Nectar-Inhabiting Bacteria Affect Olfactory Responses of an Insect Parasitoid by Altering Nectar Odors.

Floral nectar is ubiquitously colonized by a variety of microorganisms among which yeasts and bacteria are the most common. Microorganisms inhabiting floral nectar can alter several nectar traits, including nectar odor by producing microbial volatile organic compounds (mVOCs). Evidence showing that mVOCs can affect the foraging behavior of insect pollinators is increasing in the literature, whereas the role of mVOCs in altering the foraging behavior of third-trophic level organisms such as insect parasitoids is largely overlooked. Parasitoids are frequent visitors of flowers and are well known to feed on nectar. In this study, we isolated bacteria inhabiting floral nectar of buckwheat, Fagopyrum esculentum (Polygonales: Polygonaceae), to test the hypothesis that nectar bacteria affect the foraging behavior of the egg parasitoid Trissolcus basalis (Hymenoptera: Scelionidae) via changes in odors of nectar. In behavioral assays, we found that T. basalis wasps are attracted toward nectar fermented by 4 out of the 14 bacterial strains isolated, which belong to Staphylococcus epidermidis, Terrabacillus saccharophilus (both Firmicutes), Pantoea sp. (Proteobacteria), and Curtobacterium sp. (Actinobacteria). Results of chemical investigations revealed significant differences in the volatile blend composition of nectars fermented by the bacterial isolates. Our results indicate that nectar-inhabiting bacteria play an important role in the interactions between flowering plants and foraging parasitoids. These results are also relevant from an applied perspective as flowering resources, such as buckwheat, are largely used in agriculture to promote conservation biological control of insect pests.

Plant Growth-Promoting Activities of Bacteria Isolated from an Anthropogenic Soil Located in Agrigento Province.

Bacteria producers of plant growth-promoting (PGP) substances are responsible for the enhancement of plant development through several mechanisms. The purpose of the present work was to evaluate the PGP traits of 63 bacterial strains that were isolated from an anthropogenic soil, and obtained by modification of vertisols in the Sicily region (Italy) seven years after creation. The microorganisms were tested for the following PGP characteristics: indole acetic acid (IAA), NH3, HCN and siderophore production, 1-aminocyclopropane-1-carboxylate deaminase activity (ACC) and phosphate solubilization. The results of principal component analysis (PCA) showed that Bacillus tequilensis SI 319, Brevibacterium frigoritolerans SI 433, Pseudomonas lini SI 287 and Pseudomonas frederiksbergensis SI 307 expressed high levels of IAA and production of ACC deaminase enzyme, while for the rest of traits analyzed the best performances were registered with Pseudomonas genus, in particular for the strains Pseudomonas atacamensis SI 443, Pseudomonas reinekei SI 441 and Pseudomonas granadensis SI 422 and SI 450. The in vitro screening provided enough evidence for future in vivo growth promotion tests of these eight strains.

Pathotyping Citrus Ornamental Relatives with Xanthomonas citri pv. citri and X. citri pv. aurantifolii Refines Our Understanding of Their Susceptibility to These Pathogens.

Xanthomonas citri pv. citri (Xcc) and X. citri pv. aurantifolii (Xca) are causal agents of Citrus Bacterial Canker (CBC), a devastating disease that severely affects citrus plants. They are harmful organisms not reported in Europe or the Mediterranean Basin. Host plants are in the Rutaceae family, including the genera Citrus, Poncirus, and Fortunella, and their hybrids. In addition, other genera of ornamental interest are reported as susceptible, but results are not uniform and sometimes incongruent. We evaluated the susceptibility of 32 ornamental accessions of the Rutaceae family belonging to the genera Citrus, Fortunella, Atalantia, Clausena, Eremocitrus, Glycosmis, Microcitrus, Murraya, Casimiroa, Calodendrum, and Aegle, and three hybrids to seven strains of Xcc and Xca. Pathotyping evaluation was assessed by scoring the symptomatic reactions on detached leaves. High variability in symptoms and bacterial population was shown among the different strains in the different hosts, indicative of complex host-pathogen interactions. The results are mostly consistent with past findings, with the few discrepancies probably due to our more complete experimental approach using multiple strains of the pathogen and multiple hosts. Our work supports the need to regulate non-citrus Rutaceae plant introductions into areas, like the EU and Mediterranean, that are currently free of this economically important pathogen.

