Limonene’s Bacteriostatic Activity against Ralstonia solanacearum by Compromised Membrane Integrity: A Transcriptomic and Metabolomic Analysis

Ralstonia solanacearum, the causative agent of bacterial wilt, is a soil-borne pathogen that poses a widespread threat to plants in the Solanaceae family. To elucidate the mechanism by which limonene exerts its effects on R. solanacearum, we first assessed the impact of limonene on the physiological indicators of the pathogen and subsequently analyzed its transcriptome and metabolome. Our findings indicate that limonene has a potent inhibitory effect on R. solanacearum, and it also suppresses the formation of the bacterial community biofilm. Limonene primarily regulates the terpene biosynthesis pathway in R. solanacearum, thereby potentially affecting signal transduction in the pathogen and disrupting its normal growth and development. These results significantly enhance our understanding of limonene’s response to the induction of bacterial wilt and provide a reference for further prevention and control of R. solanacearum.

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.

Identification and Susceptibility Test of Ralstonia solanacearum Isolated from White Burley

[ Objective ] The paper was to isolate and identify Ralstonia solanacearum from white burley, and determine its susceptibility to 6 fungicides. [ Mcth- od] Using the combination method of semiselective medium （PCCG） and apolymerase chain reaction （PCR） technique, R. solanacearum in stalk of white burley from Dazhou City in Sichnan Province was isolated, and its biochemical type was identified. Through susceptibility test, the susceptibility of R. solanacearum to bismerthiazol, ethylicin, streptomycin, lime sulfur, 47% polylysine, 99% kojic acid was studied in laboratory. [Result] A total of 23 strains OfR. solanacearum were isolated, all belonging to biochemical type Ill. R. solanacearum obtained in the test were more susceptible to ethylicin, streptomycin and bismerthiazol, and ethylicin had good control effect against R. solanacearum with ECso of 0.086 ml/L. [ Conclusion ] The study provide theoretical basis for control of R. solanaceanon in white burley.

利用GFP标记的Ralstonia solanacearum鉴定马铃薯青枯病抗性

青枯病是危害马铃薯产业的重要病害,选育与推广抗青枯病品种是防控青枯病最高效的手段。目前已有的人工接种和田间抗性鉴定的方法不能有效区分处于潜伏侵染与完全抵抗侵染的材料,而利用GFP标记的Ralstonia solanacearum能够有效解决上述问题。笔者通过电击转化法将广宿主载体pBBR1MCS2-Tac-EGFP导入青枯菌P2中,成功获得了能够稳定遗传且不影响其致病力并带有绿色荧光报告的青枯菌菌株P2-Tac-EGFP。通过对感抗材料进行接种,结果表明,P2-Tac-EGFP能够有效区分感抗材料,并且P2-Tac-EGFP在感病材料的根部和茎部大量分布,而在抗病材料的根部仅有少量分布。综上所述,利用GFP标记的R.solanacearum能够快速准确地鉴定马铃薯青枯病抗性。

植物病原细菌Ralstonia solanacearum GMI1000中分泌蛋白信号肽分析

利用SignalP3.0、TMHMM2.0、TargetP1.01、LipoP1.0和PSORTb蛋白分析软件并结合L值计算,对植物病原细菌Ralstonia solanacearum GMI1000菌株基因组中的全部3440个ORFs进行了分析预测,确定其中186个ORFs所编码蛋白质的N-端有信号肽序列,且它们的氨基酸残基相对保守。其中134条具有分泌型信号肽,22条具有RR-motif型信号肽,30条具有信号肽酶Ⅱ型信号肽。对各类信号肽及其结构域的长度作了系统的分析。未发现Prepilin-like信号肽和细菌素和信息素信号肽。

