Combination of Pseudomonas aeruginosa and Pochonia chlamydosporia for Control of Root-Infecting Fungi in Tomato

A plant growth‐promoting rhizobacterium, Pseudomonas aeruginosa strain IE‐6, and a fungal antagonist, Pochonia chlamydosporia, were tested for their ability to inhibit mycelial growth of root‐infecting fungi under laboratory conditions including Macrophomina phaseolina, Fusarium oxysporum, F. solani and Rhizoctonia solani. Biocontrol effectiveness of the bacterium and the fungus alone or in combination was also determined for the control of root‐infecting fungi under field conditions. In a dual‐culture plate assay, the colonies of P. chlamydosporia and P. aeruginosa met each other and no further growth of either organism occurred. Against M. phaseolina, F. solani and R. solani, an ethyl acetate extract of the culture filtrates of P. aeruginosa inhibited fungal growth greater than the hexane extract, but against F. oxysporum the hexane extract caused greater inhibition of fungal growth. By contrast, against M. phaseolina, F. oxysporum and F. solani, the hexane extract of P. chlamydosporia was more effective in the inhibition of fungal growth than the ethyl acetate fraction. Ethyl acetate extracts of P. aeruginosa at 1.0 mg/ml not only inhibited the radial colony growth of R. solani but also lysed the fungal mycelium. P. aeruginosa produced siderophores and hydrogen cyanide under laboratory conditions. Field experiments conducted in 1997 and repeated in 1998 revealed that Pochonia chlamydosporia and P. aeruginosa significantly suppressed the root‐infecting fungi M. phaseolina, F. oxysporum, F. solani and R. solani and that the combination of the two caused greater inhibition of the fungal pathogens than either alone. Application of P. chlamydosporia and P. aeruginosa as a soil drench also resulted in enhanced growth of tomato plants.     

Biology of Pseudomonas stutzeri

Pseudomonas stutzeri is a nonfluorescent denitrifying bacterium widely distributed in the environment, and it has also been isolated as an opportunistic pathogen from humans. Over the past 15 years, much progress has been made in elucidating the taxonomy of this diverse taxonomical group, demonstrating the clonality of its populations. The species has received much attention because of its particular metabolic properties: it has been proposed as a model organism for denitrification studies; many strains have natural transformation properties, making it relevant for study of the transfer of genes in the environment; several strains are able to fix dinitrogen; and others participate in the degradation of pollutants or interact with toxic metals. This review considers the history of the discovery, nomenclatural changes, and early studies, together with the relevant biological and ecological properties, of P. stutzeri.     

Natural genetic transformation of Pseudomonas stutzeri by sand-adsorbed DNA

In a soil/sediment model system we have shown recently that a gram-positive bacterium with natural competence (Bacillus subtilis) can take up transforming DNA adsorbed to sand minerals. Here we examined whether also a naturally transformable soil bacterium of the gramnegative pseudomonad (Pseudomonas stutzeri) can be transformed by mineral-associated DNA. for these studies the transformation protocol of this species was further improved and characterized. The peak of competence during growth of P. stutzeri was determined to occur at the beginning of the stationary phase. The competence state was conserved during shock freezing and thawing of cells in 10% glycerol. Kinetic experiments showed that transformant formation after addition of DNA to competent cells proceeded for more than 2 h with DNA adsorption to cells being the rate limiting step. By means of the defined protocol P. stutzeri was shown to be transformed by sand-adsorbed DNA. Transformation by adsorbed or dissolved DNA occurred between 16° and 44°C. Efficiency and DNaseI-sensitivity of transformation by DNA adsorbed to sand or in liquid were comparable. It is concluded that uptake of particle-bound DNA by P. stutzeri in soil is possible. This finding adds evidence to the view that transformation occurs in natural environments where DNA is assumed to be significantly associated with mineral/particulate material and thereby is protected against enzymatic degradation.     

