Nematicidal Activity of a Nonpathogenic Biocontrol Bacterium, Pseudomonas chlororaphis O6

Bacterial culture filtrates of an aggressive rhizobacterium, Pseudomonas chlororaphis O6, displayed strong nematicidal activity. The nematicidal activity of P. chlororaphis O6 was markedly reduced in the gacS mutant of P. chlororaphis O6 grown in the presence of glycine, but no reduction of nematicidal activity in the gacS mutant was noted in the absence of glycine. The results of bioassay with P. chlororaphis O6 mutants showed that phenazine and pyrrolnitrin production was not a major factor, but the effects of glycine in the culture medium suggest that formation of hydrogen cyanide might be important. Assessments in greenhouse studies with tomatoes growing in nematode-infested soils confirmed that the application of P. chlororaphis O6 resulted in the control of the root-knot nematode. Our results demonstrated that P. chlororaphis O6 could be employed as a biocontrol agent for the control of the root-knot nematode, and the global regulator, GacS, functions as a positive regulator of the expression of nematicidal compounds and enzymes in P. chlororaphis O6.     

Detection of antibiotic-related genes from bacterial biocontrol agents with polymerase chain reaction

Pseudomonas chlororaphis PA23, Pseudomonas spp. strain DF41, and Bacillus amyloliquefaciens BS6 consistently inhibit infection of canola petals by Sclerotinia sclerotiorum in both greenhouse and field experiments. Bacillus thuringiensis BS8, Bacillus cereus L, and Bacillus mycoides S have shown significant inhibition against S. sclerotiorum on plate assays. The presence of antibiotic biosynthetic or self-resistance genes in these strains was investigated with polymerase chain reaction and, in one case, Southern blotting. Thirty primers were used to amplify (i) antibiotic biosythetic genes encoding phenazine-1-carboxylic acid, 2,4-diacetylphloroglucinol, pyoluteorin, and pyrrolnitrin, and (ii) the zwittermicin A self-resistance gene. Our findings revealed that the fungal antagonist P. chlororaphis PA23 contains biosynthetic genes for phenazine-1-carboxylic acid and pyrrolnitrin. Moreover, production of these compounds was confirmed by high performance liquid chromatography. Pseudomonas spp. DF41 and B. amyloliquefaciens BS6 do not appear to harbour genes for any of the antibiotics tested. Bacillus thuringiensis BS8, B. cereus L, and B. mycoides S contain the zwittermicin A self-resistance gene. This is the first report of zmaR in B. mycoides.     

Inactivation of gacS does not affect the competitiveness of Pseudomonas chlororaphis in the Arabidopsis thaliana rhizosphere

Quorum-sensing-controlled processes are considered to be important for the competitiveness of microorganisms in the rhizosphere. They affect cell-cell communication, biofilm formation, and antibiotic production, and the GacS-GacA two-component system plays a role as a key regulator. In spite of the importance of this system for the regulation of various processes, strains with a Gac(-) phenotype are readily recovered from natural habitats. To analyze the influence of quorum sensing and the influence of the production of the antibiotic phenazine-1-carboxamide on rhizosphere colonization by Pseudomonas chlororaphis, a gnotobiotic system based on Arabidopsis thaliana seedlings in soil was investigated. Transposon insertion mutants of P. chlororaphis isolate SPR044 carrying insertions in different genes required for the production of N-acyl-homoserine lactones and phenazine-1-carboxamide were generated. Analysis of solitary rhizosphere colonization revealed that after prolonged growth, the population of the wild type was significantly larger than that of the homoserine lactone-negative gacS mutant and that of a phenazine-1-carboxamide-overproducing strain. In cocultivation experiments, however, the population size of the gacS mutant was similar to that of the wild type after extended growth in the rhizosphere. A detailed analysis of growth kinetics was performed to explain this phenomenon. After cells grown to the stationary phase were transferred to fresh medium, the gacS mutant had a reduced lag phase, and production of the stationary-phase-specific sigma factor RpoS was strongly reduced. This may provide a relative competitive advantage in cocultures with other bacteria, because it permits faster reinitiation of growth after a change to nutrient-rich conditions. In addition, delayed entry into the stationary phase may allow more efficient nutrient utilization. Thus, GacS-GacA-regulated processes are not absolutely required for efficient rhizosphere colonization in populations containing the wild type and Gac(-) mutants.     

suhB基因对绿针假单胞菌HT66生防能力的影响

[背景]绿针假单胞菌(Pseudomonaschlororaphis)HT66是一株兼具生防安全性和吩嗪-1-甲酰胺(Phenazine-1-Carboxamide,PCN)高产的植物根际促生菌,在生物防治、生态农业及可持续发展农业领域具有广阔的应用前景.非编码RNA(ncRNA)SuhB参与了细胞中多个过程的代谢调控...     