应用计算机手段分析植物病原细菌Ralstonia solanacearum的蛋白质序列

应用SignalP 3.0,LipoP和TargetP对植物病原细菌Ralstonia solanacearum基因组中的3440个ORFs(openreading frames)进行了信号肽分析,同时系统分析了信号肽的类型及结构。结果表明,3440个ORFs中有462个ORFs所编码蛋白质具有N-端有信号肽序列,其中348个分泌类信号肽、100个信号肽具有RR-motif信号肽,14个脂蛋白类信号肽,未发现Prepilin-like信号肽和Bacteriocin and Pheromone信号肽。在这462个具有可切割信号肽的分泌蛋白中,有84.0%蛋白质为胞外分泌型(S型),13.2%为线粒体分泌型(M型),另外有2.8%为其它类型的分泌蛋白。通过LipoP分析该菌的全基因组预测具有4种类型蛋白质,其中其中SpI有425个,占12.3%;SpII有80个,占2.3%;CYT有2 541个,占73.9%;TMH有414个,占12.0%。比较了Ralstonia solanacearum和Pseudomonas syringaepv.tomato信号肽的长度及氨基酸的组成,发现这两种菌在这些方面存在这很大程度的相似性。本研究通过对Ralstonia solanacearum进行分泌蛋白和信号肽结构的分析,为将来功能基因组和分泌型外源蛋白的利用提供理论基础。

繁殖体数量对外来Ralstonia solanacearum在红壤中入侵潜力的影响

外来种的繁殖体压力因可调控其他入侵影响因素(如外来种特性)而备受入侵生态学家的关注。前人研究指出剂量-响应曲线能够定量分析繁殖体数量对入侵潜力的影响,但关于该曲线形状的认识尚未统一。利用土壤微宇宙,比较了初始接种量为10^3(PP3)、10^5(PP5)、10^7(PP7)和10^9 CFU·g^-1(PP9)的青枯病菌Ralstonia solanacearum进入土壤3和42 d后的存活量,并对初始接种量和存活量之间关系进行拟合,试图探清地下部微生物入侵的剂量-响应曲线状况。研究发现,不同接种量处理外来R.solanacearum接入土壤3 d后的存活量差异显著(P<0.05),从大到小依次为PP9、PP7、PP5和PP3,初始接种量和存活量之间拟合的剂量-响应曲线为指数型;而外来Ralstonia solanacearum接入土壤42 d后,除了初始接种量为109 CFU·g^-1处理的存活量稍高外,其余处理间差异不显著,此时的剂量-响应曲线呈直线型(斜率约为0)。结果表明,外来病原菌入侵土壤时的剂量-响应关系会随其进入土壤时间的延长而改变,前期为指数型,后期转为直线型,这说明外来R.solanacearum在红壤中的入侵潜力在入侵前期随繁殖体数量增加而呈指数增长,在入侵后期不受繁殖体数量的影响。

植物病原细菌(Ralstonia solanacearum)染色体基因组分泌信号肽计算机分析

应用SignalP 3.0、TMHMM 2.0、THUMBUP、big-PI、TargetP1.01、Lipop 1.0、TatP 1.0等7种软件对植物病原细菌Ralstonia solanacearum GMI1000染色体基因组3448个氨基酸序列进行预测分析。结果表明,该物种染色体基因组分泌信号肽ORFs有178个,占其基因组编码ORFs的5.2%,其中有150个ORFs属于Ⅰ型分泌信号肽,28个ORFs属于Ⅱ型分泌信号肽。在178个ORFs中具RR-motif信号肽结构的有13个,其中11个属于Ⅰ型信号肽,2个属于Ⅱ型信号肽。通过软件预测未发现Comc型信号肽与细菌素-信息素型信号肽结构。在染色体分泌信号肽ORFs中,有116个具可预测功能,其余62个为功能未知的推定蛋白。已知功能主要集中于细胞代谢、细胞调控及转运、细胞膜结构等领域,这些功能是该物种在长期进化过程中与环境中各种因子发生互作,相互适应的结果。

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.

