Root Exudates of Tomato Plants and Their Effect on the Growth and Antifungal Activity of Pseudomonas Strains

The study of the effect of the root exometabolites of tomato plants on the growth and antifungal activity of plant growth–promoting Pseudomonas strains showed that the antifungal activity of plant growth–promoting rhizobacteria in the plant rhizosphere may depend on the sugar and organic acid composition of root exudates.     

Effect of <Emphasis Type="Italic">Pseudomonas</Emphasis> Bacteria on Peroxidase Activity in Wheat Plants when Infected with <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis>

We investigated the effect of treating soft wheat seeds (Triticum aestivum L.) with two Pseudomonas bacteria strains, isolated from earthworm coprolites, showing a significant antifungal and growth-promoting action in preliminary screening on the activity of guaiacol-dependant peroxidase under phytopathogenic load in the presence of Bipolaris sorokiniana (Sacc.) Shoemaker as a mechanism for inducing plant resistance to the pathogen. We established a statistically significant decrease (P < 0.05) in root rot disease incidence and severity during bacterization, which is indicative both of antifungal activity of the used bacterial isolates and of their successful colonizing the rhizosphere of wheat plants. We noted a response of free and weakly bound peroxidase of wheat plants to infection with B. sorokiniana: the enzyme activity increased during pathogenesis. Bacterization also increased peroxidase activity in plant leaves and roots, the greatest differences from non-bacterized plants being observed in wheat roots in the presence of the pathogen. We detected a direct link between peroxidase activity in wheat roots and leaf tissues in the absence of the pathogen and the feedback between peroxidase activity and plant infestation by the root rot pathogen. In the presence of the phytopathogen, there is a lack of correlation between peroxidase activity in wheat roots and leaves, and there is a shift of activity towards its increase in roots, which plays an important role in the development of systemic resistance against the root rot pathogen that penetrates into plants through the roots and root collar.     

西葫芦根腐病菌拮抗细菌的防病促生作用

【目的】获取促生的同时可抑制尖孢镰刀菌(Fusarium oxysporum)的拮抗菌并明确其防治效果。【方法】平板对峙生长法测定前期分离的19株促生菌对尖孢镰刀菌的抑制作用,温室条件下接种微生物菌剂对西葫芦根腐病的防治作用;田间条件下接种复合菌剂代替部分化肥对西葫芦生长的影响。【结果】可有效拮抗尖孢镰刀菌的菌株有9株,其中,菌株FX2的抑菌活性较好,抑制率达到66.80%。在温室盆栽中,接种微生物菌剂(LHS11+FX2)对西葫芦根腐病抑制率达到57.14%。在田间试验中,微生物菌剂配施化肥对西葫芦的生物量和根系形态影响显著,以85%化肥+复合菌剂处理效果较优,其对西葫芦成熟期的产量显著提高27.13%。【结论】复合菌剂(LHS11+FX2)对西葫芦根腐病具有较好的防治作用;85%化肥+复合菌剂对西葫芦的促生作用明显,在一定程度上节约了化肥投入成本,提高了增产效益。     

Recovery of plant growth-promoting rhizobacteria from sodium alginate beads after 3 years following storage at 4 °C

Two plant growth-promoting bacteria, Bacillus subtilis and Pseudomonas corrugata, immobilized in a sodium alginate based formulation were evaluated for their survival, viability and plant growth-promoting ability after 3 years of storage at 4 °C. Populations of both of the bacterial isolates recovered from the immobilized sodium alginate beads were in the order of 10⁸ cfu g⁻¹. The plant-based bioassay indicated that the plant growth promotion ability of both of the bacterial isolates was equal to those of fresh broth-based formulations. The bacterial isolates retained the root colonization, and antifungal and enzyme activities in the alginate-based formulation during storage.     

