Suppression of damping-off disease in host plants by the rhizoplane bacterium Lysobacter sp. strain SB-K88 is linked to plant colonization and antibiosis against soilborne Peronosporomycetes

We previously demonstrated that xanthobaccin A from the rhizoplane bacterium Lysobacter sp. strain SB-K88 suppresses damping-off disease caused by Pythium sp. in sugar beet. In this study we focused on modes of Lysobacter sp. strain SB-K88 root colonization and antibiosis of the bacterium against Aphanomyces cochlioides, a pathogen of damping-off disease. Scanning electron microscopic analysis of 2-week-old sugar beet seedlings from seeds previously inoculated with SB-K88 revealed dense colonization on the root surfaces and a characteristic perpendicular pattern of Lysobacter colonization possibly generated via development of polar, brush-like fimbriae. In colonized regions a semitransparent film apparently enveloping the root and microcolonies were observed on the root surface. This Lysobacter strain also efficiently colonized the roots of several plants, including spinach, tomato, Arabidopsis thaliana, and Amaranthus gangeticus. Plants grown from both sugar beet and spinach seeds that were previously treated with Lysobacter sp. strain SB-K88 displayed significant resistance to the damping-off disease triggered by A. cochlioides. Interestingly, zoospores of A. cochlioides became immotile within 1 min after exposure to a SB-K88 cell suspension, a cell-free supernatant of SB-K88, or pure xanthobaccin A (MIC, 0.01 microg/ml). In all cases, lysis followed within 30 min in the presence of the inhibiting factor(s). Our data indicate that Lysobacter sp. strain SB-K88 has a direct inhibitory effect on A. cochlioides, suppressing damping-off disease. Furthermore, this inhibitory effect of Lysobacter sp. strain SB-K88 is likely due to a combination of antibiosis and characteristic biofilm formation at the rhizoplane of the host plant.     

Mode of antagonism of a biocontrol bacterium Lysobacter sp. SB-K88 toward a damping-off pathogen Aphanomyces cochlioides

The biocontrol bacterium Lysobacter sp. SB-K88 suppresses damping-off disease in sugar beet and spinach caused by Aphanomyces cochlioides and Pythium sp. through characteristic plant colonization and antibiosis against the pathogens. This study aimed to unravel further details on mode of antagonism of SB-K88 against a damping-off pathogen A. cochlioides AC-5. The SB-K88 substantially inhibited growth and decomposed AC-5 mycelia and suppressed the release of zoospores from the hyphae. The excised root tips of sugar beet seedlings from seeds previously inoculated with SB-K88 were less attractive to AC-5 zoospores. Although aerial growth was not affected, however, root hairs of SB-K88 inoculated sugar beet seedlings were remarkably shorter and thicker than those of uninoculated control. When exposed to zoospores, the SB-K88 inhibited motility of zoospores and/or caused lysis, and then aggregated around the dead cystospores or lysed residues within 3–6 h likely to be micro-predatory behavior to a eukaryotic organism. Confocal laser scanning microscopic analysis revealed that number of lipid bodies and activities of mitochondria were markedly increased in the affected hyphae compared with control hyphae as visualized by established vital stains. Taken together, these results suggest that Lysobacter sp. SB-K88 suppresses damping-off diseases through exerting multifaceted antagonistic effects against the peronosporomycetes.     

Possible Role of Xanthobaccins Produced by Stenotrophomonas sp. Strain SB-K88 in Suppression of Sugar Beet Damping-Off Disease

Three antifungal compounds, designated xanthobaccins A, B, and C, were isolated from the culture fluid of Stenotrophomonas sp. strain SB-K88, a rhizobacterium of sugar beet that suppresses damping-off disease. Production of xanthobaccin A in culture media was compared with the disease suppression activities of strain SB-K88 and less suppressive strains that were obtained by subculturing. Strain SB-K88 was applied to sugar beet seeds, and production of xanthobaccin A in the rhizosphere of seedlings was confirmed by using a test tube culture system under hydroponic culture conditions; 3 μg of xanthobaccin A was detected in the rhizosphere on a per-plant basis. Direct application of purified xanthobaccin A to seeds suppressed damping-off disease in soil naturally infested by Pythium spp. We suggest that xanthobaccin A produced by strain SB-K88 plays a key role in suppression of sugar beet damping-off disease.     

Possible role of xanthobaccins produced by Stenotrophomonas sp. strain SB-K88 in suppression of sugar beet damping-off disease.

