Bacterial Antagonist as Seed Treatment to Control Leaf Blight Disease of Bambara Groundnut (Vigna subterranea)

Summay Soil samples were taken from 48 fields in the southern part of Thailand in which either bambara groundnut (Vigna subterranea) or groundnut (Arachis hypogeae) had been planted. Bacillus spp. were isolated using soil dilution plates and heat treatment to screen for endospore-producing bacteria. Among 342 Bacillus spp. isolates tested, 168 isolates were not antagonistic to Bradyrhizobium sp. strain NC-92 using dual culture technique. Further testing found 16 isolates of Bacillus spp. had the ability to inhibit mycelial growth of Rhizoctonia solani, a causal agent of leaf blight of bambara groundnut. Among these isolates, Bacillus spp. isolate TRV 9-5-2 had the greatest activity in anti-microbial tests against R. solani. This isolate was later identified as B. firmus. A powder formulation of B. firmus was developed by mixing bacterial endospores, talcum, sodium carboxymethylcellulose (SCMC) and polyvinylpyrolidone (PVP). The formulations contained bacterial levels ranging from 10⁸ to 10¹⁰ c.f.u./g and the viability of bacteria in all formulations remained high after 1 year storage at room temperature (26–32 °C). All formulations showed satisfactory effectiveness in vitro in suppressing mycelial growth of R. solani using dual culture technique. The application of formulations as seed treatment showed that these formulations did not cause abnormality of seedling shape and had no effect on the germination of bambara groundnut seeds.     

Effect of mycorrhization on the accumulation of rishitin and solavetivone in potato plantlets challenged with<Emphasis Type="Italic"> Rhizoctonia solani</Emphasis>

The effect of colonization with the vesicular-arbuscular mycorrhizal fungus Glomus etunicatum on the content of rishitin and solavetivone was determined in potato plants cv. Goldrush challenged with Rhizoctonia solani. Mycorrhization stimulated significantly the accumulation of both phytoalexins in roots of plantlets challenged with R. solani but did not influence phytoalexin levels in non-challenged plantlet roots. No accumulation of solavetivone or rishitin was detected in shoots. In Petri dish bioassays, rishitin and solavetivone inhibited mycelial growth of R. solani.     

水稻纹枯病菌多聚半乳糖醛酸酶基因RsPG5的克隆与功能分析

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

类芽孢杆菌QHZ11对马铃薯黑痣病的生防效果

[背景] 马铃薯黑痣病是由立枯丝核菌（Rhizoctonia solani）引起的一种典型土传病害,目前该病害生物防治的菌种资源比较有限,相应菌株生防机制的研究更是缺乏。[目的] 明确马铃薯黑痣病病原菌立枯丝核菌（R. solani） JT18的拮抗菌QHZ11对马铃薯黑痣病的生防效果,揭示QHZ11对黑痣病的部分防治机理。[方法] 在灭菌土壤中分别接种R. solani JT18（CK）,R. solani JT18和普通有机肥（Organic Fertilized,OF）,R.solaniJT18和氨基酸有机肥（AA+OF）及R. solani JT18和QHZ11生物有机肥（BOF11）,结合实时荧光定量PCR （Real-Time Fluorescence Quantitative PCR,RT-qPCR）等方法,研究马铃薯全生育期不同处理R.solaniJT18在马铃薯根际和植株不同部位的数量变化及拮抗菌QHZ11与R.solaniJT18的数量消长规律,同时比较不同处理黑痣病的病情指数及相应的防效。[结果] RT-qPCR结果表明,随马铃薯生育进程的推进,马铃薯根际、根系和匍匐茎R.solaniJT18的数量在各处理中均呈现先升高至块茎膨大期到达峰值后下降的趋势,而且各部位R.solaniJT18的数量为CK>OF>AA+OF>BOF11且根际>根系>匍匐茎;拮抗菌QHZ11的数量变化趋势与R.solaniJT18相同,但峰值在块茎形成期,并且同时期同一部位QHZ11的定殖数量均显著高于R.solaniJT18,甚至高出2个数量级,说明QHZ11占用了一定的营养资源和生态位点,严重抑制了R.solaniJT18的生长和繁殖。病情结果表明：CK病情指数最高,OF、AA+OF和BOF11处理均显著低于CK,其中BOF11处理发病最轻;生防结果则相反,为BOF11>AA+OF>OF处理,说明普通有机肥、氨基酸有机肥及生物有机肥均可不同程度地防治马铃薯黑痣病,其中以生物有机肥效果最显著。[结论] QHZ11以有机肥为载体施入土壤后,可以通过在马铃薯根际及植株不同部位竞争营养和生态位点,从而有效抑制黑痣病病原菌R.solaniJT18的生存和繁殖,起到显著的生防效果,这对QHZ11生物有机肥的应用和推广具有重要意义,并为进一步研究QHZ11的生防机制奠定了基础。     

