited. These findings offer gene identification opportunities for the rice sheath blight pathogen and a theoretical basis for controlling this disease by spray-induced gene silencing.     

一株水稻纹枯菌拮抗细菌的分离与鉴定

【目的】从土壤中分离并鉴定水稻纹枯菌拮抗细菌,测定其体外抑菌和温室防治效果。【方法】采用系列稀释法和平板对峙法筛选拮抗细菌,基于形态、生理特征及16S rDNA序列鉴定其分类地位,采用种子细菌化温室试验测定其防效。【结果】从蔬菜根际土壤中筛选出一株纹枯菌拮抗细菌,命名为kwkjT4。菌株具有明显的体外抑菌活性,对水稻纹枯病的温室防效与井冈霉素相当,初步鉴定为假紫色色杆菌(Chromobacterium pseudoviolaceum)。最适生长条件为pH 7.0,温度32°C,培养时间为36 h;抑菌活性物质产生的最适培养条件为pH 6.0,温度28°C,培养时间为48 h;表明两者并不一致。【结论】kwkjT4菌株在水稻纹枯病的生物防治中具有潜在的应用价值。这是C.pseudoviolaceum拮抗纹枯菌的首次报道。     

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.     

Induction of systemic resistance in rice by leaf extracts of Datura metel against sheath blight disease

The leaf extracts of Datura metel [both aqueous leaf extract (ALE) and ethanolic leaf extract (ELE)] were observed here to find if they can induce systemic resistance in the rice commonly found in Eastern India. The results showed that after the treatment, the enzyme activities of all the defence-related enzymes increased to a certain level even without pathogenic infection in comparison with non-treated seedlings and then, maintain at constant level throughout the study period. When treated seedlings were infected with Rhizoctonia solani, the enzyme activities were increased more than in uninfected seedlings. The elevated enzyme activities gave the indication of an induced systemic resistance in rice. The ELE of D. metel showed better induction effect than ALE.     

Morphological and pathological variations of rice sheath blight inciting south Indian Rhizoctonia solani isolates

Frequent assessment of pathogen diversity is one of the most important criteria in designing disease management programmes. A study on diversity of field isolates of Rhizoctonia solani from sheath blight-infected rice fields of south India has been carried out. A total of 236 R. solani isolates were obtained from 45 locations in the surveyed area. Sclerotial features such as colour, size and shape and distribution pattern were varying among isolates. However, no other morphological features found to differ among isolates. Majority of the R. solani isolates were fast growers as they attained complete mycelial growth within 2 days in a 90-mm Petri plate and the emergence of sclerotial structures was seen even in 4 days of incubation. Selected 10 R. solani isolates exhibited considerable variations in pathogenicity on three different rice cultivars. Vellai ponni was found to be the most susceptible rice cultivar to all the field isolates of R. solani.     

Induction of systemic resistance in rice by leaf extracts of Zizyphus jujuba and Ipomoea carnea against Rhizoctonia solani