Three antifungal compounds, designated xanthobaccins A, B, and C, were isolated from the culture fluid of Stenotrophomonas sp. strain SB-K88, a rhizobacterium of sugar beet that suppresses damping-off disease. Production of xanthobaccin A in culture media was compared with the disease suppression activities of strain SB-K88 and less suppressive strains that were obtained by subculturing. Strain SB-K88 was applied to sugar beet seeds, and production of xanthobaccin A in the rhizosphere of seedlings was confirmed by using a test tube culture system under hydroponic culture conditions; 3 microg of xanthobaccin A was detected in the rhizosphere on a per-plant basis. Direct application of purified xanthobaccin A to seeds suppressed damping-off disease in soil naturally infested by Pythium spp. We suggest that xanthobaccin A produced by strain SB-K88 plays a key role in suppression of sugar beet damping-off disease.     

External and Internal Root Colonization of Maize by Two Pseudomonas Strains: Enumeration by Enzyme-Linked Immunosorbent Assay (ELISA)

Maize root colonization by two fluorescent Pseudomonas strains M.3.1. and TR335, isolated respectively from maize and tomato roots, were studied in hydroponic conditions. Each bacterium was inoculated separately, and three different colonization areas were studied: nutrient solution, rhizoplane, and endorhizosphere. The two Pseudomonas strains established large rhizosphere populations, and rhizoplane colonization of the entire root system was similar for both strains. However, strain M.3.1. colonized the endorhizosphere more efficiently than strain TR335. Seminal root cuttings from the tip to the seed allowed the assessment of colonization of three different root areas (i.e., root cap and elongation area, root-hair zone, and mature zone). Rhizoplane colonizations of all these three areas by M.3.1. were significantly the same, whereas strain TR335 colonized the rhizoplane of the root cap and elongation area more actively than the root-hair zone and mature zone. Population size of the strain M.3.1. in the internal tissue of these areas was greater than that of strain TR335. Co-inoculations of the two strains indicated a stimulation of the population size of strain M.3.1. regardless of root area studied, whereas population size of strain TR335 remained unchanged. These results demonstrated that external and internal maize root tissues were colonized to a greater extent by a strain isolated from a maize rhizosphere than by one isolated from another rhizosphere. Received: 26 September 1996 / Accepted: 1 November 1996     

Localization of the insect pathogenic fungal plant symbionts Metarhizium robertsii and Metarhizium brunneum in bean and corn roots

Metarhizium is an insect pathogenic fungus and a plant root symbiont. Here the root association patterns (rhizoplane or endophytic colonization) were analyzed in common beans (Phaseolus vulgaris) and sweet corn (Zea mays) using M. robertsii and M. brunneum under various vermiculite treatments (control, with sucrose, with an insect) at two time points of plant growth (10 and 20 days). We observed that M. brunneum and M. robertsii preferentially endophytically colonized the hypocotyl, however, greater rhizoplane colonization was observed at the regions proximal to the hypocotyl in both plants. Vermiculite amended with an infected insect resulted in greater endophytic and rhizoplane colonization at 20 days compared to 10 days, for both plants as well as for both Metarhizium species. Regardless of the vermiculite treatment, corn was preferentially colonized compared to bean. Sucrose amendment in the vermiculite and infected insect amended vermiculite only showed differences in rhizoplane colonization. The greatest root association occurred with M. brunneum with an infected insect and that in corn after 20 days.     

Control of Pythium damping-off of sugar beet by seed treatment with crop straw powders and a biocontrol agent

Seed treatment with non-sterilized powdered straws from 39 crops was tested for the control of Pythium damping-off of sugar beet. Four straws, including flax, coriander, pea, and lentil were effective in controlling the disease in soil artificially infested with Pythium sp. “group G.” Sterilizing flax and pea straws eliminated the efficacy of these straws. Wheat straw powder coated on sugar beet seeds increased the incidence of Pythium damping-off but this effect was reversed by the co-inoculation of wheat straws with the biocontrol agent Pseudomonas fluorescens 708. Coating sugar beet seeds with P. fluorescens 708 and flax or pea straws also increased the efficiency of the bacterial strain for the control of Pythium damping-off. Pea straws and to a lesser extent lentil straws produced volatile substances that affected mycelial growth of Pythium sp. “group G” on potato dextrose agar in Petri plates when the straws were mixed with water and left to ferment for two days. Fermentation of pea straws led to the accumulation of volatile ammonia, which was produced by the reduction of the large amount of nitrate stored in the straw. Reduction of nitrate and therefore the release of volatile ammonia did not occur in sterilized pea straws. However, fermenting sterile pea straws with bacteria from different genera restored nitrate reduction and the release of volatile ammonia, suggesting that microorganisms associated with pea straws are responsible for the conversion of nitrate into volatile ammonia which in turn control Pythium damping-off disease in sugar beet.     

