Tomato seed and root exudate sugars: composition, utilization by Pseudomonas biocontrol strains and role in rhizosphere colonization

The role of tomato seed and root exudate sugars as nutrients for Pseudomonas biocontrol bacteria was studied. To this end, the major exudate sugars of tomato seeds, seedlings and roots were identified and quantified using high-performance liquid chromatographic (HPLC) analysis. Glucose, fructose and maltose were present in all studied growth stages of the plant, but the ratios of these sugars were strongly dependent on the developmental stage. In order to study the putative role of exudate sugar utilization in rhizosphere colonization, two approaches were adopted. First, after co-inoculation on germinated tomato seeds, the root-colonizing ability of the efficient root-colonizing P. fluorescens strain WCS365 in a gnotobiotic quartz sand-plant nutrient solution system was compared with that of other Pseudomonas biocontrol strains. No correlation was observed between the colonizing ability of a strain and its ability to use the major exudate sugars as the only carbon and energy source. Secondly, a Tn5lacZ mutant of P. fluorescens strain WCS365, strain PCL1083, was isolated, which is impaired in its ability to grow on simple sugars, including those found in exudate. The mutation appeared to reside in zwf, which encodes glucose-6-phosphate dehydrogenase. The mutant grows as well as the parental strain on other media, including tomato root exudate. After inoculation of germinated sterile tomato seeds, the mutant cells reached the same population levels at the root tip as the wild-type strain, both alone and in competition, indicating that the ability to use exudate sugars does not play a major role in tomato root colonization, despite the fact that sugars have often been reported to represent the major exudate carbon source. This conclusion is supported by the observation that the growth of mutant PCL1083 in vitro is inhibited by glucose, a major exudate sugar, at a concentration of 0.001%, which indicates that the glucose concentration in the tomato rhizosphere is very low.     

Analysis of pathogenicity mutants of a bacteriocin producing Xanthomonas perforans

Since the initial discovery of Xanthomonas perforans on tomato in 1991, it has completely displaced Xanthomonas euvesicatoria as the bacterial spot of tomato pathogen in Florida. Previous research has shown that X. perforans produces at least three different bacteriocin-like compounds (BcnA, BcnB, BcnC) antagonistic toward X. euvesicatoria strains. In this study pathogenicity-attenuated, bacteriocin-producing mutants of X. perforans were created to determine their potential as biological control agents for control of X. euvesicatoria. Several candidate genes were chosen based on previous studies in which mutant phenotypes exhibited reduced virulence in either X. perforans (OpgH_Xcv) or the closely related X. euvesicatoria strain 85-10 (hpaB, hpaC, xopA, xopD, avrBs2 and gumD). Each candidate gene in X. perforans was amplified and PCR-assisted deletion mutagenesis was performed in the wild-type (wt) X. perforans strain to create potential attenuation mutants. Each mutant was tested for growth rate, disease severity and antagonism toward X. euvesicatoria strains. Three mutants, XopA, opgH, and gumD were significantly less pathogenic than the wild-type strain with the opgH mutant reaching significantly lower internal populations than all other mutants except hpaC. The opgH-strain was the most affected in its ability to grow internally in plant tissue while inhibiting X. euvesicatoria populations equal to or more than the other mutant strains. This mutant strain could potentially be used as part of an effective biological control strategy.     

The effects of traits of Bacillus megaterium onseed and root colonization and their correlation withthe suppression of Rhizoctonia root rot of soybean

Bacillus megaterium strainB153-2-2 is a potential bacterial biocontrol agentagainst Rhizoctonia solani isolate 2B12(ISG-2B). To study the role of antagonism (Ant),chemotaxis (Che), motility (Mot), and sporulation(Spo) of the biocontrol agent during seed and rootcolonization and the correlation between rootcolonization and the suppression of soybean (Glycine max) root rot caused by R. solani,strain B153-2-2(Che⁺Mot⁺Ant⁺⁺Spo⁺⁺) and the sevenderived mutants with altered antagonism, chemotaxis,motility, and/or sporulation were used. The bacterialcells were introduced into soil separately either asa soybean seed coating or soil application. Two soilmixtures defined as coarse and fine soil were used. The bacterial cell chemotactic response to soybeanroot and seed exudates and antagonism to R.solani were significantly (p = 0.05) correlatedwith root and seed colonization in some but not alltreatments. The sporulation-defective mutants had lowcell populations immediately after application and,therefore, reduced root colonization. The differencesin root colonization diminished among the mutants andstrain B153-2-2 when R. solani was present inthe soil or, as seedlings grew older. Soybean seedlingroots grown in coarse soil had significantly greatercolonization by B153-2-2 or its mutants and a lowerdisease index than that in fine soil. There was asignificant positive correlation (r ² = 0.78)between root colonization by strain B153-2-2 or itsmutants and suppression of Rhizoctonia root rot.     

