Role of chemotaxis toward fusaric acid in colonization of hyphae of Fusarium oxysporum f. sp. radicis-lycopersici by Pseudomonas fluorescens WCS365

Pseudomonas fluorescens WCS365 is an excellent competitive colonizer of tomato root tips after bacterization of seed or seedlings. The strain controls tomato foot and root rot (TFRR) caused by the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici. Under biocontrol conditions, fungal hyphae were shown to be colonized by WCS365 bacteria. Because chemotaxis is required for root colonization by WCS365 cells, we studied whether chemotaxis also is required for hyphae colonization. To that end, an in vitro assay was developed to study hyphae colonization by bacteria. The results indicated that cells of the cheA mutant FAJ2060 colonize hyphae less efficiently than cells of wild-type strain WCS365, when single strains were analyzed as well as when both strains were applied together. Cells of WCS365 show a chemotactic response toward the spent growth medium of F. oxysporum f. sp. radicis-lycopersici, but those of its cheA mutant, FAJ2060, did not. Fusaric acid, a secondary metabolite secreted by Fusarium strains, appeared to be an excellent chemo-attractant. Supernatant fluids of a number of Fusarium strains secreting different levels of fusaric acid were tested as chemo-attractants. A positive correlation was found between chemo-attractant activity and fusaric acid level. No chemotactic response was observed toward the low fusaric acid-producer FO242. Nevertheless, the hyphae of FO242 still were colonized by WCS365, suggesting that other metabolites also play a role in this process. The possible function of hyphae colonization for the bacterium is discussed.     

Effects of different plant root exudates and their organic acid components on chemotaxis, biofilm formation and colonization by beneficial rhizosphere-associated bacterial strains

Aim

It is necessary to understand the roles of root exudates involved in plant-microbe interactions to inform practical application of beneficial rhizosphere microbial strains.

Methods

Colonization of Bacillus amyloliquefaciens SQR9 (isolated from cucumber rhizosphere) and Bacillus subtilis N11 (isolated from banana rhizosphere) of their original host was found to be more effective as compared to the colonization of the non-host plant. Organic acids in the root exudates of the two plants were identified by High performance liquid chromatography (HPLC). The chemotactic response and effects on biofilm formation were assessed for SQR9 and N11 in response to cucumber and banana root exudates, as well as their organic acids components.

Results

Citric acid detected exclusively in cucumber exudates could both attract SQR9 and induce its biofilm formation, whereas only chemotactic response but not biofilm formation was induced in N11. Fumaric acid that was only detected in banana root exudates revealed both significant roles on chemotaxis and biofilm formation of N11, while showing only effects on biofilm formation but not chemotaxis of SQR9.

Conclusion

The relationship between PGPR strain and root exudates components of its original host might contribute to preferential colonization. This study advances a clearer understanding of the mechanisms relevant to application of PGPR strains in agricultural production.     

Flagella-driven chemotaxis towards exudate components is an important trait for tomato root colonization by Pseudomonas fluorescens

Motility is a major trait for competitive tomato root-tip colonization by Pseudomonas fluorescens. To test the hypothesis that this role of motility is based on chemotaxis toward exudate components, cheA mutants that were defective in flagella-driven chemotaxis but retained motility were constructed in four P. fluorescens strains. After inoculation of seedlings with a 1:1 mixture of wild-type and nonmotile mutants all mutants had a strongly reduced competitive root colonizing ability after 7 days of plant growth, both in a gnotobiotic sand system as well as in nonsterile potting soil. The differences were significant on all root parts and increased from root base to root tip. Significant differences at the root tip could already be detected after 2 to 3 days. These experiments show that chemotaxis is an important competitive colonization trait. The best competitive root-tip colonizer, strain WCS365, was tested for chemotaxis toward tomato root exudate and its major identified components. A chemotactic response was detected toward root exudate, some organic acids, and some amino acids from this exudate but not toward its sugars. Comparison of the minimal concentrations required for a chemotactic response with concentrations estimated for exudates suggested that malic acid and citric acid are among major chemo-attractants for P. fluorescens WCS365 cells in the tomato rhizosphere.     