Engineering Pseudomonas protegens Pf-5 to improve its antifungal activity and nitrogen fixation

In agricultural production, sustainability is currently one of the most significant concerns. The genetic modification of plant growth-promoting rhizobacteria may provide a novel way to use natural bacteria as microbial inoculants. In this study, the root-colonizing strain Pseudomonas protegens Pf-5 was genetically modified to act as a biocontrol agent and biofertilizer with biological nitrogen fixation activity. Genetic inactivation of retS enhanced the production of 2,4-diacetylphloroglucinol, which contributed for the enhanced antifungal activity. Then, the entire nitrogenase island with native promoter from Pseudomonas stutzeri DSM4166 was introduced into a retS mutant strain for expression. Root colonization patterns assessed via confocal laser scanning microscopy confirmed that GFP-tagged bacterial were mainly located on root surfaces and at the junctions between epidermal root cells. Moreover, under pathogen and N-limited double treatment conditions, the fresh weights of seedlings inoculated with the recombinant retS mutant-nif strain were increased compared with those of the control. In conclusion, this study has innovatively developed an eco-friendly alternative to the agrochemicals that will benefit global plant production significantly.     

Plant defensins and virally encoded fungal toxin KP4 inhibit plant root growth

Plant defensins are small, highly stable, cysteine-rich antimicrobial proteins that are thought to constitute an important component of plant defense against fungal pathogens. There are a number of such defensins expressed in various plant tissues with differing antifungal activity and spectrum. Relatively little is known about the modes of action and biological roles of these proteins. Our previous work on a virally encoded fungal toxin, KP4, from Ustilago maydis and subsequently with the plant defensin, MsDef1, from Medicago sativa demonstrated that some of these proteins specifically blocked calcium channels in both fungi and animals. The results presented here demonstrate that KP4 and three plant defensins, MsDef1, MtDef2, and RsAFP2, all inhibit root growth in germinating Arabidopsis seeds at low micromolar concentrations. We have previously demonstrated that a fusion protein composed of Rab GTPase (RabA4b) and enhanced yellow fluorescent protein (EYFP) is dependent upon calcium gradients for localization to the tips of the growing root hairs in Arabidopsis thaliana. Using this tip-localized fusion protein, we demonstrate that all four proteins rapidly depolarize the growing root hair and block growth in a reversible manner. This inhibitory activity on root and root hair is not directly correlated with the antifungal activity of these proteins and suggests that plants apparently express targets for these antifungal proteins. The data presented here suggest that plant defensins may have roles in regulating plant growth and development. A. Allen and A.K. Snyder contributed equally.     

Isolation and identification of endophytic bacteria from root tissues of Salvia miltiorrhiza Bge. and determination of their bioactivities

Endophytic bacteria are microorganisms that live in host plants, but do not cause diseases to the hosts. This study examined the occurrence, distribution, growth-promoting and antifungal activities of endophytes in the root of Salvia miltiorrhiza Bge. Six endophytic bacterial strains, which belong to genera of Pseudomonas, Rhizobium, Bacillus and Novosphingobium, were isolated from the root of healthy S. miltiorrhiza. Cell suspension (approx. 10⁹ cell?·?ml^?1) of two isolates and cell-free fermentation filtrate of four isolates substantially promoted the growth of hypocotyl and radicle of muskmelon seeds. The cell-free fermentation filtrate of six isolates had no inhibiting effect on tested pathogenic fungi, namely Fusarium solani, F. oxysporum f. sp. vasinfectum and F. oxysporum. Six compounds were isolated from one of the six endophytic bacteria, namely, Bacillus aryabhattai, and two of these compounds displayed certain antifungal activity against three tested S. miltiorrhiza pathogens. Our work indicates that endophytic bacteria occur in the root of S. miltiorrhiza, and that associated bacterial isolates have growth-promoting effect on muskmelon seeds and are expected to be a potential source for bioactive metabolites.     