Physiological response and phenolic metabolism in tomato (Solanum lycopersicum) mediated by silicon under Ralstonia solanacearum infection

Bacterial wilt, caused by Ralstonia solanacearum（Rs） is a serious soil-borne disease and silicon can enhance tomato resistance against this disease. However, few studies have focused on the mechanisms of Si-mediated pathogen resistance from the rhizosphere perspective. In this study, two tomato genotypes, HYT（susceptible） and H7996（resistant）, were used to investigate the effects of silicon application on disease inhibition, root growth, and organic acid content in both roots and root exudates under R. solanacearum infection. The results showed that Si application significantly suppressed bacterial wilt in HYT, but had no effect in H7996. Silicon concentrations in roots, stems and leaves of tomato were significantly increased by Si treatment under R. solanacearum inoculation. In HYT, Si application increased root dry weight by 22.8-51.6% and leaf photosynthesis by 30.6-208.0%, and reduced the concentrations of citric acid in root exudates by 71.4% and in roots by 83.5%. However, organic acids did not influence R. solanacearum growth. Results also demonstrated that salicylic acid（SA） content in roots was significantly increased by silicon addition for H7996 and exogenous SA application could reduce bacterial wilt disease index. Collectively, these results suggest that Si-modulated phenolic compound metabolism in roots or root exudates, especially citric acid and SA, may be a potential mechanism in the amelioration of bacterial wilt disease by Si.

pBBR1MCS2-Tac-EGFP广宿主载体适宜标记Ralstonia solanacearum

利用标记基因追踪病原菌在植物体内的入侵和定殖,是研究病原菌-寄主互作的重要手段。本研究利用电转化法将广宿主载体pBBR1MCS2-Tac-EGFP导入青枯雷尔氏菌(Ralstonia solanacearum)GMI1000菌株中,获得了青枯雷尔氏菌带绿色荧光标记的转化子。转接试验结果表明,转化子的抗生素抗性和绿色荧光强度有良好的遗传稳定性。pBBR1MCS2-Tac-EGFP不影响GMI1000菌株的致病力,且EGFP蛋白能够在植物中稳定表达。灌根法接种试验结果表明,病原菌在第1天即完成对根系的侵染,并在第6天扩散至其他组织,随后造成植株萎蔫。研究结果表明所获转化子可用于后续的病原菌侵染机理等方面的研究。

Differential Space-Time Expression of StLTPbl Gene Between Resistant and Susceptible Potato Genotypes in Response to Ralstonia solanacearum

This study is to investigate the role of lipid transfer protein （LTP1） gene of potato （Solanum tuberosum） in bacterial wilt （Ralstonia solanacearum） resistance. A novel cDNA clone encoding nsLTP was isolated from cultivated potato （Solanum tuberosum） infected with R. solanacearum by 5＇-rapid amplification of cDNA ends （RACE）. The temporal and spatial expression of StLTPbl was studied during the early stages of potato-R, solanacearum interaction by reverse transcriptase PCR （RT-PCR） and Northern blotting. The sequence analysis of the cloned cDNA, named StLTPbl, showed 691 bp which encoded a type 1 nsLTP of 91 amino acids. Construction of a phylogenic tree showed that StLTPbl is well conserved in the coding region with high identity at the amino acid level with other Solanaceae nsLTPs. The temporal and spatial expression of StLTPbl was studied during the early stages of potato-R, solanacearum interaction. StLTPbl transcription is induced faster and transcripts accumulate to higher concentrations in resistant compared with susceptible genotypes by the pathogen. Dominant differences in the pathogen-induced gene expression pattern between the upper and lower leaves and stems were observed within the same genotypes. In situ hybridization results showed that the StLTPbl mRNA was localized in phloem cells of vascular tissues in potato leaf and stem tissues after pathogen infection. Salicylic acid, methyl jasmonate and abscisic acid could induce StLTPbl gene expression without significant difference between the upper and lower tissues. These abiotic elicitors could produce a long-lastingeffect on the StLTPbl during early stages of potato-R, solanacearum interaction. Differential expression of StLTPbl gene between resistance and susceptible potato genotypes in response to R. solanacearum suggests that this gene plays a key role in plant defense mechanisms.

