Effect of K1, K2 Anti-Bacterial Agents on Tobacco Ralstonia Solanacearum

The tobacco Ralstonia Solanacearum were both cultured on nutrient agar plates and inoculated in seedling stage of tobacco, then treated with K1 and K2, two anti-bacterial agents, at a serial con-centrations to study their inhibitory efficiency. The result indicated that K1 can inhibit R. Solanacearum growth entirely, at the concentration range from 1/50 to 1/5000. K2 can reach the same result at the concentration range from 1/50 to 1/50000. Compared with the control plates, K1, at the concentration 1/50000, had no significant differences, and the average number of colony per plate was 112-115. The immature tobacco shown wilt as soon as inoculated with R. Solanacearum, and recovered gradually after using K1, K2. The densities of microbial suspension, handled by K1, K2 within 10 hs, were both significantly lower than the controlled ones. The optical microscopy also shown that handled microbial body differed from the controlled, whose body was regular short, rod shape as opposed to the handled ones with irregular rod shape and damaged body. All the results indicated that K1 and K2 both had inhibitory effects on tobacco R. Solanacearum, and K2 was more efficient than K1.

Analysis of Meteorological Epidemic Factors and Soil Improvement Controlling Technology of Tobacco Bacterial Wilt (Ralstonia solanacarum)

[Objective] The paper was to analyze the meteorological epidemic factors for occurrence and prevalence of tobacco bacterial wilt （ Ralstonia solanaca- rum）, and to study control effects of different soil conditioners on the bacterial disease in Gacligongshan demonstration area of green, ecological, high quality tobac- co leaf production. [Method] The plots attacked by tobacco bacterial wilt over the years were selected and the incidence of the disease was periodically surveyed in tobacco growth period in 2012, 2103 and 2014, respectively. 10 d Effective accumulated temperature and rainfall were counted according to the meteorological data, and the relationship between meteorological factors and disease index was analyzed. The control effects of three kinds of soil conditioners ＂Zhuanggenfeng＂, refined fulvic acid and lime on tobacco bacterial wilt were tested. [ Result] The analysis results of meteorological factors showed that 10 d effective accumulated temperature and rainfall were positively correlated to disease index. The variation curve of 10 d effective accumulated temperature and rainfall reflected the change trend of disease index. The pH values were increased by 0.57, 0.50 and 0.72 respectively after applying ＂Zhuanggenfeng＂, refined fulvic acid and lime. The aver- age control effects on tobacco bacterial wilt were 60.74% -62. 18%, 53.05% -59.53%, and 48.59% -58.53%, respectively. [ Conclusion] 10 d Effective accumulated temperature and rainfall could be used as important reference for disease forecasting and controUing. The usage of soil conditioner has a certain preven- tion and control effect on tobacco bacterial wilt disease by forming soil conditions conducive to flue-cured tobacco growth but adverse to disease survival, which is an effective auxiliary method against the disease.

Effect of Integrated Use of Inorganic Fertilizer and Organic Manures on Bacterial Wilt Incidence (BWI) and Tuber Yield in Potato Production Systems on Hill Slopes of Central Kenya

Bacterial wilt (BW) caused by Ralstonia solanacearum is one of the most damaging diseases of potato (Solanum tuberosum L.) in Kenya and worldwide. In Kenya, potato tuber yield losses due to BW infection are estimated at 50 ~ 100%. Low soil fertility is also one of the most important constraints limiting potato production in central Kenya highlands. Farmers tackle this problem through use of inorganic fertilisers and organic manures, both of which amend the soil environment to influence bacterial wilt development. Undecomposed organic manures can also introduce the pathogen into a clean field. Between short rains 1999 and 2000, 10 on-farm extension-researcher- farmer-designed and farmer-managed trials were done at Kianjuki catchment in Embu District. The objective was to use farmers?participatory research approach and select the most suitable organic and inorganic fertiliser combination(s) with lowest BWI and acceptable usable tuber yields, and also to demonstrate use of some components of integrated disease management methods in reduction of disease incidence and spread. Seven treatments were proposed, presented to the farmers for discussion and the most relevant four were selected for evaluation. A newly released potato variety 慉sante?was planted during the short rains 1999 and long rains 2000. BWI didn抰 result in significant differences between treatments but the tuber yields were significantly different in short rains of 1999 and 2000. During short rains 2000, both BWI and tuber yields and unusable tubers differed significantly between treatments. The results confirmed that well- decomposed manures or manures from pathogen-free areas can be used in combination with inorganic fertilisers to improve soil fertility and potato tuber yields in smallholder farms without influencing BWI. Use of certified seed tubers in pathogen free field and following recommended field sanitation measures resulted in apparently bacterial wilt free crop. Considering the high cost of inorganic fertiliser and its negative effects on the environment, reduced usage at half the recommended rates combined with half rates of FYM is feasible option friendly to the farmer, soil and environment. The interviewed farmers also ranked the option as the most appropriate combination for soil fertility improvement for potato production in smallholder farms.