Characterization of antifungal metabolite produced by a new strain Pseudomonas aeruginosa PUPa3 that exhibits broad-spectrum antifungal activity and biofertilizing traits

AIM: To study the antifungal activity and plant beneficial traits of a broad-spectrum antagonistic fluorescent pseudomonad strain, PUPa3. METHODS AND RESULTS: Strain PUPa3 was isolated from the rhizosphere soil of rice and identified as Pseudomonas aeruginosa on the basis of biochemical tests and by comparison of 16S rDNA sequences. This bacterium exhibits a broad-spectrum antifungal activity towards phytopathogenic fungi. The antifungal metabolite by PUPa3 was extracted, purified and characterized using nuclear magnetic resonance (NMR) and mass spectroscopy (MS). Production of indole-3-acetic acid (IAA), siderophores, phosphatase and protease in PUPa3 was determined. Strain PUPa3 did not produce hydrogen cyanide, cellulase and pectinase. CONCLUSION: The antifungal metabolite produced by PUPa3 has been identified as phenazine-1-carboxamide (PCN) on the basis of NMR and MS data. Strain PUPa3 showed a broad-spectrum antifungal activity towards a range of phytopathogenic fungi. This bacterium also showed several plant growth-promoting traits but did not show the traits attributed to deleterious rhizobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Present study reports the production of PCN as well as IAA for the first time by a saprophytic P. aeruginosa strain PUPa3. Because of the production of siderophore, growth hormone, protease and phosphatase and its innate fungicidal potential, this strain can be used as biofertilizer and antagonist against a range of phytopathogenic fungi that infect rice, groundnut, tobacco, chili, mango, sugarcane, tea, cotton and banana.     

侧孢芽孢杆菌Bl13对番茄早疫病防治效果及机制

以对番茄早疫病原菌有良好拮抗效果的侧孢芽孢杆菌Bl13为研究对象,采用盆栽试验,通过测定番茄株高、茎粗、番茄早疫病病情指数、叶片内防御酶活性以及根区土壤微生物多样性、微生物群落结构组成等指标,探究侧孢芽孢杆菌Bl13防治番茄早疫病的效果及机制。结果表明: 接种Bl13可显著降低番茄早疫病的病情指数,提高叶片内多酚氧化酶(PPO)、过氧化物酶(POD)等防御酶活性,降低病害对植物地上部分及根系生长发育的影响。同时,改善番茄根区土壤微生物群落结构,使芽孢杆菌属、假单胞菌属等常见有益菌属相对丰度显著提高,油壶菌属、血赤壳属相对丰度显著降低。侧孢芽孢杆菌Bl13可通过提高番茄叶片内防御酶活性并增加根区中有益微生物的数量来增强植物对番茄早疫病的抗性,从而实现对番茄早疫病的防治。     

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.     

Endophytic colonization of Typha australis by a plant growth-promoting bacterium Klebsiella oxytoca strain GR-3

AIMS: To isolate and characterize endophytic diazotrophic bacteria from a semi-aquatic grass (Typha australis) which grows luxuriantly with no addition of any nitrogen source. METHODS AND RESULTS: Ten endophytic diazotrophic bacteria from surface-sterilized roots and culm of T. australis were isolated and screened for plant growth-promoting activities employing standard methods. Based on the rate of nitrogenase activity, indole acetic acid (IAA) production and phosphate (P) solubilization, one root isolate namely GR-3 was found to be the most efficient one. This isolate was identified as Klebsiella oxytoca on the basis of 16S rDNA sequence analysis. Amplification of nifH by polymerase chain reaction (PCR) and detection of dinitrogenase reductase by western blot confirmed the diazotrophic nature of GR-3. It was tagged with gusA fused to a constitutive promoter and the resulting transconjugant was inoculated onto endophyte-free rice variety Malviya dhan-36 seedlings to express cross-infection ability which resulted in a significant increase in root/shoot length and chlorophyll a content. CONCLUSIONS: Roots and culm of T. australis harbour several endophytic diazotrophic bacteria. One root isolate, identified as K. oxytoca GR-3, seems to be an efficient plant growth-promoting bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: Plant growth-promoting properties of GR-3 suggest that this promising isolate merits further investigations for potential application in agriculture.     

Effectiveness of various Pseudomonas spp. and Burkholderia caryophylli containing ACC-deaminase for improving growth and yield of wheat (Triticum aestivum L.)