Chemical characterization of root exudates from rice (Oryza sativa) and their effects on the chemotactic response of endophytic bacteria

Root exudates represent an important source of nutrients for microorganisms in the rhizosphere and seem to participate in early colonization inducing chemotactic responses of rhizospheric bacteria. We characterized the root exudates collected from rice plantlets cultured under hydroponic conditions and assessed their effects on the chemotaxis of two strains of endophytic bacteria, Corynebacterium flavescens and Bacillus pumilus, collected from the rice rhizosphere. We compared these chemotactic effects on endophytic bacteria with those on two strains of plant-growth-promoting bacteria, Azospirillum brasilense (isolated from the corn rhizosphere) and Bacillus sp. (from the rice rhizosphere). The root exudates were collected at different time intervals. The highest concentration and diversity of amino acids and carbohydrates were found during the first 2 weeks after seeding. Histidine, proline, valine, alanine, and glycine were the main amino acid residues identified during the 4 weeks of culture. The main carbohydrates identified were glucose, arabinose, mannose, galactose, and glucuronic acid. The chemotactic responses of the analyzed endophytic bacteria to root exudates were 3.9 to 5.1 times higher than those of A. brasilense and 2.2 to 2.8 times higher than Bacillus sp. Our results indicate that rice exudates may induce a higher chemotactic response for endophytic bacteria than for other bacterial strains present in the rice rhizosphere.     

Chemotaxis of Bradyrhizobium japonicum to soybean exudates.

The chemotactic response of Bradyrhizobium japonicum toward soybean seed and root exudates was examined. Assays using various isoflavones and fractionated exudate indicated that isoflavones are not the principal attractants in exudates. Likewise, induction of nod genes with isoflavones or seed exudate before assay did not enhance chemotaxis. Screening of numerous compounds revealed that only dicarboxylic acids and the amino acids glutamate and aspartate were strong attractants. The presence of glutamate, aspartate, and dicarboxylic acids in appreciable concentrations in soybean seed and root exudates indicates that these compounds likely represent natural chemoattractants for B. japonicum.     

Chemotaxis of Bradyrhizobium japonicum to soybean exudates.

The chemotactic response of Bradyrhizobium japonicum toward soybean seed and root exudates was examined. Assays using various isoflavones and fractionated exudate indicated that isoflavones are not the principal attractants in exudates. Likewise, induction of nod genes with isoflavones or seed exudate before assay did not enhance chemotaxis. Screening of numerous compounds revealed that only dicarboxylic acids and the amino acids glutamate and aspartate were strong attractants. The presence of glutamate, aspartate, and dicarboxylic acids in appreciable concentrations in soybean seed and root exudates indicates that these compounds likely represent natural chemoattractants for B. japonicum.     

Strain-specific chemotaxis of Azospirillum spp.

Chemotactic responses of three Azospirillum strains originating from different host plants were compared to examine the possible role of chemotaxis in the adaptation of these bacteria to their respective hosts. The chemotaxis to several sugars, amino acids, and organic acids was determined qualitatively by an agar plate assay and quantitatively by a channeled-chamber technique. High chemotactic ratios, up to 40, were obtained with the latter technique. The chemotactic response did not rely upon the ability of the bacteria to metabolize the attractant. Rather, it depended on the attractant concentration and stereoconfiguration. Chemotaxis was found to be strain specific. Differences were particularly observed between a wheat isolate and strains originating from the C4-pathway plants maize and Leptochloa fusca. In contrast to the other two strains, the wheat isolate was strongly attracted to D-fructose, L-aspartate, citrate, and oxalate. The other strains showed maximal attraction to L-malate. The chemotactic responses to organic acids partially correlate with the exudation of these acids by the respective host plants. Additionally, a heat-labile, high-molecular-weight attractant was found in the root exudates of L. fusca, which specifically attracted the homologous Azospirillum strain. It is proposed that strain-specific chemotaxis probably reflects an adaptation of Azospirillum spp. to the conditions provided by the host plant and contributes to the initiation of the association process.     

Characterization of the chemotaxis fli Y and che A genes in Bacillus cereus

This paper describes the first identification of chemotaxis genes in Bacillus cereus. We sequenced and studied the genomic organization and the expression of the cheA and fliY genes in two different B. cereus strains, ATCC 14579 and ATCC 10987. While cheA encodes a highly conserved protein acting as the main regulator of the chemotactic response in flagellated eubacteria, fliY, which has been previously described only in B. subtilis, is one of the three genes encoding proteins of the flagellar switch complex. Although the sequences and relative position of cheA and fliY were found to be identical in the two B. cereus strains analyzed, the restriction fragment containing both genes was located differently on the physical maps of B. cereus ATCC 14579 and ATCC 10987. Evidence is shown that the genomic organization and the expression of fliY and cheA in B. cereus differ significantly from that described for B. subtilis, which is considered a model microorganism for chemotaxis in gram-positive bacteria.     