Improvement of Bio-Efficacy to Reduce Bacterial Wilt Complex Disease in Tomatoes through Bacillus amyloliquefaciens and Pseudomonas fluorescens under Field Conditions

Bacterial wilt complex disease of tomato(Solanum lycopersicum L.)was incited jointly by bacterial wilt pathogen Ralstonia solanacearum and Meloidogyne incognita worldwide.Bio-efficacy of bacterial antagonists i.e.B.amyloliquefaciens DSBA-11 and P.fluorescens DTPF-3 was studied against the wilt disease complex in tomato at National Phytotran facility Indian Agricultural Research Institute(IARI),New Delhi,at 26±2°C.Minimum wilt disease incidence(26.00%)with the highest bio-control efficacy(64.15%),less juvenile population(19.33 J2/g of soil)of M.incognita was recorded in the combined application of DTPF-3+DSBA-11 after 30 d of inoculation under glasshouse conditions.In a field study,minimum bacterial wilt disease incidences 19.0%and 20.4%were recorded in the bleaching powder treatment followed by mixed application of DSBA-11+DTPF-3,19.6%and 21.2%wilt incidence in 2014 and 2015 respectively.However,a reduction of root-knot gall index was recorded a maximum of 59.76%and 69.62%in DSBA-11+DTPF-3 treated plants followed by 54.88%and 60.13%over control in DTPF-3 treatment in 2014 and 2015 respectively.The yield of tomato fruit was increased over control by 17.48%and 16.97%in 2014 and 2015 respectively under field conditions.A combination of P.fluorescens DTPF-3+B.amyloliquefaciens DSBA-11 suppressed bacterial wilt and root-knot diseases and also increased the yield of the tomato fruit significantly(p<0.05)under field conditions.

Silicon impacts on soil microflora under Ralstonia Solanacearum inoculation

Silicon(Si) can increase plant resistance against bacterial wilt caused by Ralstonia solanacearum and enhance plant immune response. However, whether Si alleviates soil-borne disease stress through altering soil microbial community component and diversity is not clear. In this study, effects of Si application under R. solanacearum inoculation with or without plant on soil bacterial and fungal communities were investigated through high-throughput pyrosequencing technique. The results showed that Si addition significantly reduced bacterial wilt incidence. However, Si did not reduce the amount of R. solanacearum in rhizosphere soil. Principal components analysis showed that soil microbial community composition was strongly influenced by Si addition. Total 63.7% bacterial operational taxonomic units(OTUs) and 43.8% fungal OTUs were regulated by Si addition regardless of the presence of tomato plants, indicating the independent effects of Si on soil microbial community. Si-added soil harbored a lower abundance of Fusarium, Pseudomonas, and Faecalibacterium. Our finding further demonstrated that exogenous Si could significantly influence soil microbial community component, and this may provide additional insight into the mechanism of Si-enhanced plant resistance against soil-borne pathogens.