Plants accumulate a great diversity of natural products, many of which confer protective effects against phytopathogenic attack. Earlier we had demonstrated that the leaf extracts of Zizyphus jujuba and Ipomoea carnea inhibit the in vitro mycelial growth of Rhizoctonia solani, and effectively reduce the incidence of sheath blight disease in rice.⁷ Here we demonstrate that foliar application of the aqueous leaf extracts of Z. jujuba and I. carnea followed by challenge inoculation with R. solani induces systemic resistance in rice as evident from significantly increased accumulation of pathogenesis-related proteins such as chitinase, β-1,3-glucanase and peroxidase, as well as defense-related compounds such as phenylalanine ammonia-lyase and phenolic substances. Thin layer chromatographic separation of secondary metabolites revealed presence of alkaloid and terpenoid compounds in the leaf extracts of Z. jujuba that exhibited toxicity against R. solani under in vitro condition. Thus, the enhanced sheath blight resistance in rice seedlings treated with leaf extracts of Z. jujuba or I. carnea can be attributed to the direct inhibitory effects of these leaf extracts as well as their ability to elicit systemic resistance against R. solani.Key words: sheath blight, Zizyphus jujuba, Ipomoea carnea, Rhizoctonia solani, induced systemic resistance, antimicrobial compoundsSheath blight disease of rice, caused by Rhizoctonia solani, has become a major production constraint in intensive rice cropping systems where semi-dwarf, nitrogen-responsive and high-yielding rice cultivars are grown. The disease causes an annual yield loss of upto 50%.¹ R. solani is both soil- and water-borne, and can infect more than 27 families of both monocot and dicot species.² Natural host genetic resistance to R. solani has not been recorded in cultivars or wild relatives of rice.³ Several broad spectrum fungicides have been recommended for control of sheath blight, however, chemical method of disease management is neither practical due to high cost of fungicides nor sustainable as it can affect the balance of ecosystem by destroying beneficial microbial population. In addition, the environmental pollution problems associated with indiscriminate use of synthetic pesticides have prompted investigations on exploiting bio-pesticides of plant and microbial origin.Plants accumulate an enormous variety of over 100,000 secondary metabolites,⁴ which can act as pre-existing chemical inhibitors to invading pathogens and/or help strengthen defense response of host plant. The pre-formed infectional barriers in plants are generally referred to as “phytoanticipins;” whereas, the antimicrobial compounds that are synthesized de novo in response to pathogen attack are referred to as “phytoalexins.”⁵ Because of years of selective breeding leading to removal of natural products, the endogenous levels of phytoanticipins in commonly cultivated crop species are generally low and often not sufficient to fight pathogen attack, effectively.⁴ Various weed species and wild relatives of crop plants that are not subjected to selective breeding are believed to contain higher levels of antimicrobial compounds, consistent with their ability to fight invading pathogens more effectively than cultivated crop species. Identification of such weed/plant species that are enriched with antimicrobial principles, isolation of bio-active compounds from them, and application in the form of concentrated formulations to crop plants can augment their disease resistance capability by directly inhibiting the growth of pathogen and inducing defense responses. Indeed, the antimicrobial properties of tissue extracts of several weed/plant species have been reported by a number of research groups world-wide, especially in Asia and Latin America.^6–13Earlier, we had evaluated the antimicrobial activity of leaf extracts of 16 different plant species belonging to 16 different families and demonstrated that leaf extracts of most of these plant species exhibit growth-inhibitory activities against R. solani and Xanathomoas oryzae pv. oryzae (Xoo).⁷ Among these, the leaf extracts of Datura metel were found to be the most effective in inhibiting the mycelial growth and sclerotia formation of R. solani, and the growth of Xoo, as well as in reducing the incidence of sheath blight and bacterial blight diseases caused by these pathogens, respectively, under greenhouse condition.⁷ We further demonstrated that rice seedlings treated with leaf extracts of D. metel accumulated significantly higher levels of pathogenesis-related (PR) proteins and other defense related compounds following challenge inoculation with R. solani or Xoo.⁷ Our attempts to identify biologically active compounds from D. metel revealed the presence of a withanolide compound “daturilin” that exhibited remarkable antibacterial activity against Xoo.⁷Apart from D. metel, two other plants species, Zizyphus jujuba and Ipomoea carnea, were found to possess remarkable antifungal activity against R. solani.⁷ Z. jujuba is a thorny rhamnaceous plant that is widely distributed in Europe and South-eastern Asia. I. carnea of convolvulaceae family, commonly known as morning glory, is a toxic weed found in abundance in India, Brazil, the United States and other countries.¹⁴ Both of these plant species have allelopathic effect and are commonly used in folklore medicine for curing multiple diseases.