The roles of the multiple CheW and CheA homologues in chemotaxis and in chemoreceptor localization in Rhodobacter sphaeroides

Rhodobacter sphaeroides has multiple homologues of most of the Escherichia coli chemotaxis genes, organized in two major operons and other, unlinked, loci. These include cheA1 and cheW1 (che Op1) and cheA2, cheW2 and cheW3 (che Op2). We have deleted each of these cheA and cheW homologues in-frame and examined the chemosensory behaviour of these strains on swarm plates and in tethered cell assays. In addition, we have examined the effect of these deletions on the polar localization of the chemoreceptor McpG. In E. coli, deletion of either cheA or cheW results in a non-chemotactic phenotype, and these strains also show no receptor clustering. Here, we demonstrate that CheW2 and CheA2 are required for the normal localization of McpG and for normal chemotactic responses under both aerobic and photoheterotrophic conditions. Under aerobic conditions, deletion of cheW3 has no significant effect on McpG localization and only has an effect on chemotaxis to shallow gradients in swarm plates. Under photoheterotrophic conditions, however, CheW3 is required for McpG localization and also for chemotaxis both on swarm plates and in the tethered cell assay. These phenotypes are not a direct result of delocalization of McpG, as this chemoreceptor does not mediate chemotaxis to any of the compounds tested and can therefore be considered a marker for general methyl-accepting chemotaxis protein (MCP) clustering. Thus, there is a correlation between the normal localization of McpG (and presumably other chemoreceptors) and chemotaxis. We propose a model in which the multiple different MCPs in R. sphaeroides are contained within a polar chemoreceptor cluster. Deletion of cheW2 and cheA2 under both aerobic and photoheterotrophic conditions, and cheW3 under photoheterotrophic conditions, disrupts the cluster and hence reduces chemotaxis to any compound sensed by these MCPs.     

云南滇池富磷区趋化性细菌的系统多样性

从云南滇池富磷区100份土样中筛选解磷细菌(PSB),通过组氨酸激酶编码基因(cheA)筛选趋化性PSB,并通过软琼脂平板法验证其趋化性;利用钼蓝比色法测定PSB对磷酸三钙的溶解能力;基于16S rRNA基因序列分析趋化性PSB的系统亲缘关系.结果表明： 分离到的145株PSB的溶磷圈直径在0.5～2 cm,其中37株为趋化性PSB.该37株PSB对供试的4种趋化底物均具有趋化性,而且对磷酸三钙均具有解磷活性.基于16S rRNA基因序列的系统发育分析显示,这37株趋化性PSB分属于10属,共17种细菌,其中假单胞菌属种类最多(5种9株),肠杆菌属次之(3种8株),芽孢杆菌属尽管只分离到1个种(Bacillus aryabhattai),但共分离到9个菌株.     

Assessment of the Role of Chemotaxis and Biofilm Formation as Requirements for Colonization of Roots and Seeds of Soybean Plants by <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> BNM339

This article correlates colonization with parameters, such as chemotaxis, biofilm formation, and bacterial growth, that are believed to be connected. We show here, by using two varieties of soybean plants that seeds axenically produced exudates, induced a chemotactic response in Bacillus amyloliquefaciens, whereas root exudates did not, even when the exudates, also collected under axenic conditions, were concentrated up to 200-fold. Root exudates did not support bacterial cell division, whereas seed exudates contain compounds that support active cell division and high cell biomass at stationary phase. Seed exudates of the two soybean varieties also induced biofilm formation. B. amyloliquefaciens colonized both seeds and roots, and plant variety significantly affected bacterial root colonization, whereas it did not affect seed colonization. Colonization of roots in B. amyloliquefaciens occurred despite the lack of chemotaxis and growth stimulation by root exudates. The data presented in this article suggest that soybean seed colonization, but not root colonization, by B. amyloliquefaciens is influenced by chemotaxis, growth, and biofilm formation and that this may be caused by qualitative changes of the composition of root exudates.     

