Emilio Stefani

Molecular characterisation of an endophyte showing a strong antagonistic activity against Pseudomonas syringae pv. actinidiae

Background and aimsEndophytic bacteria have been often studied as biological control agents of plant pathogens and many of their secondary metabolites involved in antagonism are Non Ribosomal Peptides (NRPs). In this study, the molecular basis of the biocontrol properties of the endophyte Pseudomonas synxantha, isolated from Actinidia chinensis, against Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial kiwi canker, has been investigated.MethodsAntagonism-deficient mutants of P. synxantha strain DLS65 were generated by insertion of minitransposon mTn5-GNm in its genome. Southern blot analysis allowed the selection of single transposon insertion-mutants. Amplification of the transposon flanking regions by means of arbitrary and single primer PCR in selected mutants was perfomed to obtain amplicons for sequencing purposes.ResultsSequencing results of the amplicons obtained from three antagonism-deficient mutants led to the localization of the transposonin three genes, which implies their involvement in the antagonism of P. synxantha: an acyl-homoserine lactone acylase gene (pvdQ), a glucose-6-phosphate dehydrogenase gene (zwf) and an mbtH-like gene were identified. It is known from the literature that these three genes are involved directly or indirectly in NRPs synthesis.ConclusionsWe suggest that a molecule with antibiotic properties, produced by NRP synthetases, contributes to the antagonistic activity of P. synxantha.

Plant Growth Promoting and Biocontrol Activity of Streptomyces spp. as Endophytes

There has been many recent studies on the use of microbial antagonists to control diseases incited by soilborne and airborne plant pathogenic bacteria and fungi, in an attempt to replace existing methods of chemical control and avoid extensive use of fungicides, which often lead to resistance in plant pathogens. In agriculture, plant growth-promoting and biocontrol microorganisms have emerged as safe alternatives to chemical pesticides. Streptomyces spp. and their metabolites may have great potential as excellent agents for controlling various fungal and bacterial phytopathogens. Streptomycetes belong to the rhizosoil microbial communities and are efficient colonizers of plant tissues, from roots to the aerial parts. They are active producers of antibiotics and volatile organic compounds, both in soil and in planta, and this feature is helpful for identifying active antagonists of plant pathogens and can be used in several cropping systems as biocontrol agents. Additionally, their ability to promote plant growth has been demonstrated in a number of crops, thus inspiring the wide application of streptomycetes as biofertilizers to increase plant productivity. The present review highlights Streptomyces spp.-mediated functional traits, such as enhancement of plant growth and biocontrol of phytopathogens.

Isolation of bacterial endophytes from Actinidia chinensis and preliminary studies on their possible use as antagonists against Pseudomonas syringae pv. actinidiae

BACKGROUND: The bacterial canker, caused by Pseudomonas syringae pv. actinidiae, is the most severe disease of cultivated Actinidia spp. The pathogen is systemic and not easily controlled by agrochemical means. OBJECTIVE: Our aim was to search, select and identify, among kiwifruit bacterial endophytes, possible antagonists able to control Pseudomonas syringae pv. actinidiae. METHODS: Several kiwifruit production areas were inspected, in order to find host plants without any disease symptom inside severely affected orchards. From those plants, endophytes were isolated, selected, tested for their ability to inhibit the growth of the pathogen and identified. RESULTS: A set of 65 different bacterial endophytes was isolated and tested: several of them were able to inhibit the growth of Pseudomonas syringae pv. actinidiae in vitro. None of the antagonists proved to possess either ice nucleation activity or transferable copper resistance. Taxonomically, antagonists belonged to the families of Pseudomonadaceae and Enterobacteriaceae. CONCLUSIONS: Effective bacterial antagonists were found as endophytes in kiwifruit plants and bearing features of safety of use and negligible risk for the crop. Such finding makes it possible to select the most prospective of them, in order to develop efficient biopesticides able control the bacterial canker in commercial orchards.

Identification and Classification of Fluorescent Pseudomonas Species from Cereals in Italy

Fatiy acid profiles were prepared from 122 bacterial strains isolated from cereals grown in different Italian areas. Almost all of them were typical fluorescent pseudomonads with the three key hydroxy fatty acids 10:0 3OH, 12:0 2OH and 12:0 3OH, and belonged to two major groups. In group 1 most of the isolates were iisted as Pseudomonas syringae pathovars including pv. atrofaciens. These results fit with those obtained using SDS-PAGE. Strains in group 2 were identified as members of the P. fluorescens complex, and some were pathogenic to durum wheat seedlings. Experimentai evidence suggests that, apart from P. atrofaciens, a second pathogen may be associated with basal glume rot/leaf blight of cereais and play a roie in the disease epidemiology.

Performance of diagnostic tests for the detection and identification of Pseudomonas syringae pv. actinidiae (Psa) from woody samples

The aim of this study was to characterise the performance of new molecular methods for the detection and identification of Pseudomonas syringae pv. actinidiae (Psa) and to provide validation data in comparison to the assays mentioned in official diagnostic protocols and being currently used. Eleven molecular tests for the Psa detection were compared in an inter-laboratory comparison where each laboratory had to analyse the same panel of samples consisting of thirteen Psa-spiked kiwifruit wood extracts. Laboratories had to perform also isolation from the wood extracts. Data from this interlaboratory test performance study (TPS) was statistically analysed to assess the performance of each method. In order to provide complete validation data, both for detection and identification, this TPS was supplemented by a further study of identification from pure culture of phylogenetically closely related Pseudomonas spp., Psa, and bacterial strains associated with kiwifruit. The results of both these studies showed that simplex-PCRs gave good results, whereas duplex-PCR and real-time PCR were the most reliable tools for detection and identification of Psa. Nested and multiplex-PCR gave false-positive results. The use of the most reliable detection test is suggested for routine analyses, but when Psa-free status needs to be accurately assessed, it is recommended that at least two detection tests are used. This work provides a wide comparison of the available diagnostic methods, giving new information for a possible revision of the official diagnostic protocols (e.g. European and Mediterranean Plant Protection Organization (EPPO) protocol PM7/120 for the detection of Psa).

Pseudomonas syringae pv. aptata Hrp Mutants which Fail to Produce Periplasmic Oligosaccharides

Periplasmic oligosaccharides, extracted from Pseudomonas syringae pv. aptata and purified by column chromatography, modify the homologous interaction between tobacco leaves and P. syringae pv. tabaci. The osmoregulating oligosaccharides can act as signal molecules to plant cells, but the molecular basis of this function is not known. Genetical analysis of pathogenicity determinants in P. s. pv. aptata strain NCPPB2664 allowed the identification of hrp genes which are essential in governing the ability of the bacteria to cause disease on host plants (such as sugar beet, lettuce), and the ability to induce the hypersensitive response on non-host plants (such as tobacco). The current study addresses whether a correlation between the pathogenicity and the production of oligosaccharides exists. To test the hypothesis that in the early stages of plant-bacteria interactions the hrp genes might be involved in the production of the periplasmic oligosaccharides, the latter has been studied in P. syringae pv. aptata Hrp- mutants.