^15–18 The aqueous and methanol leaf extracts of Z. jujuba and I. carnea have been found to be highly effective in reducing in vitro mycelial growth, and therefore, sclerotia production of R. solani.⁷ In the greenhouse experiments, rice seedlings sprayed with leaf extracts of Z. jujuba and I. carnea exhibited 44 and 34% reduction in severity of sheath blight disease over the control, respectively.⁷ While these findings are encouraging, the mechanisms by which the leaf extracts of Z. jujuba and I. carnea modulate defense responses in rice have not yet been explored.Plants are endowed with defense genes which remain quiescent or are expressed at basal levels in healthy plants. Activation of defense genes results in induction of systemic resistance in host plant; this defense response, designated as induced systemic resistance (ISR), plays an important role in development of disease resistance.¹⁹ The onset of ISR in plants correlates with accumulation of phytoalexins and increased activity of PR proteins such as chitinases, β-1,3-glucanases and peroxidases;^20–23 consequently, PR proteins are generally used as ISR markers.¹⁹ The classical inducers of ISR include both biotic and abiotic factors, including disease causing microorganisms themselves,^24,25 plant growth promoting rhizobacteria,^22,26 chemicals^27,28 and natural plant products.^{7,10,12,13,29,30} Plant products have been considered as one of the major groups of compounds that induce ISR. To date, extracts of at least a few plant species have been reported to contain allelopathic substances which can act as elicitors and induce systemic resistance in host plants resulting in reduction or inhibition of disease development.^7,10,12,13In the present study, with the objective of understanding the mechanisms of disease suppression by leaf extracts of Z. jujuba and I. carnea, we investigated their ability to induce ISR in rice by analyzing the activities of ISR markers including PR-proteins and other defense enzymes involved in phenylpropanoid metabolism. The changes in activities of chitinase, β-1,3-glucanase, peroxidase, phenylalanine ammonia-lyase (PAL) and phenolic compounds induced in rice seedlings that were elicited with leaf extracts (at 1:10 dilution; w/v) of Z. jujuba or I. carnea and infected with R. solani were analyzed, and compared to changes in non-elicited and uninfected seedlings. Rice seedlings that were both elicited with leaf extracts of Z. jujuba or I. carnea and infected with R. solani accumulated significantly higher levels (2–5-fold) of ISR markers as compared to non-elicited and/or uninfected seedlings (Fig. 1). About two-fold increase in activities of ISR markers was also observed in seedlings that were either infected but not elicited or elicited but not infected; however, this increase was significantly lower than the changes in seedlings that were both elicited and infected (Fig. 1). Although the activity of all ISR markers began to increase around or after 24 h post-infection, at least two distinct induction patterns were observed. For instance, the activities of chitinase and phenolic substances gradually increased to reach maximum levels at 164 h post-infection (Fig. 1A and E); whereas, the activities of β-1,3-glucanase, peroxidase and PAL reached maximum levels at 72 to 96 h post-infection and decreased thereafter (Fig. 1B–D). The leaf extracts of Z. jujuba were found slightly more effective in inducing ISR markers than the leaf extracts of I. carnea. There was no significant change in the activity of ISR markers in control seedlings sprayed with sterile distilled water (Fig. 1). Collectively, these results suggested that the leaf extracts of Z. jujuba and I. carnea have the ability to induce systemic resistance in rice seedlings infected with R. solani. The fungitoxicity of the leaf extracts of Z. jujuba and I. carnea ⁷ combined with their ability to elicit ISR is possibly responsible for low sheath blight disease incidence observed in rice seedlings treated with these leaf extracts.⁷Open in a separate windowFigure 1Activity of ISR markers and defense-related compounds in rice seedlings elicited with the leaf extracts of Zizyphus jujuba or Ipomoea carnea and challenge inoculated with Rhizoctonia solani. Total activity of chitinase (A), β-1,3-glucanase (B), peroxidase (C) phenylalanine ammonia-lyase (PAL; D) and phenolic substances (E) was analyzed in rice seedlings. The inoculation of rice seedlings with R. solani was performed 45 days after planting. Spraying of leaf extracts (1:10 dilution; w/v) of Z. jujuba or I. carnea was performed two days prior to inoculation. Tissue samples (sheath) from elicited and/or infected seedlings were collected for analysis at various time intervals.The in vitro antimicrobial and in vivo disease inhibitory effects of natural plant products are generally attributed to the allelopathic substances present in them. However, very few attempts have been made to purify and characterize active principles from bio-active natural plant products. We have previously identified a withanolide compound from leaf extracts of D. metel which exhibited antibacterial activity against Xoo.⁷ Both Z. jujuba and I. carnea are rich source of secondary metabolites including alkaloids, terpenoids, flavonoids and phenolic compounds.^31–35 To determine the composition of bio-active ingredients within the leaf extracts of Z. jujuba and I. carnea, we performed thin layer chromatographic separation of alkaloid, terpenoid and phenolic compounds. The partially purified compounds, as reported in