Growth-promotion of strawberry plants inoculated with Azospirillum brasilense

Azospirillum brasilense (strains REC3, RLC1, PEC5) were root inoculated in strawberry plants of the cultivars ‘Milsei’, ‘Selva’ and ‘Camarosa’ to assess plant growth-promoting effects. The bacteria were able to promote plant growth (expressed as root length, root area, and dry weight of root and shoot), depending on the genotypes of plants and bacteria used, whereas the stolon production (3–4) depended only on the strawberry cultivar. To explain whether root exudates plays any role on the growth-promotion observed herein, total protein and sugar were determined, and chemotaxis properties were evaluated. The strains showed positive chemotaxis toward the root exudates, being influenced by the total sugars content, suggesting that the latter plays an important role in the chemotaxis effect and may contribute to enhance the root capacity to recruit azospirilla from rhizosphere, thus improving the growth-promoting effect exerted by these bacteria.     

香蕉林土壤可培养放线菌分离、鉴定及其抑菌活性

【背景】香蕉枯萎病是香蕉的顽固性疾病,制约着香蕉产业的发展,因此,筛选出对香蕉枯萎病菌(尖孢镰刀菌古巴专化型4号生理小种,简称Foc4)具有抑制活性的生防菌株具有重要意义。【目的】分离香蕉林土壤样品中放线菌并进行物种的初步鉴定,测定其对包括香蕉枯萎病致病菌的7种病原菌的拮抗活性,获得高活性菌株,以获得解决香蕉枯萎病的生物防治策略。【方法】采集多份广西地区香蕉林土壤样品,采用超声波等手段对其预处理,设置多种特异性培养基从中分离放线菌资源,对获得的放线菌进行基于16SrRNA基因序列的物种鉴定,以7种病原菌为靶标,采用平板对峙法从中筛选抑菌活性菌株,最后采用菌丝生长速率法对Foc4的抑菌率进行测定。【结果】从香蕉林土壤中分离出138株放线菌均为链霉菌,其中5株为潜在新种,分别为X1085、X1052、X2052、X3059和X4046;筛选出具有抑菌活性的菌株77株,阳性率为55.8%。20株对Foc4具有抑制活性,其中4株拮抗效果明显,抑制率大于80%,菌株X4050的抑菌率高达93.76%。【结论】初步明确了香蕉林土壤中可培养放线菌的物种信息,其中部分放线菌为未知物种,活性分析显示一半...     

Isolation of fluorescent pseudomonads from the rhizosphere of banana plants antagonistic towards root necrosing fungi

Total aerobic bacteria and fluorescent pseudomonads were counted in bulk and rhizospheric soils of banana plants of 14 plantations in Martinique (French West Indies). Fluorescent Pseudomonas isolates were then identified and investigated for in vitro antagonism towards Cylindrocladium sp., a fungal pathogen of banana roots. Total aerobic bacteria and fluorescent pseudomonads were significantly more abundant in rhizospheric soils than in bulk soils. Among 58 fluorescent Pseudomonas isolates, 41 were identified as Pseudomonas fluorescens biovar V and 17 as Ps. putida biovar A. Six strains exhibited an antagonism towards Cylindrocladium isolates. Among them, Ps. putida strain 93.1 totally blocked fungal growth. No relationship was established between the antifungal effect and enzyme or hydrogen cyanide production by bacteria, suggesting that siderophores and other compounds were involved in fungal inhibition. Antagonistic fluorescent pseudomonads represent a potential for the biological control of banana root infections by Cylindrocladium sp.     

Inhibition of Rhizoctonia by endospore forming bacteria indigenous to landscape planting beds

Endospore forming bacteria were collected from root samples of 35 genera of bedding plants growing in established commercial landscape beds in Central Florida. One hundred and twenty-nine bacterial strains associated with 14 species were identified using fatty acid analysis (Microbial Identification System, MIDI). All strains were evaluated for in vitro inhibition of damping off disease caused by the fungus Rhizoctonia solani. The strongest inhibition of Rhizoctonia by soluble exudates in cocultivation was observed with strains belonging to six species: B. cereus, B. pumilus, B. thuringiensis, L. sphaericus, B. amyloliquefaciens, and B. subtilis. Most strains that inhibited Rhizoctonia growth in cocultivation also inhibited growth via volatile compounds. All 129 strains were evaluated for ability to protect impatiens plants from subsequent challenge infection by Rhizoctonia solani. Certain strains of endospore forming bacteria also enhanced plant growth. It is apparent from this study that the Bacillus community associated with bedding plants in established planting beds produce soluble antifungal compounds, volatile antifungal compounds, and enhance plant growth. In developing biological control, it may be a more practical approach to promote or enhance a natural, multifaceted community of Bacillus strains within our planting beds.     