Genome Sequence of a Highly Efficient Aerobic Denitrifying Bacterium,Pseudomonas stutzeri T13

Pseudomonas stutzeri T13 is a highly efficient aerobic denitrifying bacterium. Information about the genome of this aerobic denitrifying bacterium has been limited until now. We present the draft genome of P. stutzeri T13. The results could provide further insight into the aerobic denitrification mechanism in strain T13.     

High Frequency of Natural Genetic Transformation of Pseudomonas stutzeri in Soil Extract Supplemented with a Carbon/Energy and Phosphorus Source

Agar medium (SME) prepared from aqueous soil extract was used to examine genetic transformation of Pseudomonas stutzeri JM302 (his-1) by homologous his⁺ DNA in a plate transformation assay. Growth studies indicated that SME was strongly limited in carbon and nitrogen sources. Transformation was observed on SME supplemented with pyruvate, phosphate, and ammonium. A 25-fold increase of the transformation frequency was obtained with nitrogen limitation when SME was supplemented with only pyruvate plus phosphate. Similar results were obtained with artificial soil extract medium prepared on the basis of the chemical analysis of the soil extract. On a standard minimal medium, transformation frequencies also increased (10- to 60-fold) when ammonium, phosphate, or pyruvate was growth limiting. Limitation of two or three nutrients did not stimulate transformation. The size of the inoculum (2 × 10³ to 2 × 10⁷ cells) was irrelevant to the enhanced transformation under nitrogen limitation on SME or standard minimal medium. We further show that P. stutzeri can use a variety of carbon and energy sources for competence development. It is concluded that genetic transformation of P. stutzeri is possible in the chemical environment of soil upon supply of nutrients and may be strongly stimulated by a growth-limiting concentration of single nutrients including sources of C, N, or P.     

Development and relations of <Emphasis Type="Italic">Fusarium culmorum</Emphasis> and <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> in soil

The development of Fusarium culmorum and Pseudomonas fluorescens in soil, and the relations between them, were studied using membrane filters containing the fungus, the bacterium, or both microorganisms; the filters were incubated in soil. F. culmorum was identified by indirect immunofluorescence; the GUS-labeled strain was used to visualize P. fluorescens. It was found that F. culmorum introduced in soil can develop as a saprotroph, with the formation of mycelium, macroconidia, and a small amount of chlamydospores. Introduction of glucose and cellulose resulted in increased density of the F. culmorum mycelium and macroconidia. P. fluorescens suppressed the development of the F. culmorum mycelium in soil, but stimulated chlamydospore formation. Decreased mycelial density in the presence of P. fluorescens was more pronounced in soil without additions and less pronounced in the case of introduction of glucose or cellulose. F. culmorum had no effect on P. fluorescens growth in soil.     

Observations on the pathogenesis of the imperfect fungus,Fusarium solani,in the California brown shrimp,Penaeus californiensis

The pathogenesis of Fusarium solani infections was studied using a highly susceptible species, Penaeus californiensis. In two experiments, F. solani infections were established in artificially wounded and infected juvenile and adults of P. californiensis, while similar control groups were wounded but not artificially infected. In the first experiment, the progress of F. solani infections in 15-g cultured juveniles of P. californiensis was followed by gross inspection and by light and electron microscopical study of the wound area. Developing F. solani infections were produced with a success rate of 100% within 14 days postinfection, and deaths due to disease of greater than 50% of the artificially infected group had occurred by 24 days postinfection. Infections were histologically characterized by (1) expansion of lesions into the tissue surrounding the point of entry, (2) destruction of invaded tissues by host granuloma formation and possibly by fungal enzymes and toxins, and (3) hemocyte encapsulation of hyphae with eventual melanization of the encapsulating hemocytes. In the second study, wild adult P. californiensis were artificially infected with F. solani, and changes in several of their hemolymph constituents [alkaline phosphatase, serum glutamic oxaloacetic transaminase (SGOT), glucose, total protein, hemocyte count, and hematopoietic tissue mitotic index] were determined as the disease developed. Significant differences occurred in these hemolymph parameters of shrimp with advanced F. solani infections compared to those of uninfected, unwounded control shrimp or those with early F. solani infections. Hemolymph from severely infected shrimp was hypoproteinemic, contained lower numbers of circulating hemocytes, and frequently failed to coagulate.     