The role of antibiosis and induced systemic resistance, mediated by strains of Pseudomonas chlororaphis, Bacillus cereus and B. amyloliquefaciens, in controlling blackleg disease of canola

Antibiotic-producing Pseudomonas chlororaphis strains DF190 and PA23, Bacillus cereus strain DFE4 and Bacillus amyloliquefaciens strain DFE16 were tested for elicitation of induced systemic resistance (ISR) and direct antibiosis in control of blackleg in canola caused by the fungal pathogen Leptosphaeria maculans. Inoculation of bacteria 24 h and 48 h prior to the pathogen was crucial for disease control. In systemic induction studies, the bacteria and culture extracts had lower but significant suppression of the blackleg lesion. When inoculated at the same wound site as the pathogen pycnidiospores, the bacterial culture extracts had significantly higher reduction of blackleg lesion development. However, localized plant defense-related enzyme activity at the site of inoculation was not induced by all the bacteria. Direct antifungal activity at the infection site seems to be the dominant mechanism mediating control of L. maculans. A Tn5-gacS mutant of strain PA23 exhibited a complete loss of antifungal and biocontrol activity, which was restored upon addition of the gacS gene in trans. Interestingly, a phenazine-minus derivative of PA23 that produces elevated levels of pyrrolnitrin exhibited the same or higher blackleg disease suppression compared to the wild type. These findings suggest that direct antifungal activity, possibly mediated by pyrrolnitrin, and some low level of induced systemic resistance is involved in P. chlororaphis biocontrol of blackleg.     

The GacS sensor kinase controls phenotypic reversion of small colony variants isolated from biofilms of Pseudomonas aeruginosa PA14

The GacS/GacA two-component regulatory system in pseudomonads regulates genes involved in virulence, secondary metabolism and biofilm formation. Despite these regulatory functions, some Pseudomonas species are prone to spontaneous inactivating mutations in gacA and gacS. A gacS(-) strain of Pseudomonas aeruginosa PA14 was constructed to study the physiological role of this sensor histidine kinase. This loss-of-function mutation was associated with hypermotility, reduced production of acylhomoserine lactones, impaired biofilm maturation, and decreased antimicrobial resistance. Biofilms of the gacS(-) mutant gave rise to phenotypically stable small colony variants (SCVs) with increasing frequency when exposed to silver cations, hydrogen peroxide, human serum, or certain antibiotics (tobramicin, amikacin, azetronam, ceftrioxone, oxacilin, piperacillin or rifampicin). When cultured, the SCV produced thicker biofilms with greater cell density and greater antimicrobial resistance than did the wild-type or parental gacS(-) strains. Similar to other colony morphology variants described in the literature, this SCV was less motile than the wild-type strain and autoaggregated in broth culture. Complementation with gacS in trans restored the ability of the SCV to revert to a normal colony morphotype. These findings indicate that mutation of gacS is associated with the occurrence of stress-resistant SCV cells in P. aeruginosa biofilms and suggests that in some instances GacS may be necessary for reversion of these variants to a wild-type state.     

Biocontrol traits of Pseudomonas spp. are regulated by phase variation

Of 214 Pseudomonas strains isolated from maize rhizosphere, 46 turned out to be antagonistic, of which 43 displayed clear colony phase variation. The latter strains formed both opaque and translucent colonies, designated as phase I and phase II, respectively. It appeared that important biocontrol traits, such as motility and the production of antifungal metabolites, proteases, lipases, chitinases, and biosurfactants, are correlated with phase I morphology and are absent in bacteria with phase II morphology. From a Tn5luxAB transposon library of Pseudomonas sp. strain PCL1171 phase I cells, two mutants exhibiting stable expression of phase II had insertions in gacS. A third mutant, which showed an increased colony phase variation frequency was mutated in mutS. Inoculation of wheat seeds with PCL1171 bacteria of phase I morphology resulted in efficient suppression of take-all disease, whereas disease suppression was absent with phase II bacteria. Neither the gacS nor the mutS mutant was able to suppress take-all, but biocontrol activity was restored after genetic complementation of these mutants. Furthermore, in a number of cases, complementation by gacS of wild-type phase II sectors to phase I phenotype could be shown. A PCL1171 phase I mutant defective in antagonistic activity appeared to have a mutation in a gene encoding a lipopeptide synthetase homologue and had lost its biocontrol activity, suggesting that biocontrol by strain PCL1171 is dependent on the production of a lipopeptide. Our results show that colony phase variation plays a regulatory role in biocontrol by Pseudomonas bacteria by influencing the expression of major biocontrol traits and that the gacS and mutS genes play a role in the colony phase variation process. Therefore phase variation not only plays a role in escaping animal defense but it also appears to play a much broader and vital role in the ecology of bacteria producing exoenzymes, antibiotics, and other secondary metabolites.     

