Comparative genomic analysis of Ralstonia solanacearum reveals candidate genes for host specificity

Background

Ralstonia solanacearum is a vascular soil-borne plant pathogen with an unusually broad host range. This economically destructive and globally distributed bacterium has thousands of distinct lineages within a heterogeneous and taxonomically disputed species complex. Some lineages include highly host-adapted strains (ecotypes), such as the banana Moko disease-causing strains, the cold-tolerant potato brown rot strains (also known as R3bv2) and the recently emerged Not Pathogenic to Banana (NPB) strains.

Results

These distinct ecotypes offer a robust model to study host adaptation and the emergence of ecotypes because the polyphyletic Moko strains include lineages that are phylogenetically close to the monophyletic brown rot and NPB strains. Draft genomes of eight new strains belonging to these three model ecotypes were produced to complement the eleven publicly available R. solanacearum genomes. Using a suite of bioinformatics methods, we searched for genetic and evolutionary features that distinguish ecotypes and propose specific hypotheses concerning mechanisms of host adaptation in the R. solanacearum species complex. Genome-wide, few differences were identified, but gene loss events, non-synonymous polymorphisms, and horizontal gene transfer were identified among type III effectors and were associated with host range differences.

Conclusions

This extensive comparative genomics analysis uncovered relatively few divergent features among closely related strains with contrasting biological characteristics; however, several virulence factors were associated with the emergence of Moko, NPB and brown rot and could explain host adaptation.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1474-8) contains supplementary material, which is available to authorized users.     

StructureActivity Relationships of Antimicrobial Gallic Acid Derivatives from Pomegranate and Acacia Fruit Extracts against Potato Bacterial Wilt Pathogen

Bacterial wilts of potato, tomato, pepper, and or eggplant caused by Ralstonia solanacearum are among the most serious plant diseases worldwide. In this study, the issue of developing bactericidal agents from natural sources against R. solanacearum derived from plant extracts was addressed. Extracts prepared from 25 plant species with antiseptic relevance in Egyptian folk medicine were screened for their antimicrobial properties against the potato pathogen R. solancearum by using the disc‐zone inhibition assay and microtitre plate dilution method. Plants exhibiting notable antimicrobial activities against the tested pathogen include extracts from Acacia arabica and Punica granatum. Bioactivity‐guided fractionation of A. arabica and P. granatum resulted in the isolation of bioactive compounds 3,5‐dihydroxy‐4‐methoxybenzoic acid and gallic acid, in addition to epicatechin. All isolates displayed significant antimicrobial activities against R. solanacearum (MIC values 0.5–9 mg/ml), with 3,5‐dihydroxy‐4‐methoxybenzoic acid being the most effective one with a MIC value of 0.47 mg/ml. We further performed a structure–activity relationship (SAR) study for the inhibition of R. solanacearum growth by ten natural, structurally related benzoic acids.     

Direct Delivery of Inoculum to Shoot Tissue Interferes with Genotypic Resistance to Ralstonia solanacearum in Tomato Seedlings

Employing known susceptible and resistant genotypes and pure bacterial inoculum (0.1 OD; 10⁸ CFU/ml^?1), five different inoculation methods were tried to assess the response of tomato genotypes to Ralstonia solanacearum. This included seed‐soaking inoculation, seed‐sowing followed by inoculum drenching, or at 2‐week stage through petiole‐excision inoculation, soaking of planting medium with inoculum either directly or after imparting seedling root‐injury. Seed‐based inoculations or mere inoculum drenching at 2 weeks did not induce much disease in seedlings. Petiole inoculation induced 90–100% mortality in susceptible checks but also 50–60% mortality in normally resistant genotypes within 7–10 days. Root‐injury inoculation at 2‐week seedling stage appeared the best for early and clearer distinction between resistant and susceptible lines. The observations suggest a role played by the root system in governing genotypic resistance to the pathogen. Direct shoot inoculation is to be adopted only for selecting highly resistant lines or to thin down segregating populations during resistance breeding.     

