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 specifi…

First Report of Leaf Spot and Blight Caused by Ralstonia pickettii on Bird of Paradise Tree in Italy

Bird of Paradise tree (Strelitzia alba (L. f.) Skeels) is an ornamental perennial tropical plant grown in southern Italy. In the summer of 2006 and 2007, a widespread, severe leaf disease was observed on seedlings and 1- to 2-year-old plants in two glasshouses located in eastern Sicily. Disease incidence ranged from 10 to 25%. Symptoms on the leaves consisted of dark brown-to-black stripes of varying length and found between the lateral veins. Lesions sometimes coalesced into a large area of necrotic tissue. Symptomatic tissues were ground in a drop of sterile distilled water (SDW) with a scalpel. Suspensions were streaked on King's medium B (KB), nutrient agar, and yeast extract nutrient …

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

AbstractFloral 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 buckwhe…

Draft genome sequence of Pseudomonas mediterranea strain CFBP 5447T, a producer of filmable medium-chain-length polyhydroxyalkanoates

ABSTRACT Pseudomonas mediterranea strain CFBP 5447 T is a phytopathogenic bacterium isolated from tomato plants affected by pith necrosis disease. Moreover, its ability to produce medium-chain-length polyhydroxyalkanoates (mcl-PHAs) in culture from different carbon sources and valuable microbial products, such as cyclic lipopeptides, has been well documented. Here, we report the first draft genome sequence of this species.

Fungal Contaminants and Mycotoxins in Nuts

Contamination by fungi and mycotoxins in nuts has achieved much attention in recent years. In fact, the fungal metabolites produced by the species of Aspergillus, Penicillium (aflatoxins and ochratoxins), Fusarium (trichothecenes, zearalenones and fumonisins) and Alternaria (alternariotoxins) with toxic and/or carcinogenic effects are considered a threat to human and animal health. In this chapter we will discuss the main fungal taxa and related mycotoxins most frequently associated with these materials. In this regard, the first results on the level of contamination by fungi and mycotoxins in samples of almonds and pistachios of different origins will be reported. The main strategies to re…

Characterization of Colletotrichum strains associated with olive anthracnose in Sicily

Anthracnose caused by Colletotrichum spp. is the most damaging olive fruit disease in many countries, including Italy. This disease has been sporadically detected in Sicily, but new agronomic practices can increase risk of olive anthracnose in this region. An etiological study of the disease focused on local olive cultivars growing at the International Olive Germplasm Collection (IOGC) in Villa Zagaria, Enna, Sicily has been undertaken. During 2018 and 2019, 137 Colletotrichum strains were isolated from olives. Colony morphology, conidium characteristics, and multilocus sequence analyses aided identification of three species: C. acutatum (affecting 70% of symptomatic olives), C. gloeosporio…

Collecting and preserving plant DNA for huanglongbing diagnosis in citrus samples from China

Accurate and sensitive detection of Citrus Huanglongbing associated ‘Ca. Liberibacter’species, not currently reported in the European and Mediterranean area, is an imperative need to define certification programs, to prevent introduction of the bacteria and/or their vectors in the unaffected areas, and to delineate efficient management strategies in those areas where the disease has spread. In this study, we compared different citrus sample preparation procedures for PCR based detection methods of ‘Candidatus Liberibacter asiaticus’, in order to find out the best a way to transport and preserve samples of Shatangju mandarin and fingered citron obtained during a survey in citrus orchards in …

Epidemiological survey on 'Candidatus Phytoplasma solani' and putative vectors in Western Sicily

Bois noir (BN) associated with ‘Candidatus Phytoplasma solani’ is an important grapevine yellows disease widespread in Europe and Mediterranean regions. During a two years survey (2014-15) carried out in a 10-year-old vineyard located in San Giuseppe Jato (PA), symptoms of Bois noir were observed on cvs Chardonnay, Pinot Noir and Nero d’ Avola. Incidence of the disease increased over the two years, reaching values of about 34 % on cv Chardonnay. Ca. ‘P. solani’ was detected on symptomatic leaf samples from all cultivars with the highest percentage on cv Chardonnay followed by Pinot noir and Nero d’Avola. Further surveys were performed in order to highlight the epidemiology of the disease by…

Quorum Sensing effects on Pseudomonas corrugata plant interaction and antagonistic activity

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 &lsquo

First report of Tomato leaf curl New Delhi virus affecting zucchini squash in an important horticultural area of southern Italy

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus (family Geminiviridae) which infects species in the families Cucurbitaceae and Solanaceae (Padidam et al., 1995; Mizutani et al., 2011). Begomoviruses are transmitted by the whitefly Bemisia tabaci in a persistent manner (Rosen et al., 2015). In October 2015, severe symptoms not previously reported by growers in the horticultural area of the Province of Trapani (Sicily, Italy) were observed on zucchini squash (Cucurbita pepo) in open fields. The symptoms included yellow mosaic, severe leaf curling, swelling of veins of young leaves, shortening of internodes, roughness of the skin of fruit and reduced fruit size; the sympt…

Identification and Detection of Phoma tracheiphila, Causal Agent of Citrus Mal Secco Disease, by Real-Time Polymerase Chain Reaction.