Bioprospecting of Beneficial Bacteria Traits Associated With Tomato Root in Greenhouse Environment Reveals That Sampling Sites Impact More Than the Root Compartment.

Tomato is subject to several diseases that affect both field- and greenhouse-grown crops. To select cost-effective potential biocontrol agents, we used laboratory throughput screening to identify bacterial strains with versatile characteristics suitable for multipurpose uses. The natural diversity of tomato root-associated bacterial communities was bioprospected under a real-world environment represented by an intensive tomato cultivation area characterized by extraseasonal productions in the greenhouse. Approximately 400 tomato root-associated bacterial isolates, in majority Gram-negative bacteria, were isolated from three compartments: the soil close to the root surface (rhizosphere, R), the root surface (rhizoplane, RP), and the root interior (endorhizosphere, E). A total of 33% of the isolates produced siderophores and were able to solubilize phosphates and grow on NA with 8% NaCl. A total of 30% of the root-associated bacteria showed antagonistic activity against all the tomato pathogens tested, i.e., Clavibacter michiganesis pv. michiganensis, Pseudomonas syringae pv. tomato, Pseudomonas corrugata and Xanthomonas euvesicatoria pv. perforans, and Fusarium oxysporum f. sp. lycopersici. We found that the sampling site rather than the root compartment of isolation influenced bacterial composition in terms of analyzed phenotype. This was demonstrated through a diversity analysis including general characteristics and PGPR traits, as well as biocontrol activity in vitro. Analysis of 16S rRNA gene (rDNA) sequencing of 77 culturable endophytic bacteria that shared multiple beneficial activity revealed a predominance of bacteria in Bacillales, Enterobacteriales, and Pseudomonadales. Their in vitro antagonistic activity showed that Bacillus species were significantly more active than the isolates in the other taxonomic group. In planta activity against phytopathogenic bacteria of a subset of Bacillus and Pseudomonas isolates was also assessed.

Epidemiological Investigations and Molecular Characterization of 'Candidatus Phytoplasma solani' in Grapevines, Weeds, Vectors and Putative Vectors in Western Sicily, (Southern Italy).

Bois noir is caused by 'Candidatus Phytoplasma solani', and it is one of the most important and widespread diseases in the Euro-Mediterranean region. There are complex interactions between phytoplasma and grapevines, weeds, and vectors. These ecological relationships can be tracked according to molecular epidemiology. The aims of the 2-year study (2014-2015) were to describe incidence and spatial distribution of Bois noir in a vineyard with three grapevine varieties in Sicily, and to identify the molecular types of the tuf and vmp1 genes in these naturally infected grapevines, according to the potential reservoir plants and vectors. Disease incidence in 2015 was significantly higher in 'Chardonnay' (up to 35%) than for 'Nero d'Avola' and 'Pinot noir' (<5%). All grapevine, weed, and insect samples were infected by 'Ca. P. solani' tuf-type b. Most of the collected insects were strictly related to Vitis spp. and belonged to Neoaliturus fenestratus, Empoasca spp., and Zygina rhamni. The characterization of the vmp1 gene revealed six different vmp types in grapevines (V1, V4, V9, V11, V12, V24), three in weeds (V4, V9, V11), and four in insects (V4, V9, V11, V24). Notably, V4, V9, appear both in hosts and vectors, with V9 predominant. Virtual restriction fragment length polymorphism (RFLP) analysis based on the nucleotide sequences supported the data of the conventional RFLP. Connections between the molecular data recorded in the vineyard ecosystems and the application of innovative tools based on the geostatistical analysis will contribute to further clarification of the specific ecological and epidemiological aspects of 'Ca. P. solani' in Sicily.

Loop Mediated Isothermal Amplification: Principles and Applications in Plant Virology.

In the last decades, the evolution of molecular diagnosis methods has generated different advanced tools, like loop-mediated isothermal amplification (LAMP). Currently, it is a well-established technique, applied in different fields, such as the medicine, agriculture, and food industries, owing to its simplicity, specificity, rapidity, and low-cost efforts. LAMP is a nucleic acid amplification under isothermal conditions, which is highly compatible with point-of-care (POC) analysis and has the potential to improve the diagnosis in plant protection. The great advantages of LAMP have led to several upgrades in order to implement the technique. In this review, the authors provide an overview reporting in detail the different LAMP steps, focusing on designing and main characteristics of the primer set, different methods of result visualization, evolution and different application fields, reporting in detail LAMP application in plant virology, and the main advantages of the use of this technique.

