BackgroundN-acylhomoserine lactone (AHL)-based quorum sensing (QS) systems have been described in many plant-associated Gram-negative bacteria to control certain beneficial phenotypic traits, such as production of biocontrol factors and plant growth promotion. However, the role of AHL-mediated signalling in the endophytic strains of plant-associated Serratia is still poorly understood. An endophytic Serratia sp. G3 with biocontrol potential and high levels of AHL signal production was isolated from the stems of wheat and the role of QS in this isolate was determined.ResultsStrain G3 classified as Serratia plymuthica based on 16S rRNA was subjected to phylogenetic analysis. Using primers to conserved sequences of luxIR homologues from the Serratia genus, splIR and spsIR from the chromosome of strain G3 were cloned and sequenced. AHL profiles from strain G3 and Escherichia coli DH5α expressing splI or spsI from recombinant plasmids were identified by liquid chromatography-tandem mass spectrometry. This revealed that the most abundant AHL signals produced by SplI in E. coli were N-3-oxo-hexanoylhomoserine lactone (3-oxo-C6-HSL), N-3-oxo-heptanoylhomoserine lactone (3-oxo-C7-HSL), N-3-hydroxy-hexanoylhomoserine lactone (3-hydroxy-C6-HSL), N-hexanoylhomoserine lactone (C6-HSL), and N-heptanoyl homoserine lactone (C7-HSL); whereas SpsI was primarily responsible for the synthesis of N-butyrylhomoserine lactone (C4-HSL) and N-pentanoylhomoserine lactone (C5-HSL). Furthermore, a quorum quenching analysis by heterologous expression of the Bacillus A24 AiiA lactonase in strain G3 enabled the identification of the AHL-regulated biocontrol-related traits. Depletion of AHLs with this lactonase resulted in altered adhesion and biofilm formation using a microtiter plate assay and flow cells coupled with confocal laser scanning microscopy respectively. This was different from the closely related S. plymuthica strains HRO-C48 and RVH1, where biofilm formation for both strains is AHL-independent. In addition, QS in G3 positively regulated antifungal activity, production of exoenzymes, but negatively regulated production of indol-3-acetic acid (IAA), which is in agreement with previous reports in strain HRO-C48. However, in contrast to HRO-C48, swimming motility was not controlled by AHL-mediated QS.ConclusionsThis is the first report of the characterisation of two AHL-based quorum sensing systems in the same isolate of the genus Serratia. Our results show that the QS network is involved in the global regulation of biocontrol-related traits in the endophytic strain G3. However, although free-living and endophytic S. plymuthica share some conservation on QS phenotypic regulation, the control of motility and biofilm formation seems to be strain-specific and possible linked to the life-style of this organism.

Quorum sensing regulation, mediated by N-acyl homoserine lactone signals, produced by strain Serratia plymuthica HRO-C48 isolated from the rhizosphere of oilseed rape, was found to be responsible for this strain's ability to produce the broad spectrum antibiotic pyrrolnitrin. In this study, we have shown that some other biocontrol-related traits of strain HRO-C48, such as protection of cucumbers against Pythium apahnidermatum damping-off disease, induced systemic resistance to Botrytis cinerea grey mold in bean and tomato plants, and that colonisation of the rhizosphere also depends on AHL signalling. The results prove that quorum sensing regulation may be generally involved in interactions between plant-associated bacteria, fungal pathogens and host plants.

Biological control is being considered as an alternative or a supplemental way of reducing the use of chemicals in agriculture. An endophytic strain G3 with potential as a biocontrol agent was isolated from the stems of Triticum aestivum L. It was classified by 16S rDNA sequencing as a member of Serratia. Strain G3 displayed a broad spectrum of antifungal activity in vitro against a number of phytopathogens such as Botrytis cinerea, Cryphonectria parasitica, Rhizoctonia cerealis and Valsa sordida. Molecular mechanisms involved in biocontrol by Serratia sp. G3 was investigated for its potential application to plant health management. The results showed that G3 produces an array of antimicrobial exoproducts, including chitinase, protease, antibiotic pyrrolnitrin, and siderophores for iron competition. Moreover, it also produced the plant growth hormone indole-3-acetic acid, suggesting that multiple mechanisms and their synergistic effects may be involved in biocontrol of plant diseases. Additionally, strain G3 can produce at least ten N-acyl homoserine lactones (AHLs) signal molecules for cell to cell communication, including unsubstituted, 3-oxo, and 3-hydroxy at the C3 position through liquid chromatography-tandem mass spectrometry (LC-MS/MS), which is different from the previously reported Serraia species. For the first time, N-3-oxo-heptanoyl-homoserine lactone, one of the main molecules was reported in the genus Serratia. The role of AHL-dependent quorum sensing system in the interactions between the endophytic strain G3 and host plants and its potential application in improving biocontrol efficacy will be further explored.

Antagonism of three endophytic fungi isolated from common reed (Phragmites australis) against eight soilborne pathogenic fungi was investigated on potato dextrose agar by light microscopy, scanning electron microscopy, and transmission electron microscopy. Inhibitory zones were not observed. The microscopical studies suggested that the endophytes inhibit growth of soilborne pathogens by means of coiling around hyphae and, after penetration, the degradation of hyphal cytoplasm. Since penetration of hyphae seems to play a major role in parasitism, we studied the production of cell wall degrading enzymes by the three endophytes. Choiromyces aboriginum produced higher activities of b-1,3-glucanases compared to Stachybotrys elegans and Cylindrocarpon sp. For C. aboriginum and S. elegans, colloidal chitin was the best substrate for the induction of b-1,3-glucanases and chitinases, respectively. This result suggests that mycoparasitism by endophytes on soilborne plant pathogens can be explained by their mycoparasitic activity.

Trichoderma harzianum T88 and T. atroviride T95 were tested for their efficacy in controlling apple ring rot (caused by Botryosphaeria berengeriana f. sp. piricola) in vitro and in the field. Isolates of T88 and T95 produced both volatile and non‐volatile antibiotics that suppressed mycelial growth of the pathogen. Light and scanning electron microscopy showed that mycoparasitism by Trichoderma spp. of B. berengeriana f. sp. piricola resulted in penetration and disruption of hyphal cells, and thinning of cytoplasm of the pathogen. The culture filtrates of T88 and T95 in Czapek's liquid medium suppressed conidial germination of the pathogen, and the germination level was negatively correlated with the duration of culture of Trichoderma. In inoculation tests, 32 days after simultaneous inoculation or preinoculation with B. berengeriana f. sp. piricola and Trichoderma spp., the incidence of infected apple shoots was reduced, respectively, by 65.3–76.4% and 62.5–76.4%, and the index of infection reduced by 36.9–38.9 and 40.7–44.4, The effect of inoculating B. berengeriana f. sp. piricola 3 days after the antagonists were inoculated was greater (81.4–88.8%) than simultaneous inoculation (72.2–77.8%). Re‐isolation from inoculated apple shoots demonstrated that the pathogen had been suppressed by Trichoderma. The ability to re‐isolate the pathogen from apple after co‐inoculation and pre‐inoculation with Trichoderma spp. was reduced by 27.0–42.3% and 22.2–47.1%, respectively. The biocontrol field trial suggested that the B. berengeriana f. sp. piricola canker on apple shoots and stems and rotting fruit had been efficiently controlled by the application of spore suspensions of T. harzianum T88 and T. atroviride T95. The efficacy of control by Trichoderma is thus similar to that of routine chemical control.