Identification and evaluation of a potential biocontrol agent,Bacillus subtilis,against Fusarium sp. in apple seedlings

To find a potential biocontrol agent against Fusarium sp. in apple seedlings, an endophytic bacterium strain was isolated from apple tree tissues. The inhibitive efficiency of the isolated strain against the hyphal growth of Fusarium sp. and Rhizoctonia solani was tested. Strain Y-1 showed significant inhibitory effects against Fusarium oxysporum, F. moniliforme, F. proliferatum, F. solani and R. solani. Its antifungal activity against F. oxysporum was the highest, reaching up to 64.90 %. In vivo tests indicated that strain Y-1 effectively protects apple from F. oxysporum infections. The control effect reached 92.26 % when bacterial inoculation was performed 3 days prior to pathogen inoculation. Strain Y-1 could colonize the rhizosphere and tissues within 30 days. It was also able to induce systemic resistance in apple seedlings as shown by the activities of SOD and POD. Strain Y-1 significantly increased the root length, root wet and dry weights, and plant height of the apple seedlings compared with the control group. The homology analysis of the 16S rRNA sequence, together with morphological, physical, and biochemical analyses, revealed that strain Y-1 is Bacillus subtilis.     

Identification of a stress-induced protein in stem exudates of soybean seedlings root-infected with Fusarium solani f. sp. glycines

Sudden death syndrome of soybean (Glycine max) is caused by the soilborne fungus, Fusarium solani f. sp. glycines, that infects soybean roots. Besides root necrosis, symptoms include interveinal leaf chlorosis, necrosis and premature defoliation. It is proposed that a fungal toxin is produced in soybean roots and translocated to foliage. In this study, we isolated compounds from soybean stem exudates from plants that were either inoculated or not inoculated with F. solani f. sp. glycines. A protein with an estimated molecular mass of 17 kDa and designated as FISP 17 for F. solani f. sp. glycines-induced stress protein was identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein occurred only in F. solani f. sp. glycines-infected soybean stem exudates. The N-terminal amino acid sequence of the purified protein had 100 % identity with a starvation-associated message 22 protein, and 80 and 78 % identity with purified bean pathogenesis-related proteins, PvPR1 and PvPR2, respectively. To determine if the protein was of plant or fungal origin, a synthetic peptide was designed based on the N-terminal sequence and used to raise a polyclonal antibody from rabbit. Western blot analysis showed that the antibody only reacted with a 17-kDa protein in F. solani f. sp. glycines-infected plant exudates, but no reaction occurred with healthy plant exudates or with culture filtrates of F. solani f. sp. glycines. This is the first report of the presence of a stress-induced protein in stem exudates of soybean seedlings root-infected with F. solani f. sp. glycines.     

seed rot and root rot complex of beans (Phaseolus vulgaris L.)

Fusarium solani (76%),Phyllosticta phaseolina (12%) andAspergillus flavus (10%) were isolated from bean(Phaseolus vulgaris) seeds showing lesions. Out of these the first two caused 30 per cent and 20 per cent seed rotting respectively after artificial seed inoculations. When inoculum was added into the soil before sowing the seed, 35 per cent and 10 per cent pre-emergence rot ocurred and there was 35 per cent and 15 per cent post-emergence blight when soil with healthy seedlings was inoculated withF. solani andP. phaseolina respectively.F. solani caused similar lesions on bean seeds when inoculated after an injury.F. oxysporum was isolated from the roots of dried bean plants showing necrosis.     

Suppression of Alfalfa Growth by Concommitant Populations of Pratylenchus penetrans and two Fusarium species

Growth of alfalfa (Medicago sativa cv. Vernal) seedlings was compared after inoculation with combinations of either Pratylenchus penetrans and Fusarium soloni or P. penetrans and F. oxysporum f. sp. medicaginis. A synergistic disease interaction occurred in alfalfa when F. oxysporum and P. penetrans were added simultaneously to the soil. Alfalfa growth was suppressed at all inoculum levels of P. penetrans and F. oxysporum, but not with F. solani. Seedlings inoculated with the nematode alone gave lower yields than when inoculated with either Fusarium species alone. Fusarium oxysporum, but not F. solani, was pathogenic to alfalfa under similar experimental conditions. Fusarium oxysporum did not alter the populations of P. penetrans in alfalfa roots, whereas the presence of F. solani was associated with a diminished number of P. penetrans in the roots.     