浙皖鄂地区水稻纹枯病菌5个种群的遗传结构分析

水稻纹枯病是世界性的主要病害之一。目前对该病病原菌种群的遗传多样性研究不多,知之甚少,了解其种群的遗传结构可以增加对其进化历程的了解,以制定科学的防治策略。水稻纹枯病菌通常被认为是以无性克隆繁殖为主,但有研究报道它具有混合繁殖方式。有关我国浙皖鄂地区水稻纹枯病菌种群的遗传多样性研究尚未见报道。为了解该地区水稻纹枯病菌种群的遗传变异、基因流、繁育方式及其遗传背景,采用ITS-5.8SrDNA测序技术,分析了分离自浙江富阳(FY)、安徽绩溪(JX)和巢湖(CH)以及湖北荆州(JZ)和孝感(XG)的5个水稻纹枯病菌种群75个菌株的遗传多样性。RhizoctoniasolaniAG-1IA是采集地区水稻纹枯病菌的优势类群。ITS-5.8SrDNA序列经测定共检测到78个多态位点,碱基A、T、C、G的平均含量分别为25.4%、33.6%、21.0%和20.0%。序列的平均转换与颠换比(Ti/Tv)为1.65,其中密码子第3位点的变异最高。根据序列的核苷酸变异共定义了29种单倍型,其中单倍型H5为5个种群的共享单倍型,占样本数的61.33%。5个种群的单倍型多样性和核苷酸多样性分别为0.627和0.482%,显示水稻纹枯病菌种群具有较高的遗传多样性。种群间固定化指数Fst为-0.0253-0.0170,基因流Nm为5.56-11.12,说明种群间基因交流频繁,基因流抑制了由遗传漂变引起的遗传分化,菌丝或菌核短距离扩散和带菌种子远距离传播增加了种群间的基因交流。AMOVA分析显示,种群间的遗传变异仅占总变异的19.03%,而80.97%的变异存在于种群内部,种群间的遗传分化很低。Mantel检验发现,遗传距离与地理距离无显著相关性(r=-0.241,P=0.499)。采用UPGMA法构建的单倍型间的系统发育树表明,不同地点的单倍型分支混合分布,这进一步验证了Mantel检验的结果。单倍型的网状分析显示,水稻纹枯病菌种群曾经发生过种群暴发而不断扩散,因还未能获得足够的时间建立更加复杂的结构故而呈非典型"星状"。采用中性检验分析了水稻纹枯病菌种群遗传结构,结果表明,种群间存在很强的自然选择作用,群体符合Hardy-Weinberg遗传平衡,说明水稻纹枯病菌群体是一个随机交配群体,具有以担孢子进行有性繁殖和以菌丝或菌核进行无性繁殖的混合繁殖方式。这种生物学特性可能是导致其在较小生态范围内较高的遗传多样性水平和较低的种群遗传分化的原因。另外,水稻纹枯病菌经有性繁殖产生新的基因型,并通过无性繁殖在群体内固定繁殖,这种遗传模式极有可能导致其进化潜能提高,极易对杀菌剂产生抗性。因此,对水稻纹枯病菌的防治,除了施用化学药剂和种植抗性品种外,还需要防治农田灌水引起的病原菌(菌丝和菌核)在地区间的流动传播,减少带菌种子迁移或农用机械的交叉污染,对水稻、大豆和玉米等寄主作物的种子进行播前处理等等,这些对于水稻纹枯病菌的防治也是极为重要的。     

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.     

Rice oxalate oxidase gene driven by green tissue‐specific promoter increases tolerance to sheath blight pathogen (Rhizoctonia solani) in transgenic rice

Rice sheath blight, caused by the necrotrophic fungus Rhizoctonia solani, is one of the most devastating and intractable diseases of rice, leading to a significant reduction in rice productivity worldwide. In this article, in order to examine sheath blight resistance, we report the generation of transgenic rice lines overexpressing the rice oxalate oxidase 4 (Osoxo4) gene in a green tissue‐specific manner which breaks down oxalic acid (OA), the pathogenesis factor secreted by R. solani. Transgenic plants showed higher enzyme activity of oxalate oxidase (OxO) than nontransgenic control plants, which was visualized by histochemical assays and sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Transgenic rice leaves were more tolerant than control rice leaves to exogenous OA. Transgenic plants showed a higher level of expression of other defence‐related genes in response to pathogen infection. More importantly, transgenic plants exhibited significantly enhanced durable resistance to R. solani. The overexpression of Osoxo4 in rice did not show any detrimental phenotypic or agronomic effect. Our findings indicate that rice OxO can be utilized effectively in plant genetic manipulation for sheath blight resistance, and possibly for resistance to other diseases caused by necrotrophic fungi, especially those that secrete OA. This is the first report of the expression of defence genes in rice in a green tissue‐specific manner for sheath blight resistance.     