Fungitoxicity of some higher plants againstRhizoctonia solani

Summary Leaves ofChenopodium ambrosioides exhibited strong fungitoxicity against the mycelial growth ofRhizoctonia solani causing damping off diseases of some seedlings. Minimum inhibitory concentration of the fungitoxic constituent isolated in form of essential oil, was found to be 1000 ppm at which it was fungicidal in nature. It exhibited broad range of antifungal activity and did not show any phytotoxicity on the germination and seedling growth ofPhaseolus aureus.     

Expression of the linear DNA plasmid pRS64 in the plant pathogenic fungus Rhizoctonia solani

The plant pathogenic isolate RI-64 of anastomosis group 4 of Rhizoctonia solani possesses three linear DNA plasmids (pRS64-1, -2, and -3). Unique poly(A)^– RNA, 0.5 kb in length and hybridizable with the pRS64 DNAs was found in mycelial cells of the isolate RI-64. The overall homology at the nucleotide level between pRS64-1, -2, and -3, and the cDNA prepared from the poly(A)^– RNA was 100%, 73%, and 84%, respectively. The open reading frames found in pRS64-1, -2, and -3 (ORF1-1, ORF2-1, and ORF3-1) are 68 amino acids long. The amino acids sequence showed no significant homology with known proteins. Extracts from Escherichia coli cells expressing ORF1-1 contain a specific protein of 7 kDa. Antisera raised against the ORF1-1 product obtained from E. coli cells cross-reacted with the specific proteins found in the mycelia. The results indicate that the DNA plasmids found in R. solani contain a sequence that encodes a specific protein which may be involved in determination of plant pathogenicity.     

林下参内生生防细菌的分离鉴定及定殖能力

【背景】多年生林下参在自然环境下生长多年,其体内存在的内生菌具有更强的适应性和定殖性,可以提高植物自身抗性,抑制病原菌的生长,更好地发挥与植物的互作。【目的】筛选定殖能力强、繁殖能力快且对病原菌具有拮抗作用的优势菌株。【方法】采用常规组织分离方法,从健康林下参根部组织中分离内生菌,通过对峙试验筛选出对人参病原菌有拮抗作用的内生细菌并对其以传统的鉴定方法进行鉴定。【结果】在得到的6株内生细菌中,菌株LXS-N2对人参立枯病病原菌、人参猝倒病病原菌均有明显抑菌性,而且具有定殖性好、繁殖快的特点,通过破坏病原真菌细胞壁和细胞膜以及改变菌丝形态从而抑制病原真菌生长。【结论】经形态学观察、生理生化反应及16S rRNA基因序列分析鉴定内生菌LXS-N2为贝莱斯芽孢杆菌,具有良好的应用开发潜力。     

The effect of rhizoctonia root disease and applied nitrogen on growth,nitrogen uptake and nutrient concentrations in spring wheat