Seedling Petri-dish inoculation method:A robust,easy-to-use and reliable assay for studying plant-Ralstonia solanacearum interactions

Ralstonia solanacearum causes a lethal bacterial wilt disease in many crops,leading to huge losses in crop production every year.Understanding of plant-R.solanacearum interactions will aid to develop efficient strategies to control the disease.As a soilborne pathogen,R.solanacearum naturally infects plants via roots.A huge limitation in studying plant-R.solanacearum interactions is the large variation of R.solanacearum infection assay due to the variable soil conditions and uneven inoculum exposure.Here,we developed a robust and reliable Petri-dish inoculation method which allows consistent and stable infection in young plant seedlings.This method is easy to use,takes about only 10 days from seed germination to the completion of inoculation assay,and requires less inoculum of bacteria as well as growth chamber space.We proved the efficacy of the seedling Petri-dish inoculation method by analyzing plant defense primed by molecular patterns,resistance of defense-related plant mutants,and virulence of R.solanacearum mutants.Furthermore,we demonstrated that the seedling Petri-dish inoculation method can be applied to other host plants such as tobacco and has great potential for high-throughput screening of resistant plant germplasms to bacterial wilt in the future.

河南省花生青枯病菌的分子鉴定及其生物学特性研究

为了明确河南省花生青枯病的病原菌分类属性,对采自河南省内不同市的30个青枯病菌株在16S rDNA序列、碳水化合物利用、致病性、演化型及序列变种等方面进行了鉴定。30个青枯病菌株的16S rDNA测序结果表明,引起河南花生青枯病的病原为茄科雷尔氏菌(Ralstonia solanacearum);所有青枯病菌株可侵染茄子、辣椒、马铃薯、烟草和番茄,但不侵染姜,属于生理小种1号;根据能否利用3种双糖和3种己醇的能力,可将其中4个菌株归于生化型Ⅱ,10个菌株归于生化型Ⅲ,16个菌株归于生化型V;所有青枯病菌株均能用多重PCR扩增得到144 bp演化型Ⅰ特异条带和280 bp茄科雷尔氏菌特异条带,表明青枯病原菌均属于茄科雷尔氏菌演化型Ⅰ,即亚洲组13;利用egl基因的系统发育进化分析表明,来自河南的30个菌株与Gx525、Ah-XnJn-12-6、HA2-1聚为一支,属于序列变种14。

茄子青枯病发生率与田间生态因子的相关性分析

为了解析茄子青枯病发生率与田间生态因子的关系,在厦门同安茄子青枯病发病典型区,安装了农业生境野外数据观测装备,采集气温、地温、相对湿度、雨量、太阳辐射、光合有效辐射、光照度、CO_(2)浓度和土壤容积含水率等生态因子数据,并在营养生长期和生殖生长期调查茄子青枯病发病情况和青枯病原的分布特性。结果表明:茄子营养生长期的气温(28.6℃)、地温(30.1℃)和相对湿度(66.17%),均适宜青枯病发生;茄子在营养生长期的发病率(7.90%)和病情指数(5.40)大于生殖生长期的发病率(3.43%)和病情指数(2.47);青枯菌在发病2级的植株及根际土壤中分布最多,分别为营养生长期的81.80×10^(5) cfu·g^(−1)和生殖生长期的99.31×10^(5) cfu·g^(−1);强致病力青枯菌在发病植株和根际土壤中的分布量分别大于104 cfu·g^(−1)和105 cfu·g^(−1)。茄子青枯发病率与气温、地温、相对湿度、雨量、光照辐射和二氧化碳浓度呈极显著正相关(P<0.01),与光照度和土壤容积含水率呈显著正相关(P<0.05)。研究结果为青枯病预测预报技术的建立及病害的有效防控提供依据。

华南地区茄科作物青枯病发生与病原菌分布特性分析

调查我国华南地区番茄、茄子和辣椒等茄科作物青枯病害发病情况,采集健康植株和青枯病发病株及其根际土壤样本,进行病原菌的分离鉴定、致病力测定和分布特性分析。结果表明:番茄、茄子和辣椒发病率最高的地区分别是海南陵水、福建晋安和福建邵武,其生育期内平均发病率分别达20.01%、5.24%和23.33%。从不同地理来源的茄科作物中,共分离鉴定出86株青枯菌。进一步利用弱化指数(Attenuation index,AI)对分离的青枯菌进行致病力划分,其中7株为无致病力菌株(AI>0.75),13株为过渡型菌株(0.65≤AI≤0.75),66株为强致病力菌株(AI<0.65)。研究青枯菌的分布特性发现,对于不同寄主,青枯菌分布特性均是病株及其根际土壤分布数量显著多于健康植株及其根际土壤,且分布数量均是土>根>茎,此外,调查的茄科作物中,青枯病发病株土壤青枯菌分布数量均大于106 cfu·g^(-1),青枯病发病株根和茎青枯菌分布数量均大于105 cfu·g^(-1)。