The Nitrogen-Fixation Island Insertion Site Is Conserved in Diazotrophic Pseudomonas stutzeri and Pseudomonas sp. Isolated from Distal and Close Geographical Regions

The presence of nitrogen fixers within the genus Pseudomonas has been established and so far most isolated strains are phylogenetically affiliated to Pseudomonas stutzeri. A gene ortholog neighborhood analysis of the nitrogen fixation island (NFI) in four diazotrophic P. stutzeri strains and Pseudomonas azotifigens revealed that all are flanked by genes coding for cobalamin synthase (cobS) and glutathione peroxidise (gshP). The putative NFIs lack all the features characterizing a mobilizable genomic island. Nevertheless, bioinformatic analysis P. stutzeri DSM 4166 NFI demonstrated the presence of short inverted and/or direct repeats within both flanking regions. The other P. stutzeri strains carry only one set of repeats. The genetic diversity of eleven diazotrophic Pseudomonas isolates was also investigated. Multilocus sequence typing grouped nine isolates along with P. stutzeri and two isolates are grouped in a separate clade. A Rep-PCR fingerprinting analysis grouped the eleven isolates into four distinct genotypes. We also provided evidence that the putative NFI in our diazotrophic Pseudomonas isolates is flanked by cobS and gshP genes. Furthermore, we demonstrated that the putative NFI of Pseudomonas sp. Gr65 is flanked by inverted repeats identical to those found in P. stutzeri DSM 4166 and while the other P. stutzeri isolates harbor the repeats located in the intergenic region between cobS and glutaredoxin genes as in the case of P. stutzeri A1501. Taken together these data suggest that all putative NFIs of diazotrophic Pseudomonas isolates are anchored in an intergenic region between cobS and gshP genes and their flanking regions are designated by distinct repeats patterns. Moreover, the presence of almost identical NFIs in diazotrophic Pseudomonas strains isolated from distal geographical locations around the world suggested that this horizontal gene transfer event may have taken place early in the evolution.     

Eggs of Tylenchulus semipenetrans Inhibit Growth of Phytophthora nicotianae and Fusarium solani in vitro

In previous greenhouse and laboratory studies, citrus seedlings infested with the citrus nematode Tylenchulus semipenetrans and later inoculated with the fungus Phylophthora nicotianae grew larger and contained less fungal protein in root tissues than plants infected by only the fungus, demonstrating antagonism of the nematode to the fungus. In this study, we determined whether eggs of the citrus nematode T. semipenetrans and root-knot nematode Meloidogyne arenaria affected mycelial growth of P. nicotianae and Fusarium solani in vitro. Approximately 35,000 live or heat-killed (60°C, 10 minutes) eggs of each nematode species were surface-sterilized with cupric sulfate, mercuric chloride, and streptomycin sulfate and placed in 5-pl drops onto the center of nutrient agar plates. Nutrient agar plugs from actively growing colonies of P. nicotianae or F. solani were placed on top of the eggs for 48 hours after which fungal colony growth was determined. Live citrus nematode eggs suppressed mycelial growth of P. nicotianae and F. solani (P ≤ 0.05) compared to heat-killed eggs and water controls. Reaction of the fungi to heat-killed eggs was variable. Root-knot nematode eggs had no effect on either P. nicotianae or F. solani mycelial growth. The experiment demonstrated a species-specific, direct effect of the eggs of the citrus nematode on P, nicotianae and F. solani.     