Biocontrol of avocado dematophora root rot by antagonistic Pseudomonas fluorescens PCL1606 correlates with the production of 2-hexyl 5-propyl resorcinol

A collection of 905 bacterial isolates from the rhizospheres of healthy avocado trees was obtained and screened for antagonistic activity against Dematophora necatrix, the cause of avocado Dematophora root rot (also called white root rot). A set of eight strains was selected on the basis of growth inhibitory activity against D. necatrix and several other important soilborne phytopathogenic fungi. After typing of these strains, they were classified as belonging to Pseudomonas chlororaphis, Pseudomonas fluorescens, and Pseudomonas putida. The eight antagonistic Pseudomonas spp. were analyzed for their secretion of hydrogen cyanide, hydrolytic enzymes, and antifungal metabolites. P. chlororaphis strains produced the antibiotic phenazine-1-carboxylic acid and phenazine-1-carboxamide. Upon testing the biocontrol ability of these strains in a newly developed avocado-D. necatrix test system and in a tomato-F oxysporum test system, it became apparent that P. fluorescens PCL1606 exhibited the highest biocontrol ability. The major antifungal activity produced by strain P. fluorescens PCL1606 did not correspond to any of the major classes of antifungal antibiotics produced by Pseudomonas biocontrol strains. This compound was purified and subsequently identified as 2-hexyl 5-propyl resorcinol (HPR). To study the role of HPR in biocontrol activity, two Tn5 mutants of P. fluorescens PCL1606 impaired in antagonistic activity were selected. These mutants were shown to impair HRP production and showed a decrease in biocontrol activity. As far as we know, this is the first report of a Pseudomonas biocontrol strain that produces HPR in which the production of this compound correlates with its biocontrol activity.     

Involvement of gacS and rpoS in enhancement of the plant growth-promoting capabilities of Enterobacter cloacae CAL2 and UW4

The plant growth-promoting bacteria Enterobacter cloacae CAL2 and UW4 were genetically transformed with a multicopy plasmid containing an rpoS or gacS gene from Pseudomonas fluorescens. The transformed strains were compared with the nontransformed strains for growth, indoleacetic acid (IAA) production, antibiotic production, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, siderophore production, cell morphology, and the ability to promote canola root elongation. All transformed strains had a longer lag phase, were slower in reaching stationary phase, and attained a higher cell density than the nontransformed strains. Transformation resulted in cells that were significantly shorter than the nontransformed cells. The transformed strains also produced significantly more IAA than the nontransformed strains. Introduction of rpoS or gacS from Pseudomonas fluorescens was associated with a reduction in the production of both antibiotics, 2,4-diacetylphloroglucinol and mono-acetylphloroglucinol, produced by Enterobacter cloacae CAL2. With Enterobacter cloacae CAL2, plasmid-borne rpoS, but not gacS, increased the level of ACC deaminase activity, while introduction of rpoS in Enterobacter cloacae UW4 caused a decrease in ACC deaminase activity. Neither gacS nor rpoS significantly affected the level of siderophores synthesized by either bacterial strain. Overproduction of either GacA or RpoS in Enterobacter cloacae CAL2 resulted in a significant increase in the root lengths of canola seedlings when seeds were treated with the bacteria, and overproduction of RpoS caused an increase in canola shoot as well as root lengths.     

Effect of gacS and gacA mutations on colony architecture, surface motility, biofilm formation and chemical toxicity in Pseudomonas sp. KL28

GacS and GacA proteins form a two component signal transduction system in bacteria. Here, Tn5 transposon gacS and gacA (Gac) mutants of Pseudomonas sp. KL28, an alkylphenol degrader, were isolated by selecting for smooth colonies of strain KL28. The mutants exhibited reduced ability to migrate on a solid surface. This surface motility does not require the action of flagella unlike the well-studied swarming motility of other Pseudomonas sp. The Gac mutants also showed reduced levels of biofilm and pellicle formation in liquid culture. In addition, compared to the wild type KL28 strain, these mutants were more resistant to high concentrations of m-cresol but were more sensitive to H2O2, which are characteristics that they share with an rpoS mutant. These results indicate that the Gac regulatory cascade in strain KL28 positively controls wrinkling morphology, biofilm formation, surface translocation and H2O2 resistance, which are important traits for its capacity to survive in particular niches.     