Antibacterial Activities of Three Kinds of Plant Extracts against Ralstonia solanacearum of Cherry Tomato

Bacterial wilt has become an important obstacle restricting high quality and high yield of cherry tomato. In this paper, the antibacterial activities of ex- tracts from Allium sativum, Ginkgo biloba and Lygodium japonicum against Ralstonia solanacearum were compared. The results indicated that with the increasing concentrations of three extracts, the antibacterial effect enhanced gradually. The lowest concentrations of three extracts with the inhibitory effects significantly higher than that of pesticide control were 20% A. sativum extract, 40% G. biloba extract and 60% L japonicum extract, and the antibacterial effect of 20% A. sativum extract was significantly higher than those of other two plant extracts. The inhibition zone diameters of R. solanacearum strains B2. 1.2.2, I1.2. 1.1, KS. 1.1.1 and 1.5.3.1.2 were 9.23, 8.30, 8.12 and 9.19 mm, respectively. The antibacterial effects of three plant extracts reached the maximums when the concentration was 100%, but A. sativum extract had the strongest antibacterial effect against test strains. Therefore, A. sativum can be used as the main raw material for development of plant protectant for cherry tomato.

Quantitative Gene Expression of Peroxidase, Polyphenoloxidase and Catalase as Molecular Markers for Resistance against Ralstonia solanacearum

Brown rot or bacterial wilt of potato caused by Ralstonia solanacearum, is an economically important disease. Potato, cv. Nicola, was found to be relatively highly resistant to the infection with R. solanacearum and showed 15.12% wilt disease index, meantime, cv. Kara showed intermediate resistance with 37.40% disease index while, cv. Spunta was susceptible and showed 80.33% disease index. The role of defense-related enzymes in imparting resistance in potato against R. solanacearum was investigated by quantifying enzymes activity and gene expression of three defense- related enzymes, peroxidase, polyphenol oxidase and catalase. Peroxidase showed maximum activity 0.488 min-1·g-1 early at 12 h after pathogen inoculation in the cv. Nicola, whereas in susceptible cultivar Spunta showed lower activity of maximum 0.226 min-1·g-1 later at 48 h after inoculation. While, the moderately resistant cultivar Kara showed intermediate activity for the peak and its time. Meanwhile, polyphenol oxidase showed similar trends to that of peroxidase. On the contrary, catalase showed the highest activity values in the susceptible, cv. Spunta, while, in relatively highly resistant (cv. Nicola) and the moderately resistant (cv. Kara) showed lower values of activity and up to 96 h after inoculation. Meanwhile, gene expression of related enzymes the RT-PCR was used. At zero time, the relatively highly resistant potato cultivar, Nicola, showed the highest values of gene expression for both Peroxidase (POD) and Poly Phenol Oxidase (PPO). While, the susceptible potato cultivar, Spunta showed the lowest values. On the contrary, Catalase (CAT) gene expression was the highest in the susceptible, cv. Spunta, and was the lowest in the relatively highly resistant, cv. Nicola, while, was of intermediate values in the intermediate resistance, cv. Kara. Results show that peroxidase and polyphenol oxidase activities can be used as biochemical markers to reveal the resistance and susceptibility nature of potato cultivars against bacterial wilt disease of potato caused by R. solanacaerum.

Davana： A New Host Plant for Ralstonia solanacearum from India

Ralstonia solanacearum infecting Davana （Artemisia pallens Wall.） from commercial nurseries in India was isolated on modified semi selective media （SMSA）. Here, we report a new host for Ralstonia solanacearum i.e. davana. It has huge demand in medicinal and aromatic industries. Isolate was confirmed as race-l, biovar-3 by morphological, physiological, biochemical and pathogenicity studies. Two sets of primers （OLI 1 ＆ Y2 and Y 1 ＆ Y2） were used in this study. Further, the identity of the isolate was confirmed by serological diagnostic kit obtained from International Potato Research Center, Lima, Peru and single chain variable fragment antibody specific to Ralstonia solanacearum used to confirm the casual organism.