Characterization of antimicrobial peptides against a US strain of the rice pathogen Rhizoctonia solani

AIM: To identify antimicrobial peptides with high lytic activity against Rhizoctonia solani strain LR172, causal agent of rice sheath blight and aerial blight of soyabeans in the US. METHODS AND RESULTS: Among 12 natural and synthetic antimicrobial peptides tested in vitro, the wheat-seed peptide, purothionin, showed the strongest inhibitory activity that was similar to the antifungal antibiotics, nystatin and nikkomycin Z. Cecropin B, a natural peptide from cecropia moth, and synthetic peptide D4E1 produced the highest inhibitory activity against R. solani among linear peptides. Membrane permeabilization levels strongly correlated with antifungal activity of the peptides. Noticeable changes in membrane integrity were observed at concentrations of >/=0.5 micromol l(-1) for purothionin, 2 micromol l(-1) for cecropin B, D4E1, D2A21, melittin, and phor21, and 8 micromol l(-1) for magainin II and phor14. An increase of nuclear membrane permeabilization was observed in fungal cells treated with cecropin B, but not with purothionin. Diffusion of nuclear content was observed by fluorescent microscopy 10 min after adding a lethal concentration of cecropin B. Evaluation by electron microscopy confirmed severe cytoplasmic degradation and plasma membrane vesiculation. Purothionin and cecropin B were the most stable against proteolytic degradation when added to liquid cultures of R. solani. CONCLUSIONS: Purothionin, cecropin B, D4E1 and phor21 were shown to exhibit high in vitro lytic activity against R. solani strain LR172 for rice and soyabean. These peptides are greater than 16 amino acids long and rapidly increase fungal membrane permeabilization. Resistance to proteolysis is important for sufficient antifungal activity of antimicrobial peptides. SIGNIFICANCE AND IMPACT OF THE STUDY: Selected antimicrobial peptides offer an attractive alternative to traditional chemicals that could be utilized in molecular breeding to develop crops resistant to rice sheath blight and aerial blight of soyabean.     

Morphological,pathological and molecular characterisation of rice sheath blight disease causal organism Rhizoctonia solani AG-1 IA in Egypt

Rice sheath blight disease caused by Rhizoctonia solani is considered a distractive soil-borne disease of rice production worldwide. The study aimed to determine the causal organism of sheath blight symptoms in Egyptian rice fields. Sheath blight symptoms were first observed in a small area during 2013, 2014 and 2016 seasons, later in a wide area of rice fields in 2016 to 2018 seasons. Pathogen identification was carried out based on morphological traits and internal transcribed spacer sequencing. Thirty-six isolates were identified as R. solani fungus. The isolates exhibited a wide range of variability in their morphological traits and virulence patterns. Five isolates were sequenced and aligned with Chinese isolates with 75–100% identity. This is the first report of R. solani AG-1 IA that associated with rice sheath blight in Egypt. Initiate a breeding program for disease resistance and integrated disease management procedures are important to keep the disease under control.     

Induction of defense response against Rhizoctonia solani in cucumber plants by endophytic bacterium Bacillus thuringiensis GS1

An endophytic bacterium, Bacillus thuringiensis GS1, was isolated from bracken (Pteridium aquilinum) and found to have maximal production of chitinase (4.3 units/ml) at 5 days after culture. This study investigated the ability of B. thuringiensis GS1 to induce resistance to Rhizoctonia solani KACC 40111 (RS) in cucumber plants. Chitinase activity was greatest in RS-treated plants at 4 days. beta-1,3- Glucanase activity was highest in GS1-treated plants at 5 days. Guaiacol peroxidase (GPOD) activity increased continuously in all treated plants for 5 days. Ascorbate peroxidase (APX) activity in RS-treated plants was increased 1.5-fold compared with the control at 4 days. Polyphenol oxidase (PPO) activity in RS-treated plants was increased 1.5-fold compared with the control at 3 days. At 5 days after treatment, activity staining revealed three bands with chitinase activity (Ch1, Ch2, and Ch3) on SDSPAGE of cucumber plants treated with GS1+RS, whereas only one band was observed for RS-treated plants (Ch2). One GPOD isozyme (Gp1) was also observed in response to treatment with RS and GS1+RS at 4 days. One APX band (Ap2) was present on the native-PAGE gel of the control, and GS1- and GS1+RS-treated plants at 1 day. PPO bands (Po1 and Po2) from RS- and GS1+RS-treated plants were stronger than in the control and GS1-treated plants upon native-PAGE at 5 days. Taken together, these results indicate that the induction of PR proteins and defense-related enzymes by B. thuringiensis GS1 might have suppressed the damping-off caused by R. solani KACC 40111 in cucumber plants.     