This study assessed the possible role of different traits in selected plant growth-promoting rhizobacteria (PGPR) for improving wheat growth and yield under natural conditions. Rhizobacteria exhibiting 1-aminocyclopropane-1-carboxylate (ACC)-deaminase activity were isolated and screened for their growth-promoting activity in wheat under axenic conditions. Five isolates belonging to Pseudomonas and one Burkholderia caryophylli isolate that showed promising performances under axenic conditions were selected and characterized for in vitro ACC-deaminase activity, chitinase activity, auxin production, P solubilization, and root colonization. These isolates were then used as inocula for wheat cultivated under natural conditions in pot and/or field trials. Significant increases in root elongation, root weight, tillers per pot, 1,000-grain weight, and grain and straw yields were observed in response to inoculation with PGPR in the pot trials. Inoculation with these PGPR was also effective under field conditions and increased the wheat growth and yield significantly. However, the efficacy of the strains was inconsistent under the axenic, pot, and field conditions. Pseudomonasfluorescens (ACC50), which exhibited a relatively high in vitro ACC-deaminase activity, chitinase activity, auxin production, and P solubilization and more intensive root colonization, was the most efficient isolate under the field conditions. Therefore, these results demonstrated that ACC-deaminase activity is an efficient parameter for the selection of promising PGPR under axenic conditions. However, additional traits of PGPR, including auxin production, chitinase activity, P solubilization, and root colonization, are also important for selecting PGPR as biofertilizers.     

Studies on plant growth-regulating substances: Water uptake by discs from inulin-storing roots and tubers in response to growth-substance treatment

Discs of tissue cut from chicory and dandelion roots are shown to take up large amounts of water when treated with solutions of the auxin 2,4-dichlorophenoxyacetic acid. The effects of disc size, concentration of auxin and cold-storage of the roots on the magnitude of the response have been studied. A range of plant growth-regulating chemicals has been examined for the capacity to promote water uptake in chicory root discs. The results are considered both in relation to chemical structure and to the growth responses induced by these compounds in the wheat cylinder and pea segment tests. The evidence indicates that the water uptake response by chicory root tissues provides a useful method for assessing plant growth-promoting activity.     

Characterization of plant growth-promoting rhizobacterium <Emphasis Type="Italic">Exiguobacterium</Emphasis> NII-0906 for its growth promotion of cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis>)

A phosphate solubilizing and antagonistic bacterial strain, isolated from a Western Ghat forest soil in Kerala province, India (designated as NII-0906), showed cold tolerance and grew from 10 to 37°C (optimum temperature 30°C). It was a Gram-positive, rod shaped, 0.8–1.6 μm in size, and exhibited tolerance to a wide pH range (5–12; optimum 7.0) and salt concentration up to 7% (w/v). The isolate showed maximum similarity with Exiguobacterium marinum TF-80^T based on 16S rRNA analysis. It solubilized tricalcium phosphate under in vitro conditions. The phosphate solubilization was estimated along a temperature range (5–40°C), and maximum activity (84.7 μg mL⁻¹ day⁻¹) was recorded at 30°C after 10 days of incubation. The phosphate solubilizing activity coincided with a concomitant decrease in pH of the medium. The isolate also exhibited antifungal activity against phytopathogenic fungi in Petri dish assays and produced siderophore and hydrogen cyanide. The strain’s plant growth promotion properties were demonstrated through a cowpea-based bioassay under greenhouse conditions. The bacterial inoculation resulted in significant increment in plant root, stem and as well as in plant biomass. Further, scanning electron microscopic study revealed the root colonization in cowpea. These results could offer potential perspective for the strain to be used as plant growth-promoting rhizobacteria, which could be used as an inoculant for regional crops.     

Cell Ultrastructure in Azospirillum brasilense Biofilms

Microbiology - Due to the primary localization of both epiphytic and endophytic plant growth-promoting rhizobacteria on the surface of the plant root system, biofilm formation is an adaptive trait...     