Genes Involved in Cyclic Lipopeptide Production Are Important for Seed and Straw Colonization by Pseudomonas sp. Strain DSS73

Survival in natural bulk soil and colonization of sugar beet seeds and barley straw residues were determined for Pseudomonas sp. strain DSS73 and Tn5 mutants in amsY (encoding a peptide synthetase involved in production of the cyclic lipopeptide amphisin) and gacS (encoding the sensory kinase of the two-component GacA/GacS regulatory system). No differences in survival or growth in response to carbon amendment (citrate) were observed in bulk soil. However, both mutants were impaired in their colonization of sugar beet seeds and barley straw residues by an inoculum established in the bulk soil. The two mutants had comparable colonization phenotypes, suggesting that amphisin production is more important for colonization than other gacS-controlled traits.     

Pathogenicity of animal and plant parasitic Aphanomyces spp and their economic impact on aquaculture and agriculture

Parasitic Aphanomyces species are a global threat to agri- and aquaculture, causing multimillion USD damage every year. Via the global trade, Aphanomyces has spread across all continents with exception of South America and Antarctica, and has become a major problem in pea, sugar beet, fish and crayfish production. The widespread A. euteiches and A. cochlioides induce root rot diseases in leguminous species and sugar beet respectively. The fish pathogen A. invadans is the causative agent of Epizootic Ulcerative Syndrome in various fish species whilst A. astaci infects crayfishes causing crayfish plague. Aphanomyces have developed an efficient transmission and infection mechanism which allows a rapid colonization and disruption of the host's infected tissues. This review presents an overview on the current research on Aphanomyces genus. We summarise the latest research efforts on four pathogenic Aphanomyces species, shedding light on the biology of these microorganisms, the pathogenicity factors of these parasites, the diseases which they cause, their distribution and finally the strategies to control the diseases.     

Suppression of Rhizoctonia solani diseases of sugar beet by antagonistic and plant growth-promoting yeasts

AIMS: Isolates of Candida valida, Rhodotorula glutinis and Trichosporon asahii from the rhizosphere of sugar beet in Egypt were examined for their ability to colonize roots, to promote plant growth and to protect sugar beet from Rhizoctonia solani AG-2-2 diseases, under glasshouse conditions. METHODS AND RESULTS: Root colonization abilities of the three yeast species were tested using the root colonization plate assay and the sand-tube method. In the root colonization plate assay, C. valida and T. asahii colonized 95% of roots after 6 days, whilst Rhod. glutinis colonized 90% of roots after 8 days. Root-colonization abilities of the three yeast species tested by the sand-tube method showed that roots and soils attached to roots of sugar beet seedlings were colonized to different degrees. Population densities showed that the three yeast species were found at all depths of the rhizosphere soil adhering to taproots up to 10 cm, but population densities were significantly (P < 0.05) greater in the first 4 cm of the root system compared with other root depths. The three yeast species, applied individually or in combination, significantly (P < 0.05) promoted plant growth and reduced damping off, crown and root rots of sugar beet in glasshouse trials. The combination of the three yeasts (which were not inhibitory to each other) resulted in significantly (P < 0.05) better biocontrol of diseases and plant growth promotion than plants exposed to individual species. CONCLUSIONS: Isolates of C. valida, Rhod. glutinis and T. asahii were capable of colonizing sugar beet roots, promoting growth of sugar beet and protecting the seedlings and mature plants from R. solani diseases. This is the first successful attempt to use yeasts as biocontrol agents against R. solani which causes root diseases. SIGNIFICANCE AND IMPACT OF THE STUDY: Yeasts were shown to provide significant protection to sugar beet roots against R. solani, a serious soil-borne root pathogen. Yeasts also have the potential to be used as biological fertilizers.     

Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain.

Tn5 mutagenesis and complementation analysis were used to clone a 6-kb genomic fragment required for biosynthesis of 2,4-diacetylphloroglucinol (Phl) from fluorescent Pseudomonas sp. strain F113. A recombinant plasmid, pCU203, containing this region partially complemented a Phl production-negative mutant (F113G22) derived from strain F113. When sugar beet seeds were sown into an unsterilized soil, in which sugar beet was subject to damping-off by Pythium ultimum, the emergence of sugar beet seeds inoculated with strain F113 was significantly greater than that of seeds inoculated with F113G22. Transfer of pCU203 into eight other Pseudomonas strains conferred the ability to synthesize Phl in only one of these strains, Pseudomonas sp. strain M114. Strain M114(pCU203) showed enhanced antagonism towards P. ultimum in vitro and significantly increased the emergence of sugar beet seeds in the same soil compared with emergence induced by the parent strain M114.     