Phoma tracheiphila is the causal agent of a tracheomycotic disease of citrus called mal secco causing the dieback of twigs and branches. This pathogen is of quarantine concern; therefore, fast and reliable protocols are required to detect it promptly. A specific primer pair and a dual-labeled fluorogenic probe were used in a real-time polymerase chain reaction (PCR) with the Cepheid Smart Cycler II System (Transportable Device TD configuration) to detect this fungus in citrus samples. Real-time PCR assay was compared to modified conventional PCR assay. The sensitivity of the former was evaluated by testing P. tracheiphila DNA dilutions, and the minimum amount detectable was about 500 fg, wh…

Defense-related gene expression in two olive cultivars inoculated with Pseudomonas savastanoi pv. savastanoi

I batteri associati al nettare fiorale influiscono sulle longevità degli insetti parassitoidi

I parassitoidi adulti frequentemente visitano le piante in fioritura, ricercando nettare fiorale per coprire i loro bisogni energetici e nutrizionali. In letteratura, infatti, i benefici del nettare sulla fitness dei parassitoidi sono ampiamente documentati. Sebbene il nettare fiorale costituisca un substrato ideale per la crescita di comunità microbiche che possono alterarne le caratteristiche organolettiche, l'effetto della fermentazione batterica del nettare sui parassitoidi non è stato ancora sufficientemente esplorato. In questo lavoro è stata studiata la longevità dei parassitoidi adulti utilizzando nettare sintetico fermentato da diversi ceppi batterici appartenenti ai phyla Firmicut…

Antimicrobial activity of the extracts of Terfezia claveryi and Tirmania pinoyi against gram-positive and gram-negative bacteria causal agent of diseases in tomato

Tomato diseases caused by virus, bacteria and fungi have been reported worldwide and caused considerable economic losses. Among all diseases, attention is paid to those caused by bacteria. In this study, the extracts of two “desert truffles” Terfezia claveryi and Tirmania pinoyi were tested against six bacterial species, causal agent of economically important tomato diseases: Pseudomonas corrugata, P. mediterranea, P. syringae pv. tomato Pectobacterium carotovorum subsp. carotovorum, Xanthomonas vesicatoria and Clavibacter michiganensis subsp. michiganansis. The extracts from both fungal species, evaluated by agar well diffusion method, showed an antimicrobial activity against all the teste…

Race typing and molecular characterization of Xanthomonas campestris pv. campestris strains occurring in Italy

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 r…

Mixed infection of Pectobacterium carotovorum subsp. Carotovorum and P. carotovorum subsp. Brasiliensis in tomato stem rot in Italy

Grafted tomato plants grown in a soilless culture system in heated greenhouses in Sicily were affected by stem rot disease. Symptoms consisted of dark brown longitudinal discoloration of the basal part of the stem and petioles. Longitudinal sections of the stem revealed brown watersoaked or soft-rotted pith tissue and internal vascular discoloration. Bacterial isolates with two different colony morphologies were obtained from symptomatic tissues. Isolates were identified as Pectobacterium spp. on the basis of biochemical and molecular analyses. The analysis of the 16S rRNA gene sequence and of the concatenated sequences of the housekeeping genes rpoS and pgi revealed that the isolates of th…

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, Brevib…

A real-time PCR quantitative detection assay for Pseudomonas savastanoi pv. nerii in Nerium oleander

A real-time PCR assay based on TaqMan chemistry was developed for the detection of Pseudomonas savastanoi pathovars that cause bacterial knot disease on different plant species. Primers and probe sequences were based on the iaaL gene coding for (indole-3-acetyl)-L-lysine synthetase and previously used in conventional PCR tests. Assay specificity was tested with an extended range of strains of P. savastanoi from eight hosts, with 13 other Pseudomonas spp., and with other microorganisms naturally occurring on or in oleander plants. A pure culture cell suspension was quantifi ed over a seven log concentration range (108 to 102 cfu ml-1 ). Different protocols were developed for the detection an…

Occurrence of tomato pith necrosis caused by Pseudomonas marginalis in Italy.