Plants respond to pathogen infection by enhancing the antifungal gene expression of root-associated bacteria

Plant health and fitness widely depend on interactions with soil microorganisms. Some bacteria such as pseudomonads can inhibit pathogens by producing antibiotics, and controlling these bacteria could help improve plant fitness. In the present study, we tested whether plants induce changes in the antifungal activity of root-associated bacteria as a response to root pathogens. We grew barley plants in a split-root system with one side of the root system challenged by the pathogen Pythium ultimum and the other side inoculated with the biocontrol strain Pseudomonas fluorescens CHA0. We used reporter genes to follow the expression of ribosomal RNA indicative of the metabolic state and of the gene phlA, required for production of 2,4-diacetylphloroglucinol, a key component of antifungal activity. Infection increased the expression of the antifungal gene phlA. No contact with the pathogen was required, indicating that barley influenced gene expression by the bacteria in a systemic way. This effect relied on increased exudation of diffusible molecules increasing phlA expression, suggesting that communication with rhizosphere bacteria is part of the pathogen response of plants. Tripartite interactions among plants, pathogens, and bacteria appear as a novel determinant of plant response to root pathogens.     

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.     

Survival of Bacillus subtilis AF 1 in the bacterized peanut rhizosphere and its influence on native microflora and seedling growth

A mutant of Bacillus subtilis AF 1 (an antifungal strain with plant growth-promoting activity, resistant to 100 g streptomycin/ml) was isolated from pigeon pea rhizosphere and, when used in peanut bacterization, decreased fungal and bacterial numbers and increased the actinomycete population in the rhizosphere. Over 28 days, AF 1 declined by 0.9 log c.f.u. in sterilized soil and by 2.6 log c.f.u. in native soil. From the seedlings raised with peanut seeds bacterized with AF 1 at 6.5 log c.f.u./seed, 4.1 and 4.7 log c.f.u. of AF 1 were recovered from the rhizosphere and total underground parts, respectively. Bacterization with AF 1 had no significant growth-promoting or -inhibiting effect on the peanut.     

Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria

One of the major mechanisms utilized by plant growth-promoting rhizobacteria (PGPR) to facilitate plant growth and development is the lowering of ethylene levels by deamination of 1-aminocyclopropane-1-carboxylic acid (ACC) the immediate precursor of ethylene in plants. The enzyme catalysing this reaction, ACC deaminase, hydrolyses ACC to α -ketobutyrate and ammonia. Several bacterial strains that can utilize ACC as a sole source of nitrogen have been isolated from rhizosphere soil samples. All of these strains are considered to be PGPR based on the ability to promote canola seedling root elongation under gnotobiotic conditions. The treatment of plant seeds or roots with these bacteria reduces the amount of ACC in plants, thereby lowering the concentration of ethylene. Here, a rapid procedure for the isolation of ACC deaminase-containing bacteria, a root elongation assay for evaluating the effects of selected bacteria on root growth, and a method of assessing bacterial ACC deaminase activity are described in detail. This should allow researchers to readily isolate new PGPR strains adapted to specific environments.     

广西葛根内生细菌的分离鉴定及其促生特性

【背景】植物内生菌长期与宿主共生,对宿主生长发育产生影响。葛根作为重要的药食两用作物,葛根内生菌的研究具有重要实践意义。【目的】对广西葛根根部内生细菌进行分离、鉴定及促植物生长特性分析,旨在了解该药食同源植物内生细菌种群结构及其促生特性,为分析内生菌群体在药食同源植物产量和品质形成的作用及其内生细菌资源的开发利用提供参考。【方法】采用6种不同的培养基从广西葛根的根瘤、根系和根愈伤组织分离内生细菌,16S rRNA基因测序和系统发育分析内生细菌的分布特征和遗传多样性,采用生理生化方法测定分离菌株的固氮活性、溶磷特性、产生嗜铁素、分泌吲哚乙酸(indole-3-aceticacid,IAA)等促生特性。【结果】从葛根根瘤、根系和根部愈伤组织中共分离得到223个菌株,16S rRNA基因测序鉴定这些菌株隶属于2门4纲10科19属,其中芽孢杆菌属、假单胞菌属、土壤杆菌属、肠杆菌属为葛根优势菌群;内生细菌数量和群落组成存在明显的组织特异性,其数量表现为根瘤>根系>根愈伤组织,但其种群多样性表现为根愈伤组织>根系>根瘤。不同培养基分离出的细菌种群丰富度有差异。从供试菌株中筛...