Effect of Population Dynamics of Pseudomonas cepacia and Paecilomyces lilacinus on Colonization of Polyfoam Rooting Cubes by Rhizoctonia solani

Suspensions of Pseudomonas cepacia (strain 5.5B) and Paecilomyces lilacinus (isolate 6.2F) were applied to polyfoam rooting cubes for control of stem rot of poinsettia caused by Rhizoctonia solani. The populations of antagonists and colonization of rooting cubes by R. solani were monitored during a 3-week period. Colonization of cubes by R. solani was reduced in cubes treated with P. cepacia, but the population of P. cepacia decreased by as much as 97% during the test period. Increased colonization by R. solani was correlated with a decline in population of P. cepacia. P. lilacinus was more persistent than P. cepacia in cubes, with only a 21% reduction observed during the 3-week period. Colonization of the P. lilacinus-treated cubes by R. solani was significantly less than colonization of infested controls. No correlation existed between population of P. lilacinus and colonization of cubes by R. solani.     

Highly Different Levels of Natural Transformation Are Associated with Genomic Subgroups within a Local Population of Pseudomonas stutzeri from Soil

A highly sensitive and specific PCR-based method of monitoring 16S rRNA genes of Pseudomonas stutzeri was developed for searching P. stutzeri DNA in environmental samples. This monitoring was combined with a reliable and sensitive method for isolating P. stutzeri colony formers from soil and sediment, depending on their utilization of ethylene glycol, starch, and maltose. With these techniques, P. stutzeri populations (n = 2 to 170) were obtained from five of six sites giving positive PCR signals (including three marine sediment and two soil samples). The phylogenetic positions of isolates from the five sites, based on their 16S ribosomal DNA sequences, indicated that the environmental isolates were affiliated with different genomovars of P. stutzeri. Using the broad-host-range plasmid pNS1 with kanamycin and gentamicin resistance determinants as the transforming DNA, naturally transformable strains were identified among the isolates from all sites. For one population from soil, the genetic relationship of the 120 members was determined by randomly amplified polymorphic DNA-PCR with three PCR primers. Among the population members which are taxonomically closely related as determined by 16S sequence comparisons of group representatives, a rather high genetic diversity and a characteristic clustering into subgroups were found. Remarkably, within the population, nontransformability and different levels of transformability (a frequency between about 10⁻⁹ and 10⁻⁴ per cell) were often associated with distinct genetic subgroups. It is concluded that transformability is widespread among environmental P. stutzeri strains and that its specific level is a heritable trait that may vary strongly within a local population.     

<Emphasis Type="Italic">Exiguobacterium acetylicum</Emphasis> strain 1P (MTCC 8707) a novel bacterial antagonist from the North Western Indian Himalayas

Exiguobacterium acetylicum strain 1P (MTCC 8707) is a rhizospheric, Gram positive, rod shaped, yellow pigmented bacterium isolated from an apple orchard rhizospheric soil, on nutrient agar plates incubated at 4°C. The species level identification was arrived on the basis of 16S rRNA gene sequencing. The sequence showed 98% similarity with sequences of E. acetylicum available in the public domain. The strain was positive for siderophore and HCN production. In separate invitro assays it was found to inhibit the growth and development of Rhizoctonia solani, Sclerotium rolfsii, Pythium and Fusarium oxysporum. The volatile compound produced by the bacterium was found to be the most potent in inhibiting the hyphal development of R. solani, S. rolfsii, Pythium and F. oxysporum by 45.55, 41.38, 28.92 and 39.74% respectively. Commonly observed deformities caused by the diffusible and volatile compounds produced by the bacterium included hyphal inhibition, constriction and deformation. Under pot culture conditions the bacterium improved the germination and early growth parameters of pea (Pisum sativum) in the presence of R. solani and S. rolfsii.     