Root disease caused by Rhizoctonia solani is a common problem of spring wheat in South Australia. There are reports that nitrogen applications can reduce the incidence and severity of the disease. A glasshouse trail in pots examined the effects of disease and of applied nitrogen on wheat growth, and evaluated the utility of the basal stem nitrate concentration in diagnosing deficiency in plants with and without root disease. Plants were harvested at the mid-tillering stage. Shoot growth was increased by applied nitrogen until a maximum yield was attained, after which additional N had no effect on shoot yield. Root growth, however, responded positively only to low levels of applied N, after which it declined, and in the highest N treatment root mass was less than in the plants without applied N. Root disease caused severe reductions in plant growth, and both root and shoot mass were affected similarly. Even though growth of diseased plants responded positively to applied nitrogen the response was less than that of disease-free plants. The critical concentration of basal stem nitrate-N did not appear to be affected by root disease, and was estimated at 1200 mg kg^-1, consistent with other glasshouse data. The basal stem nitrate-N concentration, either in fresh or dried tissue, appeared a better diagnostic tool of N stress than did total shoot N concentration or content, because of sharper definition of critical concentrations. Concentrations of other nutrients in shoot tissue were affected differentially by both applied nitrogen and root disease, but generally did not reach critical levels, although phosphorus and magnesium appeared deficient in very disease-stressed plants.     

立枯丝核菌对高粱幼苗活性氧及抗氧化酶活性的影响

以立枯丝核菌AG1-IA接种‘龙杂19号’高粱幼苗,通过盆栽试验在不同侵染时间观测分析高粱幼苗生长及生理代谢相关指标,揭示立枯丝核菌侵染对高粱生长、渗透调节物质及其抗氧化酶活性的影响机理。结果表明,(1)高粱幼苗株高、根长、地上(茎和叶片)鲜质量和干质量、地下(根)鲜质量和干质量均随接种时间延长呈下降趋势,且在接菌72 h时较对照依次分别显著下降了41.0%、29.2%、50.0%、50.0%、53.3%和50.0%。(2)幼苗叶片叶绿素a含量在接菌72 h时较对照显著下降45.3%,叶片PSⅡ最大光化学效率(Fv/Fm)值随接菌时间增加而逐渐下降。(3)幼苗叶片内MDA、O^(-·)_(2)和H_(2)O_(2)含量随接菌时间增加均呈逐渐上升趋势,在接菌72 h时较对照分别显著上升244.6%、140.4%和137.0%;叶片SOD、POD、APX和CAT的活性在接菌后变化趋势不尽相同,但在接菌72 h时较对照分别显著上升了16.5%、60.3%、50.0%和36.5%。(4)幼苗叶片可溶性蛋白和可溶性糖含量随接菌时间增加均呈逐渐上升趋势,并在接菌24~72 h增幅均达到显著水平。研究发现,接种立枯丝核菌可致高粱幼苗植株产生病斑,体内活性氧过量积累,膜质发生显著氧化损伤;侵染前期高粱植株主要以积累更多的渗透调节物质来抵御立枯丝核菌带来的伤害,侵染后期随植株受到伤害加重,同时诱导植株抗氧化酶类活性显著增强,以维持高粱体内活性氧代谢稳态平衡,减少植物膜进一步氧化损伤。     

Mixtures of plant growth-promoting rhizobacteria for induction of systemic resistance against multiple plant diseases

Studies of induced systemic resistance using strains of plant growth-promoting rhizobacteria (PGPR) have concentrated on the use of individual PGPR as inducers against multiple diseases of a single crop. To date, few reports have examined the potential of PGPR strain mixtures to induce systemic resistance against diseases of several different plant hosts. The objective of this study was to select mixtures of compatible PGPR strains with the capacity to elicit induced systemic resistance in four hosts. The specific diseases and hosts tested in this study included: bacterial wilt of tomato (Lycopersicon esculentum) caused by Ralstonia solanacearum, anthracnose of long cayenne pepper (Capsicum annuum var. acuminatum) caused by Colletotrichum gloeosporioides, damping off of green kuang futsoi (Brassica chinensis var. parachinensis) caused by Rhizoctonia solani, and cucumber mosaic virus (CMV) on cucumber (Cucumis sativus). To examine compatibility, seven selected PGPR strains were individually tested for in vitro antibiosis against all other PGPR strains and against three of the tested pathogens (R. solanacearum, C. gloeosporioides, and R. solani). No in vitro antibiosis was observed among PGPR strains or against pathogens. Twenty-one combinations of PGPR and seven individual PGPR were tested in the greenhouse for induced resistance activity. Results indicated that four mixtures of PGPR and one individual strain treatment significantly reduced the severity of all four diseases compared to the nonbacterized control: 11 mixtures reduced CMV of cucumber, 16 mixtures reduced bacterial wilt of tomato, 18 mixtures reduced anthracnose of long cayenne pepper, and 7 mixtures reduced damping off of green kuang futsoi. Most mixtures of PGPR provided a greater disease suppression than individual PGPR strains. These results suggest that mixtures of PGPR can elicit induced systemic resistance to fungal, bacterial, and viral diseases in the four hosts tested.     