The LuxR Regulators PcoR and RfiA Co-regulate Antimicrobial Peptide and Alginate Production in Pseudomonas corrugata.

Cyclic lipopeptides (CLPs) are considered as some of the most important secondary metabolites in different plant-associated bacteria, thanks to their antimicrobial, cytotoxic, and surfactant properties. In this study, our aim was to investigate the role of the Quorum Sensing (QS) system, PcoI/PcoR, and the LuxR-type transcriptional regulator RfiA in CLP production in the phytopatogenic bacterium, Pseudomonas corrugata based on our previous work where we reported that the pcoR and rfiA mutants were devoid of the CLPs cormycin and corpeptin production. Due to the close genetic link between the QS system and the RfiA (rfiA is co-transcribed with pcoI), it was difficult to ascertain the specific regulatory role in the expression of target genes. A transcriptional approach was undertaken to identify the specific role of the PcoR and RfiA transcriptional regulators for the expression of genes involved in CLP production. The RNA-seq-based transcriptional analysis of the wild-type (WT) strain CFBP 5454 in comparison with GL2 (pcoR mutant) and GLRFIA (rfiA mutant) was performed in cultural conditions favoring CLP production. Differential gene expression revealed that 152 and 130 genes have significantly different levels of expression in the pcoR and rfiA mutants, respectively. Of these, the genes linked to the biosynthesis of CLPs and alginate were positively controlled by both PcoR and RfiA. Blast homology analysis showed that 19 genes in a large CLP biosynthetic cluster involved in the production of three antimicrobial peptides, which span approximately 3.5% of the genome, are strongly over-expressed in the WT strain. Thus, PcoR and RfiA function mainly as activators in the production of bioactive CLPs, in agreement with phenotype analysis of mutants. RNA-seq also revealed that almost all the genes in the structural/biosynthetic cluster of alginate exopolysaccharide (EPS) are under the control of the PcoR-RfiA regulon, as supported by the 10-fold reduction in total EPS yield isolated in both mutants in comparison to the parent strain. A total of 68 and 38 gene expressions was independently regulated by PcoR or RfiA proteins, respectively, but at low level. qPCR experiments suggest that growth medium and plant environment influence the expression of CLP and alginate genes.

Grape and environmental mycoflora monitoring in old, traditionally cultivated vineyards on Mount Etna, southern Italy.

BACKGROUND: Grape contamination by several fungal species occurs during a vineyard's preharvest and harvest. Agronomic management and microclimatic conditions can affect fungi occurrence and epidemiology, thus explaining qualitative differences in mycoflora composition, including the presence of phytopathogenic or mycotoxigenic fungi. In this study a two-year grape, air and soil mycoflora monitoring programme was undertaken in vineyards on Mount Etna (eastern Sicily, Italy). The mycoflora composition was investigated at pea berry and veraison phenological phases from air and soil and at ripening from sample grapes. RESULTS: Mycoflora in air and soil varied according to the phenological stage. In the air samples, penicillia were dominant over aspergilli at the pea berry phase, but their ratio was inverted at early veraison. Black aspergilli (BA) were isolated from the vine environment and grape samples, where BA were represented mainly by Aspergillus niger aggregate, which showed no or low ochratoxin A (OTA) production. Aspergillus carbonarius was either not identified or identified at low frequency, although most of the isolates produced OTA. CONCLUSION: Monitoring focused on the environmental mycoflora composition and highlighted the good health profile of various Sicilian autochthonous grape cultivars. In addition, data suggest that the lower relative humidity occurring at the highest altitudes reduces BA incidence. © 2016 Society of Chemical Industry.

Transcriptome analysis of Pseudomonas mediterranea and P. corrugata plant pathogens during accumulation of medium-chain-length PHAs by glycerol bioconversion.