落叶型大丽轮枝菌T-DNA插入突变体库的构建

采用ATMT技术建立大丽轮枝菌落叶型菌株XJ2008菌株的T-DNA插入突变体文库,共获得6 043个突变体。从中随机挑选104个突变体,以野生型XJ2008菌株为参照,评价其致病性、菌落生长速率、分生孢子及微菌核的产生能力等。结果表明,有12.5%的突变体丧失产孢能力,4.8%的突变体的生长速率显著减慢,8.7%的突变体的生长速率显著加快,12.5%的突变体丧失产生微菌核的能力,47.1%的突变体的致病性显著低于野生型菌株XJ2008,且突变体2-736、2-740、2-745的病情指数分别约为野生型菌株XJ2008的0.184、0.168和0.197倍。该突变体库突变体遗传稳定性好,性状多样性丰富。     

Mutations in lipopolysaccharide biosynthetic genes impair maize rhizosphere and root colonization of Rhizobium tropici CIAT899

Three transposon mutants of Rhizobium tropici CIAT899 affected in lipopolysaccharide (LPS) biosynthesis were characterized and their maize rhizosphere and endophytic root colonization abilities were evaluated. The disrupted genes coded for the following putative products: the ATPase component of an O antigen ABC-2 type transporter ( wzt ), a nucleotide-sugar dehydratase ( lpsβ2 ) and a bifunctional enzyme producing GDP-mannose ( noeJ ). Electrophoretic analysis of affinity purified LPS showed that all mutants lacked the smooth LPS bands indicating an O antigen minus phenotype. In the noeJ mutant, the rough LPS band migrated faster than the parental band, suggesting a truncated LPS core. When inoculated individually, the wzt and noeJ mutants colonize the rhizosphere and root to a lower extent than the parental strain while no differences were observed between the lpsβ2 mutant and the parental strain. All mutants were impaired in competitive rhizosphere and root colonization. Pleiotropic effects of the mutations on known colonization traits such as motility and growth rate were observed, but they were not sufficient to explain the colonization behaviours. It was found that the LPS mutants were sensitive to the maize antimicrobial 6-methoxy-2-benzoxazolinone (MBOA). Only the combined effects of altered growth rate and susceptibility to maize antimicrobials could account for all the observed colonization phenotypes. The results suggest an involvement of the LPS in protecting R. tropici against maize defence response during rhizosphere and root colonization.     

Response to carbon-starvation in Pseudomonas aeruginosa strain IE-6S+: analysis of general cross protection,production of some nematocidal compounds in vitro,and the biological control of Meloidogyne javanica in tomato

Adaptation to nutrient-limited conditions by repeated culture on soil agar media was found to induce resistance to osmotic, oxidation, thermal and pH stress as well as carbon-limited culture conditions in Pseudomonas aeruginosa strain IE-6S⁺. Culture filtrate of the resistant strains obtained from 10% strength King's medium B (KMB) caused greater (32–54%) mortality of Meloidogyne javanica juveniles compared with their parental strain. When 10% strength KMB was amended with 1% (w/v) glucose, the ability to cause nematode mortality was substantially enhanced by adapted strains, while activity of the parental strain was repressed. Two of the four starved bacteria IE-6S⁺PBK1 and IE-6S⁺KUC2 grown in KMB liquid medium amended with glucose synthesized salicylic acid (5.1 and 5.8 g ml^–1, respectively) and hydrogen cyanide (picrate paper turned yellow to brownish red for both strains) in greater quantities compared to wild type strain (SA = 4.4 g ml^–1, picrate paper turned orange-yellow). Neither wild type strain IE-6S⁺ nor its adapted strains were capable of utilizing tomato root exudates as a sole carbon source. Strains adapted to carbon-limiting conditions exhibited enhanced colonization in the rhizosphere and inner root tissues of tomato compared to their exponentially growing counterpart. Pre-adapted bacterial inoculants applied in the soil also caused greater (15%) reduction in nematode penetration compared to the parental strain or controls.     