Colonization study of gfp-tagged Achromobacter marplatensis strain in sugar beet

This study details the introduction of a gfp marker into an endophytic bacterial strain (Achromobacter marplatensis strain 17, isolated from sugar beet) to monitor its colonization of sugar beet (Beta. vulgaris L.). Stability of the plasmid encoding the gfp was confirmed in vitro for at least 72 h of bacterial growth and after the colonization of tissues, under nonselective conditions. The colonization was observed using fluorescence microscopy and enumeration of culturable endophytes in inoculated sugar beet plants that grew for 10 or 20 days. gfp-Expressing strains were re-isolated from the inner tissues of surface-sterilized roots and stems of inoculated plants, and the survival of the Achromobacter marplatensis 17:gfp strain in plants 20 days after inoculation, even in the absence of selective pressure, suggests that it is good colonizer. These results also suggest that this strain could be a useful tool for the delivery of enzymes or other proteins into plants. In addition, the study highlights that sugar beet plants can be used effectively for detailed in vitro studies on the interactions between A. marplatensis strain 17 and its host, particularly if a gfp-tagged strain of the pathogen is used.     

Root Colonization by Pseudomonas sp. DSMZ 13134 and Impact on the Indigenous Rhizosphere Bacterial Community of Barley

Over the last few decades, the ability of rhizosphere bacteria to promote plant growth has been considered to be of scientific, ecological, and economic interest. The properties and mechanisms of interaction of these root-colonizing bacteria have been extensively investigated, and plant protection agents that are based on these bacterial strains have been developed for agricultural applications. In the present study, the root colonization of barley by Pseudomonas sp. DSMZ 13134, that is contained in the commercially available plant protection agent Proradix®, was examined using the fluorescence in situ hybridization method with oligonucleotide probes and specific gfp-tagging of the inoculant strain in combination with confocal laser scanning microscopy. In the first phase of root colonization, the inoculant strain competed successfully with seed and soil-borne bacteria (including Pseudomonads) for the colonization of the rhizoplane. Pseudomonas sp. DSMZ 13134 could be detected in all parts of the roots, although it did not belong to the dominant members of the root-associated bacterial community. Gfp-tagged cells were localized particularly in the root hair zone, and high cell densities were apparent on the root hair surface. To investigate the impact of the application of Proradix® on the structure of the dominant root-associated bacterial community of barley, T-RFLP analyses were performed. Only a transient community effect was found until 3 weeks post-application.     

Effect of the polysaccharide ofAgrobacterium radiobacter on the growth of plants and occurrence of damping-off in sugar beet

Agrobacterium radiobacter, strain B6 (a strain isolated in this laboratory, which limited the occurrence of damping-off of sugar beet and influenced growth of plants in hot-house and field experiments) was found to produce an acidic exopolysaccharide in a mineral medium with various carbon sources. Hydrolyzates of the polysaccharide contained glucose, galactose, glycerol, succinic acid and pyruvic acid, whose quantitative content varied according to the carbon source used. The polysaccharide isolated from the medium containing glucose exhibited the highest physiological activity. Seeds germinated best and sugar beet roots were found to grow most rapidly in a medium containing 0.2 % (W/W) of the polysaccharide. The roots exposed for 3 d in this medium grew 2.7-fold as compared with non-treated plants. Higher sumbers of microorganisms were detected on the surface of roots treated with the polysaccharide. Growth of roots was also stimulated when immersing the seeds (30 min) in a 0.2 –0.4 % solution of this polysaccharide. After a two-fold treatment the roots were less damaged by the fungusPythium ultimum. Plants from seeds treated with the polysaccharide grew in the field soil more rapidly than the non-treated plants but worse than after bacterization of the seeds byA. radiobacter B6 and were only partially protected against the damping-off of sugar beet.     

Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain.

Tn5 mutagenesis and complementation analysis were used to clone a 6-kb genomic fragment required for biosynthesis of 2,4-diacetylphloroglucinol (Phl) from fluorescent Pseudomonas sp. strain F113. A recombinant plasmid, pCU203, containing this region partially complemented a Phl production-negative mutant (F113G22) derived from strain F113. When sugar beet seeds were sown into an unsterilized soil, in which sugar beet was subject to damping-off by Pythium ultimum, the emergence of sugar beet seeds inoculated with strain F113 was significantly greater than that of seeds inoculated with F113G22. Transfer of pCU203 into eight other Pseudomonas strains conferred the ability to synthesize Phl in only one of these strains, Pseudomonas sp. strain M114. Strain M114(pCU203) showed enhanced antagonism towards P. ultimum in vitro and significantly increased the emergence of sugar beet seeds in the same soil compared with emergence induced by the parent strain M114.     