Pseudomonas corrugata and P. mediterranea are soil inhabitant bacteria, generally living as endophytes on symptomless plants and bare soil, but also capable of causing plant diseases. They share a similar genome size and a high proteome similarity. P. corrugata produces many biomolecules which play an important role in bacterial cell survival and fitness. Both species produce different medium-chain-length PHAs (mcl-PHAs) from the bioconversion of glycerol to a transparent film in P. mediterranea and a sticky elastomer in P. corrugata. In this work, using RNA-seq we investigated the transcriptional profiles of both bacteria at the early stationary growth phase with glycerol as the carbon source. Quantitative analysis of P. mediterranea transcripts versus P. corrugata revealed that 1756 genes were differentially expressed. A total of 175 genes were significantly upregulated in P. mediterranea, while 217 were downregulated. The largest group of upregulated genes was related to transport systems and stress response, energy and central metabolism, and carbon metabolism. Expression levels of most genes coding for enzymes related to PHA biosynthesis and central metabolic pathways showed no differences or only slight variations in pyruvate metabolism. The most relevant result was the significantly increased expression in P. mediterranea of genes involved in alginate production, an important exopolysaccharide, which in other Pseudomonas spp. plays a key role as a virulence factor or in stress tolerance and shows many industrial applications. In conclusion, the results provide useful information on the co-production of mcl-PHAs and alginate from glycerol as carbon source by P. mediterranea in the design of new strategies of genetic regulation to improve the yield of bioproducts or bacterial fitness.

Pseudomonas corrugata crpCDE is part of the cyclic lipopeptide corpeptin biosynthetic gene cluster and is involved in bacterial virulence in tomato and in hypersensitive response in Nicotiana benthamiana.

Pseudomonas corrugata CFBP 5454 produces two kinds of cyclic lipopeptides (CLPs), cormycin A and corpeptins, both of which possess surfactant, antimicrobial and phytotoxic activities. In this study, we identified genes coding for a putative non-ribosomal peptide synthetase and an ABC-type transport system involved in corpeptin production. These genes belong to the same transcriptional unit, designated crpCDE. The genetic organization of this locus is highly similar to other Pseudomonas CLP biosynthetic clusters. Matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis revealed that transporter and synthetase genomic knock-out mutants were unable to produce corpeptins, but continued to produce cormycin A. This suggests that CrpCDE is the only system involved in corpeptin production in P. corrugata CFBP 5454. In addition, phylogenetic analysis revealed that the CrpE ABC transporter clustered with the transporters of CLPs with a long peptide chain. Strains depleted in corpeptin production were significantly less virulent than the wild-type strain when inoculated in tomato plants and induced only chlorosis when infiltrated into Nicotiana benthamiana leaves. Thus, corpeptins are important effectors of P. corrugata interaction with plants. Expression analysis revealed that crpC transcription occurs at high cell density. Two LuxR transcriptional regulators, PcoR and RfiA, have a pivotal role in crpC expression and thus in corpeptin production.

N-acyl-homoserine-lactone quorum sensing in tomato phytopathogenic Pseudomonas spp. is involved in the regulation of lipodepsipeptide production.

Pseudomonas corrugata and Pseudomonas mediterranea are two closely related phytopathogenic bacteria both causal agents of tomato pith necrosis. P. corrugata produces phytotoxic and antimicrobial cationic lipodepsipeptides (LDPs) which are thought to act as major virulence factors. Previous studies have demonstrated that P. corrugata CFBP 5454 has an N-acyl homoserine lactone (AHL) quorum sensing (QS) system PcoI/PcoR and that LDP production occurs at high population densities. No molecular studies on virulence have thus far been reported for P. mediterranea. In this study, we show that P. mediterranea also produces LDPs as well as possessing an AHL-dependent QS system, designated PmeI/PmeR, which is highly homologous to the PcoI/R system of P. corrugata producing and responding to C(6)-AHL. Downstream of pmeI, a partial DNA sequence revealed the presence of a homolog of the rfiA gene of P. corrugata which encodes a transcriptional regulator involved in bacterial virulence. Pathogenicity tests and MALDI-TOF spectra of wild-type strains of both bacterial species and their respective QSs and rfiA derivative mutants revealed that, in the absence of LDPs, the strains induce very weak symptoms indicating that LDPs may act as major virulence factors. Mutational analysis of both QS systems suggests that their mode of action is in places different.