Rice leaf extract synthesized silver nanoparticles: An in vitro fungicidal evaluation against Rhizoctonia solani,the causative agent of sheath blight disease in rice

Silver nanoparticles (Ag NP) were synthesized using rice leaf extract and optimized synthetic conditions were found to be 0.4 % leaf extract, 0.6 mM AgNO₃ and 30 min of autoclaving. Produced NP were characterized using UV–vis, DLS, zeta potential, XRD, TEM and FTIR. Ag NP formation was established from UV–vis spectra and NP showed zeta potential value of −27.4 mV. NP were spherical, polydisperse and average size was 16.5 ± 6.2 nm. Antifungal activity of Ag NP was assessed by poisoned food technique and resazurin broth dilution against mycelium and sclerotia of fungus R. solani, the causative agent of sheath blight disease in rice. Results confirmed effective hyphal growth inhibition and % growth inhibition was dose dependent (2.5–10 μg/mL). Ag NP showed enhanced mycelial inhibition (81.7–96.7 %) at 10 μg/mL. MIC values of Ag NP were in the range of 5–10 and 15–20 μg/mL towards fungal mycelium and sclerotia, respectively. Ag NP treatment (20 μg/mL) completely inhibited the disease incidence at 20 μg/mL. Ag NP treatment (10 μg/mL) caused 1.3 and 1.5 times enhancement in seedling vigor index. Hence, Ag NP can be utilized towards management and control of various fungal diseases of crops.     

Suppression of Rhizoctonia solani and induction of host plant resistance by Paenibacillus kribbensis PS04 towards controlling of rice sheath blight

We have identified a promising antagonistic micro-organism for suppressing Rhizoctonia solani and triggering induced resistance in rice. The isolate PS04 was identified as Paenibacillus kribbensis. PS04 and its crude metabolites showed inhibitory effects against R. solani both in vitro and in vivo, and with high stability against temperature and pH. Strain PS04 is a great biocontrol agent.     

Optimization of different application methods of multi-facial bacterial and fungal antagonists against sheath blight pathogen of rice,Rhizoctonia solani AG1-IA

Relative effectiveness of seven different application methods of five native bacterial and fungal biocontrol agents (BCAs) and one fungicide (azoxystrobin) was evaluated against Rhizoctonia solani, under pot and field conditions on rice cultivar Pusa Sugandha-5. Plants grown in pots infected with R. solani suffered a 30%–49% decrease in plant growth and yield of grain. However, treatment with BCAs reduced the adverse effect of the pathogen but significantly varied with the treatment schemes. Amongst the treatments, soil application (SA) at 20 + 40 days after planting (DAP) followed by foliar application (FA) at 60 DAP was recorded as most efficacious, and reduced the severity of disease by 42–68%, resulting in a 21–36% plant growth promotion and yield enhancement. Treatment comprising SA 20 + FA 40 DAP was next in effectiveness but statistically equal to seed priming (SP) + SA 40 DAP of treatment. Amongst the BCAs, Pseudomonas putida was shown to be the most efficient, trailed by Trichoderma harzianum, P. fluorescens, T. viride and Bacillus subtilis. Field trials under naturally infested fields have also validated the effectiveness of P. putida. The SA 20 + 40 + FA 60 DAP with P. putida and T. harzianum were found quite effective and decreased the disease severity and incidence (40–81%), and improved the grain yield (42–72%). Relatively lower ShB control was recorded with SA 20 + FA 60 DAP, however, it was statistically at par with SA 20 DAP treatment and equal to SA 20 + FA 40 DAP.