Survival and epiphytic fitness of a nonpathogenic mutant of Xanthomonas campestris pv. glycines

Xanthomonas campestris pv. glycines is the causal agent of bacterial pustule disease of soybeans. The objective of this work was to construct a nonpathogenic mutant derived from the pathogenic wild-type strain YR32 and to evaluate its effectiveness in preventing growth of its parent on the soybean phyllosphere. A mini-Tn5-derived transposon was used to generate nonpathogenic mutants. Southern hybridization and pulsed-field gel electrophoresis confirmed the presence of a single transposon in each of the nonpathogenic mutants. One of the nonpathogenic mutants, M715, failed to induce a hypersensitive response in tomato leaves. An ice nucleation gene (inaZ) carried in pJL1703 was introduced into strain YR32 as a reporter gene to demonstrate that the presence of M715 could reduce colonization of the soybean phyllosphere by YR32. de Wit serial replacement analysis showed that M715 competed equally with its wild-type parental strain, YR32. Epiphytic fitness analysis of YR32 in the greenhouse indicated that the population dynamics of strains YR32, YR32(pJL1703), and M715 were similar, although the density of the mutant was slightly less than that of its parent. The M715 mutant was able to survive for 16 days after inoculation on soybean leaves and maintained population densities of approximately 10(4) to 10(5) cells g (fresh weight) of leaf(-1). Therefore, M715 shows promise as an effective biocontrol agent for bacterial pustule disease in soybeans.     

水稻内生菠萝泛菌YJ76吲哚产量上调突变株的鉴定及生理性状

菠萝泛菌(Pantoea ananatis)YJ76是从水稻"越富"品种中分离的优势内生菌,与宿主水稻互作时具有多种促生作用,其分泌的吲哚作为细菌种内及种间的信号分子参与调控多种生理生化行为。[目的]筛选获得与吲哚调控相关的突变株,鉴定突变位点并研究突变基因对菌株的生存适应性以及对宿主水稻定殖和促生的影响,为研究吲哚调控通路奠定基础。[方法]用双亲本接合法构建YJ76的mTn5转座子插人突变文库,以染色体步移TAIL-PCR技术鉴定突变基因,最后探究基因突变对菌体产生的影响。[结果]筛选到1株吲哚产量大幅上升的YJ76突变株M04,鉴定突变位点为一个长度195 bp未报道过的新基因,将其命名为ipc(indole production control),基因突变后增强了YJ76对重金属、四环素和酸的抗性,也增强了菌体对宿主水稻定殖和促生的能力。[结论]吲哚产量上调的ipc突变株能够提高菌体生存适应性并增强其对宿主水稻定殖和促生的能力。     

Root colonization by phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 is essential for biocontrol of tomato foot and root rot

The phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicislycopersici. To test whether root colonization is required for biocontrol, mutants impaired in the known colonization traits motility, prototrophy for amino acids, or production of the site-specific recombinase, Sss/XerC were tested for their root tip colonization and biocontrol abilities. Upon tomato seedling inoculation, colonization mutants of strain PCL1391 were impaired in root tip colonization in a gnotobiotic sand system and in potting soil. In addition, all mutants were impaired in their ability to control tomato foot and root rot, despite the fact that they produce wild-type levels of phenazine-1-carboxamide, the antifungal metabolite previously shown to be required for biocontrol. These results show, for what we believe to be the first time, that root colonization plays a crucial role in biocontrol, presumably by providing a delivery system for antifungal metabolites. The ability to colonize and produce phenazine-1-carboxamide is essential for control of F. oxysporum f. sp. radicis-lycopersici. Furthermore, there is a notable overlap of traits identified as being important for colonization of the rhizosphere and animal tissues.     

Impact of two fluorescent pseudomonads and an arbuscular mycorrhizal fungus on tomato plant growth,root architecture and P acquisition

The ability of fluorescent pseudomonads and arbuscular mycorrhizal fungi (AMF) to promote plant growth is well documented but knowledge of the impact of pseudomonad-mycorrhiza mixed inocula on root architecture is scanty. In the present work, growth and root architecture of tomato plants (Lycopersicon esculentum Mill. cv. Guadalete), inoculated or not with Pseudomonas fluorescens 92rk and P190r and/or the AMF Glomus mosseae BEG12, were evaluated by measuring shoot and root fresh weight and by analysing morphometric parameters of the root system. The influence of the microorganisms on phosphorus (P) acquisition was assayed as total P accumulated in leaves of plants inoculated or not with the three microorganisms. The two bacterial strains and the AMF, alone or in combination, promoted plant growth. P. fluorescens 92rk and G. mosseae BEG12 when co-inoculated had a synergistic effect on root fresh weight. Moreover, co-inoculation of the three microorganisms synergistically increased plant growth compared with singly inoculated plants. Both the fluorescent pseudomonads and the myco-symbiont, depending on the inoculum combination, strongly affected root architecture. P. fluorescens 92rk increased mycorrhizal colonization, suggesting that this strain is a mycorrhization helper bacterium. Finally, the bacterial strains and the AMF, alone or in combination, improved plant mineral nutrition by increasing leaf P content. These results support the potential use of fluorescent pseudomonads and AMF as mixed inoculants for tomato and suggest that improved tomato growth could be related to the increase in P acquisition.     