青枯菌Ⅲ型效应蛋白调控植物免疫研究进展

青枯菌(Ralstonia solanacearum)通常从寄主植物根部侵染维管束系统,引发青枯病(Bacterial wilt),造成植物产生不可逆的萎蔫、死亡。青枯病是一种毁灭性土传细菌病害,目前已成为制约我国茄科作物生产的主要病害之一。Ⅲ型分泌系统(TypeⅢsecretion system,T3SS)在青枯菌侵染寄主过程中发挥重要作用,青枯菌利用T3SS向寄主细胞分泌大量Ⅲ型效应蛋白(TypeⅢeffectors,T3Es)以干扰寄主的免疫反应。国内外学者致力于探索青枯菌如何通过识别寄主体内的靶标蛋白来干扰寄主细胞、调控寄主免疫功能。因此,对青枯菌T3Es靶向识别的植物蛋白进行鉴定有助于了解青枯菌T3Es的生物学功能、青枯菌的致病过程,同时也有助于挖掘寄主中青枯病抗性相关蛋白及其作用机制。尽管大多数T3Es功能仍然未知,但目前已报道的青枯菌T3Es可通过多种分子机制干扰植物先天免疫、诱导植物发生超敏反应、影响植物代谢和激素信号传导通路等来调节寄主抗青枯病能力。该文综述青枯菌T3Es的特征、功能鉴定方法及其参与调控青枯菌致病性及植物免疫机制的研究进展,并对青枯菌T3Es与寄主互作机制研究进行展望,有助于更加深入地了解青枯菌的致病机理及寄主免疫系统的应答机理,为青枯菌诱导寄主抗性机制的解析及青枯病的防治提供有效参考。

福建猕猴桃细菌性枯萎病的病原菌鉴定

为明确近年来在福建省宁德市蕉城区石后乡猕猴桃苗圃新发生的一种细菌性病害病的原菌,并进一步探讨其发生流行规律,采用稀释分离法对采集病株进行病原菌的分离和纯化,对获得的细菌菌株进行致病性测定,通过柯赫氏法则验证、常规的细菌生物学特性、生理生化反应、Biolog鉴定、16S rDNA和内切葡聚糖酶基因(egl)序列分析对病原菌进行分类鉴定。结果表明,从发病猕猴桃病株上获得9株细菌菌株,将其接种在健康的猕猴桃植株后,其发病症状与田间自然发病症状基本一致,且从接种后病株的茎秆和根部又重新分离到与原菌落形态相同的细菌。柯赫氏法则证实这9株细菌菌株为猕猴桃细菌性枯萎病的致病菌。这9株菌株在NA培养基上的菌落形态一致,均呈不规则型、扁平,并逐渐向四周扩散,不产生粘稠状物质,不会使培养基变色;在TTC培养基上,菌落颜色呈暗红色,产生可扩散至培养基中的红褐色色素,其流动性较差;在YDC培养基上,菌落呈乳白色。经综合分析,这9株细菌菌株均被鉴定为假茄科雷尔氏菌(Ralstonia pseudosolanacearum),生化型Ⅲ和演化型Ⅰ(亚洲组),序列变种14。这是首例青枯菌侵染猕猴桃的报道。以上研究结果为制定猕猴桃细菌性枯萎病精准有效的综合防控提供了理论依据。