The role of volatile and non-volatile antibiotics produced by Pseudomonas chlororaphis strain PA23 in its root colonization and control of Sclerotinia sclerotiorum

Pseudomonas chlororaphis strain PA23 has demonstrated excellent biocontrol in the canola phyllosphere. This bacterium produces the non-volatile antibiotics phenazine and pyrrolnitrin as well as the volatile antibiotics nonanal, benzothiazole and 2-ethyl-1-hexanol. In vitro experiments were conducted to study the effects of different mutations on the production of these three organic volatile antibiotics by PA23. In planta experiments in the greenhouse investigated the role of the non-volatile antibiotics on root colonization and biocontrol ability of PA23 against Sclerotinia sclerotiorum on sunflower. Analysis of phenazine- and pyrrolnitrin-deficient Tn mutants of PA23 revealed no differences in production of the three volatile antibiotics. On all sampling dates, PA23 applied alone or in combination with the mutants showed significantly higher (P = 0.05) root bacterial number and Sclerotinia wilt suppression (P = 0.05). Decline of the bacterial population seemed to be inversely proportional to/or negatively correlated with the number of antibiotics produced by PA23 but the relative importance of phenazine or pyrrolnitrin on root colonization and/or wilt suppression was not clear. In several cases, the strains producing at least one antibiotic maintained relatively higher bacterial numbers than non-producing strains. However, by 6 weeks after sowing, there was a rapid and significant (P = 0.05) increase in the proportion of introduced bacteria capable of producing at least one antibiotic over the total bacterial population. Furthermore, combining certain mutants with PA23 reduced the root colonization and biocontrol ability of PA23. Strain PA23-314 (gacS mutant) showed competitive colonization in comparison to the other mutants for most sampling dates.     

Thiamine-auxotrophic mutants of Pseudomonas fluorescens CHA0 are defective in cell-cell signaling and biocontrol factor expression

In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 x 10(-8) M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens.     

Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24

Pseudomonas fluorescens 2P24 is a biocontrol agent isolated from a wheat take-all decline soil in China. This strain produces several antifungal compounds, such as 2,4-diacetylphloroglucinol (2,4-DAPG), hydrogen cyanide and siderophore(s). Our recent work revealed that strain 2P24 employs a quorum-sensing system to regulate its biocontrol activity. In this study, we identified a quorum-sensing system consisting of PcoR and PcoI of the LuxR–LuxI family from strain 2P24. Deletion of pcoI from 2P24 abolishes the production of the quorum-sensing signals, but does not detectably affect the production of antifungal metabolites. However, the mutant is significantly defective in biofilm formation, colonization on wheat rhizosphere and biocontrol ability against wheat take-all, whilst complementation of pcoI restores the biocontrol activity to the wild-type level. Our data indicate that quorum sensing is involved in regulation of biocontrol activity in P. fluorescens 2P24.     

Biological control of Sclerotinia sclerotiorum (Lib.) de Bary, the causal agent of white mold, by Pseudomonas species on canola petals

Sclerotinia sclerotiorum is an important pathogen on canola. Due to the public concern over pesticide use, alternative methods of disease control, such as biological control, should be considered. Several bacterial strains were isolated from canola and soja plants. Inhibition of S. sclerotiorum by bacterial strains in vitro was assayed on PDA medium in dual culture test. Eight Pseudomonas sp. strains (PB-3, PB-4, PB-5, PB-6, PB-7, PB-8, PB-10 and PB-11) caused inhibition zone against 5. sclerotiorum hyphal growth. The biocontrol potential of the bacteria was tested in a plant assay. Disease suppression was investigated using a petal inoculation technique. Canola petals were pretreated with bacteria, and then inoculated with 5. sclerotiorum ascospores 24 h later. Greenhouse experiment showed that application of Pseudomonas sp. strains (1 x 10(8) cfu ml(-1)) effectively suppressed S. sclerotiorum (1 x 10(5) ascospores ml(-1)) on petals and all of them achieved significant (P<0.01) disease suppression. Fourteen days after inoculation, strain PB-3 had 88/7% disease control and strain PB-4 had 69/9% disease control. Result from all studies indicates PB-3 to be effective biocontrol against S. sclerotiorum of canola. PB-3, PB-4, PB-7, PB-8, PB-10 and PB-11 were identified as Pseudomonas fluorescens biovar III. PB-5 and PB-6 was identified as Pseudomonas fluorescens biovar II. Strains PB-3, PB-4, PB-6, PB-10 and PB-11 produced protease and HCN. Strain PB-5 produce protease; no HCN.