青枯雷尔氏菌胞外多糖合成缺失突变株构建及其生物学特性

【目的】由青枯雷尔氏菌(Ralstonia solanacearum)引起的植物青枯病是一种毁灭性土传病害。胞外多糖(extracellular polysaccharides,EPS)是青枯雷尔氏菌关键的致病因子之一。通过构建胞外多糖缺失突变株,研究胞外多糖在青枯病致病中的作用。【方法】从青枯雷尔氏菌FJAT-91的基因组中克隆出胞外多糖合成结构基因epsD同源臂,克隆至自杀性质粒p K18mobsacB,再将庆大霉素抗性基因(Gm)插入同源臂中间,获得重组质粒p K18-epsD。将重组质粒转化至青枯雷尔氏菌FJAT-91感受态细胞中,通过同源重组敲除epsD基因,获得EPS合成缺失的突变株FJAT-91Δeps 。研究突变株与野生菌株在菌落形态、胞外多糖合成、运动能力、定殖能力的差异性。【结果】突变菌株FJAT-91ΔepsD与出发菌株FJAT-91相比:胞外多糖产量显著减少,生长较慢;泳动能力(swimming motility)和群集运动能力(swarming motility)显著降低;在番茄苗根部和茎部的定殖能力显著降低;弱化指数(AI)为0.905,鉴定为无致病力菌株。【结论】胞外多糖在青枯雷尔氏菌的致病中起着关键的作用,本课题研究成果为开发植物疫苗提供了优良的材料与研究基础。     

Reduced rhizosphere colonization ability ofAgrobacterium tumefaciens chromosomal virulence (chv) mutants

Mutations in the chromosomal virulence (chv) region ofA. tumefaciens strain A723 reduce virulence, motility, and ability of the bacteria to bind to plant cells. We conducted experiments to assess the ability ofchv mutants to colonize the rhizosphere ofPisum sativum. The mutation had no effect on ability of bacteria to grow with a defined number of root cap cells as the sole carbon and nitrogen source. Ten days after inoculation, there were up to 103-fold more wild type thanchv mutant bacteria present in the rhizosphere of inoculated plants.     

Salmonella Typhimurium SPI-1 genes promote intestinal but not tonsillar colonization in pigs

Salmonella Pathogenicity Island 1 (SPI-1) genes are indispensable for virulence of Salmonella Typhimurium in several animal species. The role of SPI-1 in the pathogenesis of Salmonella Typhimurium infections of pigs, however, is not well described. The interactions of a porcine Salmonella Typhimurium field strain and its isogenic mutants with disruptions in the SPI-1 genes hilA, sipA and sipB with porcine intestinal epithelial cells were characterized in vitro and in a ligated intestinal loop model in pigs. HilA and SipB were essential in the invasion of porcine intestinal epithelial cells in vitro. A sipA mutant was impaired for invasion using a polarized cell line, but fully invasive in a non-polarized cell line. All SPI-1 mutants induced a significant decrease in influx of neutrophils in the porcine intestinal loop model compared with the wild type strain. Pigs were orally inoculated with 10(8) colony forming units of both the wild type Salmonella Typhimurium strain and its isogenic sipB::kan mutant strain. The sipB mutant strain was significantly impaired to invade the intestinal, but not the tonsillar tissue, one day after inoculation and was unable to efficiently colonize the intestines and the GALT, but not the tonsils, 3 days after inoculation. This study shows that SPI-1 plays a crucial role in the invasion and colonization of the porcine gut and in the induction of influx of neutrophils towards the intestinal lumen, but not in the colonization of the tonsils.     

The sss colonization gene of the tomato-Fusarium oxysporum f. sp. radicis-lycopersici biocontrol strain Pseudomonas fluorescens WCS365 can improve root colonization of other wild-type pseudomonas spp.bacteria