Rice domestication influences the composition and function of the rhizosphere bacterial chemotaxis systems

Plant and Soil - Specific soil bacteria can sense and respond to the selective rhizosphere recruitment of root exudates using unique systems of chemotaxis that mediate plant-microbe and...     

Chemotaxis of a Ralstonia sp. SJ98 toward different nitroaromatic compounds and their degradation

A Ralstonia sp. SJ98, isolated by a chemotactic enrichment technique, was capable of utilizing different nitroaromatic compounds (NACs). It utilized p-nitrophenol, 4-nitrocatechol, o-nitrobenzoic acid, and p-nitrobenzoic acid as the sole source of carbon and energy. It was observed that Ralstonia sp. SJ98 was chemotactic to the above-mentioned NACs as tested by the drop assay, swarm plate assay, and capillary assay. However, it failed to show chemotactic behavior toward those compounds which were not degraded by the microorganism. This is the first report which shows the chemotaxis of a microorganism toward different NACs and their subsequent degradation. Some of the intermediates of the NACs' degradative pathways have been identified using TLC, GC, and GC-MS studies. The results presented here indicate a correlation between chemotaxis and biodegradation of NACs.     

Plant Growth-Promoting Chitinolytic Paenibacillus elgii Responds Positively to Tobacco Root Exudates

Bacterial strains from chitin/chitosan-rich soils, from two industries, were screened for their chitinolytic, antifungal, and mineral phosphate solubilization abilities. The isolate SMA-1-SDCH02, positive for all three properties, was selected and identified as Paenibacillus elgii based on morphological and biochemical characters and supported by 16S rRNA gene sequence analysis. P. elgii enhanced the growth of groundnut in terms of shoot height, root length, total chlorophyll, and fresh and dry weight when applied alone or in combination with chitosan. The plant growth-promoting activity of P. elgii was seen in tobacco in a specially designed gnotobiotic setup indicating its capability to promote growth of at least groundnut and tobacco. Metabolite changes in the bacteria, studied using attenuated total reflectance-infrared (ATR-IR) spectroscopy, revealed split bands of amide I at the 1659- and 1636-cm⁻¹ regions when grown in minimal media amended with tobacco root exudates. The difference in ATR-IR bands in the presence of tobacco root exudates indicated production of compounds with differences in functional groups.     

Coexpression of the long and short forms of CheA, the chemotaxis histidine kinase, by members of the family Enterobacteriaceae.

CheA is the histidine protein kinase of a two-component signal transduction system required for bacterial chemotaxis. Motile cells of the enteric species Escherichia coli and Salmonella typhimurium synthesize two forms of CheA by utilizing in-frame initiation sites within the gene cheA. The full-length protein, CheAL, plays an essential role in the chemotactic signaling pathway. In contrast, the function of the short form, CheAs, remains elusive. Although CheAs lacks the histidine residue that becomes phosphorylated in CheAL, it exhibits both kinase activity and the ability to interact with and enhance the activity of CheZ, a chemotaxis protein that accelerates dephosphorylation of the two-component response regulator CheY. To determine whether other members of the family Enterobacteriaceae express CheAs and CheZ, we analyzed immunoblots of proteins from clinical isolates of a variety of enteric species. All motile, chemotactic isolates that we tested coexpressed CheAL, CheAs, and CheZ. The only exceptions were closely related plant pathogens of the genus Erwinia, which expressed CheAL and CheZ but not CheAs. We also analyzed nucleotide sequences of the cheA loci from isolates of Serratia marcescens and Enterobacter cloacae, demonstrating the presence of in-frame translation initiation sites similar to those observed in the cheA loci of E. coli and S. typhimurium. Since coexpression of CheAs and CheZ appears to be limited to motile, chemotactic enteric bacteria, we propose that CheAs may play an important role in chemotactic responses in some environmental niches encountered by enteric species.