Combinatorial effect of endophytic and plant growth promoting rhizobacteria against wilt disease of Capsicum annum L. caused by Fusarium solani

Combination of biocontrol agents that are compatible with each other is a strategic approach to control the plant disease and pest. The present study was designed to evaluate the protective effects of compatible endophytic bacterial strains (Bacillus subtilis; EPCO16 and EPC5) and rhizobacterial strain (Pseudomonas fluorescens; Pf1) against chilli wilt disease caused by Fusarium solani. Our results showed that B. subtilis (EPCO16 and EPC5) and P. fluorescens (Pf1) were compatible and effectively inhibited the growth of the F. solani. The application of endophytic and rhizobacterial strains, singly and in combination in green house and field conditions were found to be effective in controlling the chilli Fusarium wilt disease by inducing systemic resistance (ISR) as evidenced by enhanced activities of PO, PPO, PAL, β-1,3-glucanase, Chitinase and Phenolic involved in the synthesis of phytolaexins thereby promoting the growth of plants. However, combinations of EPCO16 + EPC5 + Pf1 bacterial strains were more effective than single agents. These findings suggest that synergistic interactions of biocontrol agents may be responsible for the management of chilli wilt disease caused by F. solani.     

Cloning of Genes Involved in the Synthesis of Pyrrolnitrin from Pseudomonas fluorescens and Role of Pyrrolnitrin Synthesis in Biological Control of Plant Disease

A soil isolate of Pseudomonas fluorescens (BL915) was shown to be an effective antagonist of Rhizoctonia solani-induced damping-off of cotton. Investigation of the biological basis of this antagonism revealed that the strain produces pyrrolnitrin, a secondary metabolite known to inhibit R. solani and other fungi. Mutants of strain BL915 that did not produce pyrrolnitrin and did not suppress damping-off of cotton by R. solani were generated by exposure to N-methyl-N′ -nitro-N-nitrosoguanidine. A gene region that was capable of restoring pyrrolnitrin production to the non-pyrrolnitrin-producing mutants and of conferring this ability upon two other P. fluorescens strains not otherwise known to produce this compound or to be capable of suppressing damping-off caused by R. solani was isolated from strain BL915. The non-pyrrolnitrin-producing strains (mutants of BL915 and the other two P. fluorescens strains) which synthesized pyrrolnitrin after the introduction of the gene region from strain BL915 were also shown to be equal to strain BL915 in their ability to suppress R. solani-induced damping-off of cotton. These results indicate that we have isolated from P. fluorescens BL915 a gene(s) that has a role in the synthesis of pyrrolnitrin and that the production of this compound has a role in the ability of this strain to control damping-off of cotton by R. solani.     

Association of Criconemella xenoplax and Fusarium spp. with Root Necrosis and Growth of Peach

Criconemella xenoplax, Fusarium solani, and F. oxysporum caused necrosis of Nemaguard peach feeder roots in greenhouse tests. Root necrosis was more extensive in the presence of either fungus than wtih C. xenoplax alone. Shoot growth and plant height were less for plants inoculated with F. oxysporum or F. solani than for plants inoculated with the fungi plus C. xenoplax. Neither synergistic nor additive effects on root necrosis or plant growth occurred between C. xenoplax and the fungal pathogens.     

Chitosan as a Component of Pea-Fusarium solani Interactions

Chitosan, a polymer of β-1,4-linked glucosamine residues with a strong affinity for DNA, was implicated in the pea pod-Fusarium solani interaction as an elicitor of phytoalexin production, an inhibitor of fungal growth and a chemical which can protect pea tissue from infection by F. solani f. sp. pisi. Purified Fusarium fungal cell walls can elicit phytoalexin production in pea pod tissue. Enzymes from acetone powders of pea tissue release eliciting components from the F. solani f. sp. phaseoli cell walls. Hydrochloric acid-hydrolyzed F. solani cell walls are about 20% glucosamine. The actual chitosan content of F. solani cell walls is about 1%. However, chitosan assays and histochemical observations indicate that chitosan content of F. solani spores and adjacent pea cells increases following inoculation. Dormant F. solani spores also accumulate chitosan. Concentrations of nitrous acid-cleaved chitosan as low as 0.9 microgram per milliliter and 3 micrograms per milliliter elicit phytoalexin induction and inhibit germination of F. solani macroconidia, respectively. When chitosan is applied to pea pod tissue with or prior to F. solani f. sp. pisi, the tissue is protected from infection.