We show that the disease tomato foot and root rot caused by the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici can be controlled by inoculation of seeds with cells of the efficient root colonizer Pseudomonas fluorescens WCS365, indicating that strain WCS365 is a biocontrol strain. The mechanism for disease suppression most likely is induced systemic resistance. P. fluorescens strain WCS365 and P. chlororaphis strain PCL1391, which acts through the production of the antibiotic phenazine-1-carboxamide, were differentially labeled using genes encoding autofluorescent proteins. Inoculation of seeds with a 1:1 mixture of these strains showed that, at the upper part of the root, the two cell types were present as microcolonies of either one or both cell types. Microcolonies at the lower root part were predominantly of one cell type. Mixed inoculation tended to improve biocontrol in comparison with single inoculations. In contrast to what was observed previously for strain PCL1391, mutations in various colonization genes, including sss, did not consistently decrease the biocontrol ability of strain WCS365. Multiple copies of the sss colonization gene in WCS365 improved neither colonization nor biocontrol by this strain. However, introduction of the sss-containing DNA fragment into the poor colonizer P. fluorescens WCS307 and into the good colonizer P. fluorescens F113 increased the competitive tomato root tip colonization ability of the latter strains 16- to 40-fold and 8- to 16-fold, respectively. These results show that improvement of the colonization ability of wild-type Pseudomonas strains by genetic engineering is a realistic goal.     

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.     

Establishment of phosphate-solubilizing strains of Azotobacter chroococcum in the rhizosphere and their effect on wheat cultivars under green house conditions

A pot experiment was conducted in the green house to investigate the establishment of phosphate solubilizing strains of Azotobacter chroococcum, including soil isolates and their mutants, in the rhizosphere and their effect on growth parameters and root biomass of three genetically divergent wheat cultivars (Triticum aestivum L.). Five fertilizer treatments were performed: Control, 90 kg N ha^—1, 90 kg N + 60 kg P₂O₅ ha^—1, 120 kg N ha^—1 and 120 kg N + 60 kg P₂O₅ ha^—1. Phosphate solubilizing and phytohormone producing parent soil isolates and mutant strains of A. chroococcum were isolated and selected by an enrichment method. In vitro phosphate solubilization and growth hormone production by mutant strains was increased compared with soil isolates. Seed inoculation of wheat varieties with P solubilizing and phytohormone producing A. chroococcum showed better response compared with controls. Mutant strains of A. chroococcum showed higher increase in grain (12.6%) and straw (11.4%) yield over control and their survival (12—14%) in the rhizosphere as compared to their parent soil isolate (P4). Mutant strain M37 performed better in all three varieties in terms of increase in grain yield (14.0%) and root biomass (11.4%) over control.     

Interaction between arbuscular mycorrhizal fungus Glomus mosseae and plant growth promoting fungus Phoma sp. on their root colonization and growth promotion of cucumber (Cucumis sativus L.)

Interaction between arbuscular mycorrhizal fungus Glomus mosseae and plant growth promoting fungus Phoma sp. was studied for its effect on their root colonization and plant growth of cucumber. Two isolates of Phoma sp. (GS8-2 and GS8-3) were tested with G. mosseae. The percent root length colonized by G. mosseae was not adversely affected by the presence of Phoma isolates. In contrast, the root colonization of both isolates GS8-2 and GS8-3 in 4-week-old plants was significantly reduced (80.7% and 84.3%, respectively) by added G. mosseae. Inoculating plants with each Phoma isolate significantly increased the shoot dry weight. However, dual inoculation of each Phoma isolate with G. mosseae had no significant effect on growth enhancement.     

苹果树腐烂病菌细胞色素P450基因Vmcyp5的功能

【目的】研究表明,细胞色素P450(CYP)在死体营养型真菌的毒素合成代谢中发挥重要作用,预测可能与病原菌致病相关。论文对苹果树腐烂病菌(Valsa mali)毒素合成基因簇中的1个上调表达的CYP基因Vmcyp5进行生物学功能研究,明确CYP基因对病原菌致病力影响,为细胞色素P450基因家族对苹果树腐烂病菌致病机理的进一步研究提供依据。【方法】通过Double-joint PCR和PEG介导的原生质体转化技术获得具有G418抗性的突变体,并对突变体进行PCR检测及Southern blotting验证得到单拷贝敲除突变体。将目的基因片段重新导入敲除突变体,筛选获得互补突变体。最终对野生型菌株及敲除突变体、互补突变体进行菌落、产孢及致病力观察,利用SPSS软件对数据进行差异显著性分析,并利用q RT-PCR技术分析突变体黑色素基因簇的表达水平。【结果】通过基因敲除技术获得1个Vmcyp5基因的敲除突变体。与野生型菌株相比,Vmcyp5基因的敲除突变体菌落呈白色,产孢量减少51.3%。q RT-PCR分析发现敲除突变体黑色素基因簇基因表达量降低。重要的是,敲除突变体致病力较野生型菌株降低24.5%。互补突变体菌落颜色、产孢及致病力近似恢复至野生型菌株水平。【结论】Vmcyp5基因与病原菌黑色素合成、子实体的产生和致病力相关。