Evaluation of garlic intercropping for enhancing the biological control of Ralstonia solanacearum in flue-cured tobacco fields

Tobacco bacterial wilt, Ralstonia solanacearum, is an important disease affecting the root and stem. The disease causes extensive damage to flue-cured tobacco all over the word. Field trials were conducted in 2008 and 2009 at Longyan, Fujian Province, China, to evaluate garlic intercropping for enhancing the biological control of R. solanacearum in flue-cured tobacco fields. The results of the study demonstrate that tobacco bacterial wilt was clearly inhibited by intercropping garlic in 2008 and 2009. The appearance of the disease in intercropped fields was delayed for about 15 days. The total number of R. solanacearum in root system soils was significantly lower in intercropped fields than in monocultured fields in 2008. These numbers were between 138×10⁴ and 161×10⁴ cfu g^–1 dry soil in intercropped fields. The corresponding values in monocultured fields were 357×10⁴ cfu g^–1 dry soil. The monetary value of tobacco leaves was obviously higher in intercropped fields than in monocultured ones. The per cent increase in monetary values in the intercropped fields was between 14 and 34%. Consequently, intercropping tobacco with garlic might be very useful for enhancing biological control of R. solanacearum in flue-cured tobacco fields.     

Potato bacterial wilt suppression and plant health improvement after application of different antioxidants

Bacterial wilt caused by Ralstonia solanacearum is a devastating disease that often threatens potato production and exportation. The potential of four antioxidants (seaweed extract (SWE), yeast, chitosan and ascorbic acid (ASA)) in controlling the disease was evaluated in vitro, under glasshouse and field conditions. The field experiment was conducted in two naturally infested locations: Wardan, Giza (sandy soil), and Talia, Minufiya (silty clay soil). Only chitosan showed antibacterial properties against the pathogen in vitro. SWE, yeast and chitosan showed disease suppression under both glasshouse and field conditions. The disease suppression was accompanied by an increase in the ratio of soil copiotrophic to oligotrophic bacteria. The three antioxidants increased plant nitrogen content, decreased soil OM content and decreased C/N ratio. Disease suppression after chitosan application was clearly observed only in Wardan area, which was characterized by a higher soil alkalinity. A high percentage of antagonistic fluorescent strains similar to Pseudomonas putida group were detected for chitosan‐treated plants in Wardan area (sandy soil). ASA drastically decreased the count of the pathogen in soil, but was conducive to the pathogen in plant tissues. A remarkable increase in microbial (bacterial and fungal) soil and rhizosphere diversity as indicated by PCR‐DGGE analysis for bacterial 16S rRNA and fungal 18S rRNA was recorded. In Talia area (silty clay soil), the soil microbial community was more stable and was in general resistant to the disease where the soils were characterized by high electrical conductivity. SWE, yeast and ASA significantly increased crop production in Talia area only.     

茄科雷尔氏菌脂酰-CoA合成酶的功能鉴定

【目的】茄科雷尔氏菌是一种常见的农作物致病菌,引起植物青枯病。研究其脂肪酸代谢途径将有助于寻找新的抗菌药物靶点,为防治青枯病害提供新的思路。【方法】利用大肠杆菌FadD序列,进行同源比对发现茄科雷尔氏菌GMI1000中RSc2857(RsFadD)具有较高的相似性,推测其具有脂酰-CoA合成酶活性。采用PCR扩增方法获得RsfadD基因,连入表达载体pBAD24M后互补大肠杆菌fadD突变株,并检测转化子的生长情况。RsfadD与pET-28b连接后,在大肠杆菌BL(DE3)中表达,并利用Ni-NTA纯化获得带有组氨酸标签的RsFadD,体外测定RsFadD的活性。利用同源重组方法,获得RsfadD敲除突变株,分析突变株的生长性状。【结果】RsfadD异体互补大肠杆菌fadD突变株,恢复突变株在以脂肪酸为碳源的基础培养基上生长。体外活性测定RsFadD具有脂酰-CoA合成酶活性,对不同链长的脂肪酸都具有活性,但活性低于大肠杆菌FadD。RsfadD突变株在添加不同链长脂肪酸的基础培养上仅能微弱生长,而在丰富培养基上生长无差异。【结论】茄科雷尔氏菌中RsfadD编码脂酰-CoA合成酶,在脂肪酸利用过程中发挥重要作用。但RsfadD突变株在基础培养基上微弱生长,说明茄科雷尔氏菌基因组中还有其他的脂酰-CoA合成酶基因。以上研究结果为进一步研究茄科雷尔氏菌中脂酰-CoA合成酶以及脂肪酸利用机制奠定了基础。     

Pseudomonads: major antagonistic endophytic bacteria to suppress bacterial wilt pathogen, <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in the eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