Morphological, Pathological, Biochemical and Molecular Characterization of <i>Ralstonia solanacearum</i>Isolates in Bangladesh

One thirty samples (fifty-five potato tubers, twenty-seven potato stems, three chili stems, twenty-eight soil samples, five weed samples, three banana leaves, and nine water samples) were examined and one hundred six (106) Bangladeshi isolates of Ralstonia solanacearum were isolated and identified. Isolation was made on selective media (Tetrazolium chloride media) and R. solanacearum was identified based on morphological, pathological and biochemical properties and polymerase chain reaction (PCR) by using the species-specific primers. Studies showed that 81.54% (106) samples were positive on tetrazolium chloride solid medium. Among them 90 isolates were virulent and rest of them were avirulent. Fifty isolates were selected for chemical characterization based on hypersensitivity test. R. solanacearum is gram negative, aerobic facultative bacteria on the basis of chemical characterization. Fifty tested isolates expressed as race 3 while in biovar test forty-eight showed as biovar III and the rest two showed as biovar I. In nine tested isolates from the three districts a species-specific band of 280 bp was amplified in PCR that confirmed the identity of R. solanacerum.

Advances on the Molecular Mechanism of the Interaction between Ralstonia solanacearum and Hosts

Ralstonia solanacearum is an important model phytopathogenic bacterium that causes bacterial wilt disease on many plant species and leads to serious economic losses. The interactions between R. solanacearum and host plants have become a model system for the study of plants and pathogens interactions. This paper reviews the advances on the molecular mechanisms between R. solanacearum and hosts interaction including the formation of plant innate immunity, the suppression of plant innate immunity by this pathogen and the activation of effector-triggered immunity. Furthermore, we made a prospect on how to utilize the interaction mechanism between R. solanacearum and hosts to control the disease.

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.

Detection and Analysis of Number of Ralstonia solanacearum in Soil before Winter Tillage in Fuzhou Tobacco-growing Area, Jiangxi Province

[Objective]The paper was to optimize the tobacco planting area in Fuzhou City and to prevent the outbreak of tobacco bacterial wilt in large area.[Method]At the end of 2017,soil samples were collected from plots planned to be planted with tobacco in the following year in Yihuang,Guangchang,Lichuan and Le’an counties.[Result]Among 352 plots,116 plots were infected by Ralstonia solanacearum,while 236 plots were free of the pathogen,and the infected plots accounted for 32.95% of total plots.Among them,75 plots exceeded the order of magnitudes of 103,accounting for 21.31% of total plots and 64.66% of infected plots.It is suggested that the plots with an order of magnitude above 103 should be pretreated with quicklime or purple soil,or conducted crop rotation,or seeds must be directly abandoned;the dosage of biocontrol agents should be increased in planting.The plots with an order of magnitude below 103 should be pretreated with quicklime or purple soil,and the dosage of biocontrol agents should be increased in planting.[Conclusion]The results provide reliable theoretical basis and data support for soil improvement and bacterial wilt control.

Rapid Method for Isolation of PCR Amplifiable Genomic DNA of <i>Ralstonia solanacearum</i>Infested in Potato Tubers

The aim of the present study was to develop a very fast and simple genomic DNA isolation method for Ralstonia solanacearum which infest potato tubers. One hundred potato tubers were collected and ten composite samples were prepared having 10 tubers each. Four different DNA isolation methods were used for bacterial genomic DNA isolation present in tubers. PCR with R. solanacearum specific primers and pathogenicity tests were performed. Out of four methods two gave PCR amplifiable DNA. The simplest method was boiling the cell lysate for 5 min, vortexing for 2 min then extraction with phenol chloroform method. This method provides significant amount of DNA which is free from contaminants thus rendering the DNA amicable to PCR amplification. The developed method would be useful for quick and sensitive detection of this pathogen in seed potatoes and would be beneficial to stop the further spread of pathogen.