The transcriptional activator rfiA is quorum-sensing regulated by cotranscription with the luxI homolog pcoI and is essential for plant virulence in Pseudomonas corrugata.

The gram-negative phytopathogen Pseudomonas corrugata has an acyl-homoserine lactone (AHL) quorum-sensing (QS) system called PcoI/PcoR that is involved in virulence on tomato. This work identifies, downstream of pcoI, a gene designated rfiA, which we demonstrate is directly linked to QS by cotranscription with pcoI. The deduced RfiA protein contains a DNA-binding domain characteristic of the LuxR family but lacks the autoinducer-binding terminus characteristic of the QS LuxR-family proteins. We also identified, downstream of rfiA, an operon designated pcoABC, encoding for the three components of a tripartite resistance nodulation-cell-division (RND) transporter system. The expression of pcoABC is regulated by RfiA. We found that lipodepsipeptide (LDP) production is cell density dependent and mutants of pcoI, pcoR, and rfiA are unable to inhibit the growth of the LDP-sensitive microorganisms Rhodotorula pilimanae and Bacillus megaterium. P. corrugata rfiA mutants were significantly reduced in their ability to cause necrosis development in tomato pith. In addition, it was established that PcoR in the absence of AHL also played a role in virulence on tomato. A model for the role of PcoI, PcoR, and RfiA in tomato pith necrosis is presented.

Pseudomonas corrugata contains a conserved N-acyl homoserine lactone quorum sensing system; its role in tomato pathogenicity and tobacco hypersensitivity response.

Pseudomonas corrugata is a phytopathogenic bacterium, causal agent of tomato pith necrosis, yet it is an ubiquitous bacterium that is part of the microbial community in the soil and in the rhizosphere of different plant species. Although it is a very heterogeneous species, all the strains tested were able to produce short chain acyl homoserine lactone (AHL) quorum sensing signal molecules. The main AHL produced was N-hexanoyl-L-homoserine lactone (C(6)-AHL). An AHL quorum sensing system, designated PcoI/PcoR, was identified and characterized. The role of the quorum sensing system in the expression of a variety of traits was evaluated. Inactivation of pcoI abolished the production of AHLs. The pcoR mutant, but not the pcoI mutant, was impaired in swarming, unable to cause a hypersensitivity response on tobacco and resulted in a reduced tomato pith necrosis phenotype. The pcoI mutant showed a reduced antimicrobial activity against various fungi and bacteria when assayed on King's B medium. These results demonstrate that the AHL quorum sensing in Ps. corrugata regulates traits that contribute to virulence, antimicrobial activity and fitness. This is the first report of genes of Ps. corrugata involved in the disease development and biological control activity.

Pseudomonas salomonii sp. nov., pathogenic on garlic, and Pseudomonas palleroniana sp. nov., isolated from rice.

A total of 26 strains, including 15 strains isolated from garlic plants with the typical symptoms of 'Café au lait' disease and 11 strains isolated from diseased or healthy rice seeds and sheaths infested by Pseudomonas fuscovaginae, were compared with 70 type or reference strains of oxidase-positive pathogenic or non-pathogenic fluorescent pseudomonads. The strains were characterized by using a polyphasic taxonomic approach. Numerical taxonomy of phenotypic characteristics showed that the garlic and rice strains were related to each other. However, they clustered into separate phenons, distinct from those of the other strains tested, and were different in several nutritional tests. On the basis of DNA-DNA hybridization, the garlic and rice strains constituted two distinct DNA hybridization groups, indicating that they belonged to separate species. The two groups of strains were also well differentiated by siderotyping. Garlic strains were pathogenic to garlic plants and either weakly pathogenic or non-pathogenic on rice; rice strains were either weakly pathogenic or non-pathogenic on rice and non-pathogenic on garlic. A phylogenetic analysis of 16S rRNA gene sequences confirmed that the two groups of strains belonged to the y-Proteobacteria and to the genus Pseudomonas. The names Pseudomonas salomonii sp. nov. and Pseudomonas palleroniana sp. nov. are respectively proposed for the garlic strains and the rice strains. The type strains are P. salomonii CFBP 2022(T) ( = ICMP 14252(T) = NCPPB 4277(T)) and P. palleroniana CFBP 4389(T) (= ICMP 14253(T) = NCPPB 4278(T)).