Developing a bacteriophage cocktail for biocontrol of potato bacterial wilt

Bacterial wilt is a devastating disease of potato and can cause an 80% production loss. To control wilt using bacteriophage therapy, we isolated and characterized twelve lytic bacteriophages from different water sources in Kenya and China. Based on the lytic curves of the phages with the pathogen Ralstonia solanacearum, one optimal bacteriophage cocktail, P1, containing six phage isolations was formulated and used for studying wilt prevention and treatment efficiency in potato plants growing in pots. The preliminary tests showed that the phage cocktail was very effective in preventing potato bacterial wilt by injection of the phages into the plants or decontamination of sterilized soil spiked with R. solanacearum. Eighty percent of potato plants could be protected from the bacterial wilt(caused by R. solanacearum reference strain GIM1.74 and field isolates), and the P1 cocktail could kill 98% of live bacteria spiked in the sterilized soil at one week after spraying.However, the treatment efficiencies of P1 depended on the timing of application of the phages, the susceptibility of the plants to the bacterial wilt, as well as the virulence of the bacteria infected,suggesting that it is important to apply the phage therapy as soon as possible once there are early signs of the bacterial wilt. These results provide the basis for the development of bacteriophagebased biocontrol of potato bacterial wilt as an alternative to the use of antibiotics.

A bacterial effector protein uncovers a plant metabolic pathway involved in tolerance to bacterial wilt disease

Bacterial wilt caused by the soil-borne plant pathogen Ralstonia solanacearum is a devastating disease worldwide.Upon plant colonization,R.solanacearum replicates massively,causing plant wilting and death;collapsed infected tissues then serve as a source of inoculum.In this work,we show that the plant metabolic pathway mediated by pyruvate decarboxylases(PDCs)contributes to plant tolerance to bacterial wilt disease.Arabidopsis and tomato plants resp ond to R.solanacearum infection by in creasing PDC activity,and plants with deficient PDC activity are more susceptible to bacterial wilt.Treatment with either pyruvic acid or acetic acid(substrate and product of the PDC pathway,respectively)enhances plant tolerance to bacterial wilt disease.An effector protein secreted by R.solanacearum,RipAK,interacts with PDCs and inhibits their oligomerization and enzymatic activity.Collectively,our work reveals a metabolic pathway involved in plant resistance to biotic and abiotic stresses,and a bacterial virulence strategy to promote disease and the completion of the pathogenic life cycle.

The relative importance of soil moisture in predicting bacterial wilt disease occurrence

Soil-borne plant diseases cause major economic losses globally.This is partly because their epidemiology is difficult to predict in agricultural fields,where multiple environmental factors could determine disease outcomes.Here we used a combination of field sampling and direct experimentation to identify key abiotic and biotic soil properties that can predict the occurrence of bacterial wilt caused by pathogenic Ralstonia solanacearum.By analyzing 139 tomato rhizosphere soils samples isolated from six provinces in China,we first show a clear link between soil properties,pathogen density and plant health.Specifically,disease outcomes were positively associated with soil moisture,bacterial abundance and bacterial community composition.Based on soil properties alone,random forest machine learning algorithm could predict disease outcomes correctly in 75%of cases with soil moisture being the most significant predictor.The importance of soil moisture was validated causally in a controlled greenhouse experiment,where the highest disease incidence was observed at 60%of maximum water holding capacity.Together,our results show that local soil properties can predict disease occurrence across a wider agricultural landscape,and that management of soil moisture could potentially offer a straightforward method for reducing crop losses to R.solanacearum.

番茄青枯病病原菌生物学特性及致病机制研究进展

由青枯雷尔氏菌引起的青枯病给番茄产业发展带来了严重挑战,青枯病的侵染造成番茄产量和质量严重受损。在总结番茄青枯病病原菌生物学特性的基础上,分析了番茄青枯病病原菌的致病机理及其对作物产量和质量的危害,阐述了青枯菌的致病效应蛋白,提出了番茄青枯病生物防治方法,以期为全面了解和有效防治番茄青枯病提供参考。