Endophytic bacteria of eggplant, cucumber and groundnut were isolated from different locations of Goa, India. Based on in vitro screening, 28 bacterial isolates which effectively inhibited Ralstonia solanacearum, a bacterial wilt pathogen of the eggplant were characterized and identified. More than 50% of these isolates were Pseudomonas fluorescens in which a vast degree of variability was found to exist when biochemical characteristics were compared. In greenhouse experiments, the plants treated with Pseudomonas isolates (EB9, EB67), Enterobacter isolates (EB44, EB89) and Bacillus isolates (EC4, EC13) reduced the wilt incidence by more than 70%. All the selected isolates reduced damping off by more than 50% and improved the growth of seedlings in the nursery stage. Most of the selected antagonists produced an antibiotic, DAPG, which inhibited R. solanacearum under in vitro conditions and might have been responsible for reduced wilt incidence under in vivo conditions. Also production of siderophores and IAA in the culture medium by the antagonists was recorded, which could be involved in biocontrol and growth promotion in crop plants. From our study we conclude that Pseudomonas is the major antagonistic endophytic bacteria from eggplants which have the potential to be used as a biocontrol agent as well as plant growth-promoting rhizobacteria. Large scale field evaluation and detailed knowledge on antagonistic mechanism could provide an effective biocontrol solution for bacterial wilt of solanaceous crops.     

Conversion of volatile fatty acids into polyhydroxyalkanoate by Ralstonia eutropha

Aims:  The aims of this study were to optimize condensed corn solubles (CCS) as a medium for growth of Ralstonia eutropha and to determine the effects of individual volatile fatty acids (VFAs) on polyhydroxyalkanoate (PHA) production .
Methods and Results:  A CCS medium of concentration 240 g l⁻¹ with a carbon : nitrogen ratio of 50 : 1 was developed as the optimal medium. Cultures were grown in 1-l aerated flasks at 250 rev min⁻¹ at 30°C for 120 h. Comparable growth rates were observed in CCS vs a defined medium. At 48 h, VFAs were fed individually at different levels. Optimal levels of all the acids were determined to maximize PHA production. An overall comparison of the VFAs indicated that butyric and propionic acids provided the best results.
Conclusion:  An optimized CCS medium supported growth of R. eutropha . Butyric and propionic acids were the most efficient carbon sources to maximize PHA production when added at the 5 g l⁻¹ level.
Significance and Impact of the Study:  The study shows that a byproduct of ethanol industry can be effectively used as a low cost medium for PHA production, thus partly reducing the cost of commercialization of biopolymers.     

Detection of Ralstonia solanacearum in Soil and Weeds from Commercial Tomato Fields Using Immunocapture and the Polymerase Chain Reaction

A detection assay for Ralstonia solanacearum in soil and weeds was developed by combining immunocapture and the polymerase chain reaction (IC‐PCR). Anti‐R. solanacearum polyclonal antibodies were produced in a white female rabbit and Dynal^® super‐paramagnetic beads were coated with purified immunoglobulinG (IgG). Using IC‐PCR, the 718 bp target DNA was amplified at a detection threshold of approximately 10⁴ colony‐forming units (CFU) bacteria per millilitre of suspension. DNA was not amplified in soil suspensions derived from autoclaved and non‐autoclaved soils, which contained R. solanacearum at 1–10⁵ CFU/g soil. However, a positive PCR result was obtained when bacteria in the soil suspensions were first enriched in nutrient broth. IC‐PCR detected R. solanacearum in tomato stems 24 h after inoculation by stem puncture with a suspension containing approximately 10⁵ CFU/ml. IC‐PCR detected the bacterium in 28 of 55 (51%) weeds and 10 of 32 (31%) soil samples. Of the weeds, Physalis minima, Amaranthus spinosus and Euphorbia hirta had the highest incidence of infection. R. solanacearum was not detected in soil taken from fallow fields, but it was discovered in some weed species. Symptomless tomato and pepper plants collected from the fields in which tomato bacterial wilt had previously occurred were found to contain R. solanacearum. These discoveries suggest that weeds and latent hosts may play a role in the survival of R. solanacearum between cropping cycles.     

花生根分泌物的鉴定及其化感作用

采用改进的根系分泌物循环收集系统收集花生根系分泌物,利用气相色谱/质谱联用仪(GC-MS)鉴定其结构,并研究了花生根系分泌物对花生青枯病原菌的化感作用.结果表明,花生根系分泌物中主要含有丙三醇、苯甲酸、3,5-二甲基苯甲醛、苯乙酮、硬脂酸、棕榈酸和乳酸等7种物质.7种根系分泌物中只有苯乙酮在浓度低于0.1g·L-1时,对花生青枯病原菌生长才有明显的促进作用.同时还发现,苯乙酮浓度超过0.1 gL-1后对花生青枯病原菌有显著的抑制作用.这一结论为利用苯乙酮调控花生青枯病害的发生提供了可靠的依据.