Detoxification of oxalic acid by pseudomonas fluorescens strain pfMDU2: implications for the biological control of rice sheath blight caused by Rhizoctonia solani

Rhizoctonia solani isolates varying in their virulence were tested for their ability to produce oxalic acid (OA) in vitro. The results indicated that the virulent isolates produced more OA than the less virulent isolates. In order to isolate OA-detoxifying strains of Pseudomonas fluorescens, rhizosphere soil of rice was drenched with 100 mM OA and fluorescent pseudomonads were isolated from the OA-amended soil by using King's medium B. These isolates were tested for their antagonistic effect towards growth of R. solani in vitro. Among them P. fluorescens PfMDU2 was the most effective in inhibiting the mycelial growth of R. solani. P. fluorescens PfMDU2 was capable of detoxifying OA and several proteins were detected in the culture filtrate of PfMDU2 when it was grown in medium containing OA. To investigate whether the gene(s) involved in OA-detoxification resides on the plasmids in P. fluorescens PfMDU2, a plasmid-deficient strain of P. fluorescens was generated by plasmid curing. The plasmid-deficient strain (PfMDU2P-) failed to grow in medium containing OA and did not inhibit the growth of R. solani. Both PfMDU2 and PfMDU2P- were tested for their efficacy in controlling sheath blight of rice under greenhouse conditions. Seed treatment followed by soil application of rice with P. fluorescens strain, PfMDU2, reduced the severity of sheath blight by 75% compared with the control, whereas PfMDU2P- failed to control sheath blight disease.     

The role of rhizosphere bacteria in herbicide-mediated increase in Rhizoctonia solani-induced cotton seedling damping-off

A study was carried out to determine the role of rhizosphere-residing bacteria in the observed pendimethalin- and prometryn-mediated increase in cotton seedling damping-off incidence caused by Rhizoctonia solani. Judging from the results, pendimethalin-mediated increase in disease incidence may be due to a decrease in the populations of indigenous cotton root colonizing bacteria in the presence of pendimethalin. Prometryn-mediated increase in disease may be due to a decrease in the populations of indigenous root colonizing bacteria as well as increased susceptibility of cotton seedlings to R. solani infection in the presence of prometryn. Pendimethalin and prometryn neither affected the growth of R. solani nor caused any visible change in seedling physical characteristics.     

A comparison ofin vitro andin vivo effect of clay minerals,humic acid and micronutrients on the activity of fungicides againstRhizoctonia solani

Activity of nine fungicides against mycelial growth ofRhizoctonia solani in potato dextrose broth and in pot tests as seed treatment against cowpea seedling rot in infested soil was differentially in fluenced by clay minerals, humic acid and micronutrients. Humic acid, extracted from farmyard manure, considerably lowered the activity, bothin vitro andin vivo, of all fungicides except chloroneb. Montmorillonite caused substantial decrease in disease control by fungicides but enhanced the toxicity of 2-methoxyethyl mercury chloride (MEMC), and quintozene in culture. Kaolinite inactivated carbendazim, benomyl and thiophanatemethyl in nutrient broth but had little effect on disease control by these fugicides. The six micronutrients altered the activity of fungicides to varying levels often without definite correlation betweenin vitro andin vivo results. The results ofin vitro growth inhibition tests are largely inapplicable to dieseas control tests in infested soil mainly due to the differences in the ambient conditions of the two systems.     

Disease cycle involved in damping-off of groundnut caused byRhizoctonia solani

Summary By taking into consideration of the results of various studies on damping-off of groundnut caused byRhizoctonia solani, the mechanisms leading to ultimate collapse and death of the seedlings, are described.     

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

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