番茄青枯病生防芽胞杆菌的筛选与鉴定

为了有效利用芽胞杆菌资源,本研究采用抑菌圈法从不同地理来源的芽胞杆菌中筛选出24个对青枯雷尔氏菌具有拮抗作用的菌株。其中,6个菌株对青枯雷尔氏菌的抑菌圈直径大于14.00 mm,菌株FJAT-11709的抑菌圈直径最大,为14.78 mm。盆栽试验比较了6个菌株对番茄青枯病的防治效果,结果表明,菌株FJAT-20261和FJAT-19700防效最好,分别达72.73%和67.77%。通过形态特征、生理生化测定及16S r RNA基因序列分析,菌株FJAT-20261和FJAT-19700分别被鉴定为耐寒短杆芽胞杆菌和特基拉芽胞杆菌。本文报道这2种芽胞杆菌对青枯雷尔氏菌具有拮抗作用,为青枯病的生物防治提供了新的菌株资源。

烟草根际细菌铜绿假单胞菌swu31-2的定殖能力及其对烟草青枯病的防治作用

从重庆黔江烟草田间分离获得一株烟草青枯菌拮抗菌株Pseudomonas aeruginosaswu31-2(简称swu31-2)。采用逐步提高药物浓度的方法,筛选获得了抗链霉素300μg/mL对烟草青枯病菌拮抗活性稳定的swu31-2突变菌株。采用灌根接种法,研究其在烟草根、茎和叶表面及内部的定殖能力及其对烟草青枯病的防治作用。结果表明,swu31-2能在烟草各组织的表面及内部定殖。该菌株在烟草各组织内部的数量均表现为"由增到减"的趋势。在接种后第8天定殖数量达到最高峰,随后有所下降;到第20天各组织内的数量仍然维持较高水平(105cfu/g以上)。同时,在接种20 d后,烟草的根、茎和叶的表面仍然可以检测到swu31-2的存在。盆栽试验结果表明,swu31-2的菌液和活性物质对烟草青枯病均有一定的防治效果。其中先施swu31-2菌液和活性物质粗提物的防效好于农用链霉素(51.25%),分别为60.87%和60.32%,而后施菌液和活性物质粗提物的防效也分别达39.50%和20.90%。

利用无致病力青枯菌株防治番茄青枯病的研究

自发突变的无致病力番茄青枯菌株Tm3具有产生细菌素的能力，对番茄青枯菌株Tm46有较强的抑菌作用，且能诱导烟草植株产生过敏性反应．盆栽和小区试验结果表明，Tm3具有较好的防治番茄青枯病的能力．

水杨酸诱导桉树抗青枯病的作用及相关酶活性变化

在毒性测定中,1～20mmol水杨酸(salicylicacid,SA)对桉树青枯病菌的生长无抑制作用。通过淋根,1～5mmol的SA可以诱导桉树苗显著地增强对青枯病的抗性,但以5mmol最佳。过氧化物酶(POX)和多酚氧化酶(PPO)的活性,在0 1～5mmolSA范围内,随着浓度的升高相应增强;SA淋根3～9d后挑战接种青枯病菌均能诱导桉树显著地提高对青枯病的抗性,但以间隔5～7d效果最好,且POX和PPO的酶活性与抗病性的变化趋势相符,均在第7天升至最高值,分别比对照高2倍和1倍。POX酶灵敏快速,更能准确地反映植株抗病性的变化;叶片注射SA不能诱导桉树抗青枯病。

抗烟草青枯病菌的枯草芽孢杆菌SH7的筛选与鉴定

由青枯雷尔氏菌(Ralstonia solanacearum)侵染引起的烟草青枯病是我国烟区的主要病害之一,严重影响烟草的产量和品质。从云南和贵州烟草青枯病重病区采集600份健康烟草根基土壤,并从中筛选出1株对R.solanacearum具有较强拮抗活性的细菌菌株SH7,该菌株在室内平板抑菌试验中,对R.solanacearum抑菌带宽可达13.2 mm。预先用SH7无菌发酵液保护烟株根部,对R.solanacearum防效达到66.0%。SH7发酵液经70%(NH4)2SO4饱和度沉淀并透析后得到的蛋白粗提液经平板抑菌活性检测,对R.solanacearum具有很强的抑菌活性,抑菌带宽为15.7 mm。初步确定SH7菌株产生的主要抑菌活性物质为蛋白多肽类物质。经菌体形态学、生理生化测定和16s rDNA序列测定,结果表明:SH7菌株为枯草芽孢杆菌(Bacillus subtilis)。