Spatial Peculiarities in the Colonization of the Plant Rhizoplane by Microscopic Fungi

Spatial peculiarities in the colonization of the tomato, cucumber, and barley rhizoplanes by microscopic fungi were studied. The apical zone of roots was colonized with a limited number of Rstrategists (the order Mucorales, Fusariumsp., Aspergillus niger, and Mycelia sterilia). The fungal population of the root hairs and the basal zone of roots was 2- to 3-fold denser due to the prevalence of Kstrategists. Fusaria, Fusarium oxysporumin particular, colonized roots in earlier terms than the genera Trichoderma, Penicillium, Gliocladium, and others. The F. oxysporumpopulation was at a maximum in the rhizoplane zone nearest the root tip.     

Growth inhibition and excessive branching in <Emphasis Type="Italic">Aphanomyces cochlioides</Emphasis> induced by 2,4-diacetylphloroglucinol is linked to disruption of filamentous actin cytoskeleton in the hyphae

We observed that 2,4-diacetylphloroglucinol (DAPG), a major antimicrobial metabolite produced by a rhizoplane bacterium Pseudomonas fluorescens ECO-001 inhibited mycelial growth of a damping-off phytopathogen Aphanomyces cochlioides AC-5 through inducing excessive branching and curling in the hyphae. This study aimed to unravel the mode of action of DAPG caused excessive branching, curling and growth inhibition of AC-5 hyphae by detecting localized changes in the cortical filamentous actin (F-actin) organization by rhodamine-conjugated phalloidin. Confocal laser scanning microscopic observations revealed that both living bacteria and DAPG severely disrupted the organization of F-actin in the A. cochlioides hyphae in a similar manner. Furthermore, an inhibitor of F-actin polymerization, latrunculin B also induced similar growth inhibition, excessive branching and caused disruption of F-actin in the AC-5 hyphae. Our results suggested that growth inhibition and excessive branching induced in A. cochlioides by DAPG is likely to be linked to the disruption of F-actin cytoskeleton in the affected hyphae. This is the first report on disruption of cytoskeleton of a eukaryotic A. cochlioides by a well-known biocontrol metabolite DAPG secreted from a prokaryotic bacterium ECO-001.     

Suppression of sugar beet damping-off caused by Rhizoctonia solani using bacterial and fungal antagonists

Rhizoctonia damping-off caused by Rhizoctonia solani Kühn, is one of the most damaging sugar beet diseases. It causes serious economic damage wherever sugar beets are grown. Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Suppression of damping-off disease caused by R. solani was carried out by four isolates of Bacillus subtilis (Ehrenberg) Cohn as well as three isolates of each of Trichoderma harzianum Rifai and Trichoderma hamatum (Bonord.) Bainier. The effect of Bacillus and Trichoderma isolates against R. solani was investigated in vitro and tested on sugar beet plants under greenhouse conditions. Isolates of Bacillus and Trichoderma were able to inhibit the growth of R. solani in dual culture. Furthermore, Trichoderma isolates gave high antagonistic effect than isolates of B. subtilis. Under greenhouse conditions, coating seeds by T. harzianum and B. subtilis separately, reduced seedling damping-off significantly. However, applications of T. harzianum increased the percentage of surviving plants more than B. subtilis in comparison to control. The obtained results indicate that T. harzianum and B. subtilis are very effective biocontrol agents that offer potential benefit in sugar beet damping-off and should be harnessed for further biocontrol applications.     

Optimization of in vivo crosslinking technique for the study of AlpB-protein interactions in Lysobacter sp. XL1 cells

The bacterium Lysobacter species strain XL1 is known as a producer of extracellular lytic enzymes, which are capable of degrading cell wall components of other bacteria and simple eukaryotes. This ability determines the ecological, medical and agricultural relevance of Lysobacter sp. XL1. However, the molecular mechanism of secretion of lytic exoenzymes from Lysobacter cells is yet unknown, which in turn necessitates the search of protein–protein interactions that occur during exoenzyme secretion. The current paper is concerned with investigation of protein complexes that are likely formed during the secretion of AlpB lytic protease from the cells of Lysobacter sp. XL1. In this study, we have optimized the method of stabilization of protein complexes formed in the intact cells of Lysobacter sp. XL1 by using crosslinking reagent dithiobis(succinimidylpropionate) (DSP) and detected DSP-linked protein complexes by the monoclonal antibodies against AlpB propeptide.