Dipartimento di Scienze e Tecnologie Fitosanitarie. Universita` degli Studi di Catania. Via Santa Sofia 100, 95123 Catania, ItalyIn 2006, a serious outbreak of tomato pith necrosis (TPN) with approxi-mately 90% disease incidence was observed in two greenhouses in Sicily.Adult fruiting plants showed chlorosis and slight wilting of shoot apices.Internalstem browning alongthe entire length oftheplant was observed.Pith tissues were soft but not rotted or hollowed. Only fluorescent colo-nies developed on King’s B medium (KB) after isolation from infected tis-sues. Ten pure isolates were all levan, oxidase, potato soft-rotting andarginine dihydrolase positive and were negative for tobacco hypersens…

Poly(hydroxyalkanoate) synthase genes in pseudomonads strains, isolation and heterologous expression

Draft genome sequence of Pseudomonas corrugata, a phytopathogenic bacterium with potential industrial applications

Pseudomonas corrugata was first described as the causal agent of a tomato disease called 'pith necrosis' yet it is considered as a biological resource in various fields such as biocontrol of plant diseases and production of industrially promising microbial biopolymers (mcl-PHA). Here we report the first draft genome sequence of this species. © 2014 Elsevier B.V.

Unusual occurrence of Schizophyllum comune on young topgrafted citrus

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), …

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

International audience; 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 nu…

The transcriptional activator rfiA is quorum-sensing regulated by cotranscription with the luxI homolog pcol 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 pcol, a gene designated rfiA, which we demonstrate is directly linked to QS by cotranscription with pcol. 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 pco…

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,…

Clonal populations of Clavibacter michiganensis subsp michiganensis are responsible for the outbreaks of bacterial canker in greenhouse tomatoes in Italy

Clavibacter michiganensis subsp. michiganensis (Cmm) strains, collected in greenhouses from 17 farms during tomato bacterial canker outbreaks occurring between 2005 and 2008 in Sicily, were analysed by a multiphasic approach. Population studies were conducted to investigate the possible sources of inocula. Cmm strains were characterized by PCR assays targeting virulence genes, fingerprinting techniques, metabolic profiles and virulence. These strains were comparatively analysed with Cmm strains isolated in other parts of Italy over a period of 15 years. Chromosomal genes encoding virulence determinants tomA, ppaA, chpC, and the plasmid-encoded genes pat-1 and celA were detected by PCR in al…

Role of secondary metabolites in the biocontrol activity of Pseudomonas corrugata and Pseudomonas mediterranea

In this study, the Pseudomonas corrugata strain CFBP 5454 and the P. mediterranea strain CFBP 5447 were shown to produce diffusible compounds that inhibit the in vitro growth of plant pathogenic fungi and bacteria and antifungal volatile compounds. In addition, both bacterial strains were found to produce cyanide. Mutant derivatives in LuxR transcriptional regulators, i.e. P. corrugata GL2 (pcoR mutant) and GLRFIA (rfiA mutant), and P. mediterranea PSMER (pmeR mutant) and PSRFIA (rfiA mutant) impaired in cyclic lipopeptide (CLP) production, showed a diffusible compound-mediated reduced activity, depending on the biocontrol strain, challenge microorganism and culture medium. The volatile com…

Cellulolytic activity in Aspergillus spp. contaminating livestock feeds and raw materials

The contamination by Aspergillus spp. have become a global concern in food and feedstuffs and can lead to a reduction in yield and quality of agricultural products with significant economic losses. Most species of Aspergillus produce cellulose-degrading enzymes and some of them also have mycotoxigenic activity. This study aimed i) to evaluate the Aspergillus contamination in feeds (16) and row materials (32) collected in Sicily; ii) to isolate and identify fungi belonging to the genus Aspergillus and iii) to analyze their ability to produce cellulolytic enzymes. Aspergillus spp. contamination was evaluated on PDA using serial ten-fold dilution and spread plate technique (Mirabile et al., 20…

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 sou…

Multilocus sequence typing analysis of Italian Xanthomonas campestris pv. campestris strains suggests the evolution of local endemic populations of the pathogen and do not correlate with race distribution

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in Brassicaceae. It is widespread in Italy and severe outbreaks occur under conditions that favour disease development. In this study a multilocus sequence typing approach (MLST) based on the partial sequence of seven loci was applied to a selection of strains representative of the main areas of cultivation and hosts. The aim was to investigate whether the long tradition of brassica crops in Italy has influenced the evolution of different Xcc populations. All loci were polymorphic; 14 allelic profiles were identified of which 13 were unique to Italian strains. Based on the seven loci, the most common genotype withi…

First outbreak of Pepper vein yellows virus infecting sweet pepper in Italy

Sweet pepper (Capsicum annum) is an economically important crop worldwide, including Sicily where about 4,000 hectares are grown each year. In October 2015, severe symptoms not previously reported by growers in the horticultural area of the province of Trapani (Sicily, Italy) were observed on sweet pepper plants in eight different greenhouses. Symptoms included upward leaf curling, internodal shortening and interveinal yellowing. Symptoms were more evident in the upper part of the plants. These symptoms were reminiscent of those caused by poleroviruses. In the greenhouse, symptoms were evident in about 35% of the plants. Three samples per greenhouse (24 in total) were collected for analysis.

Over-expression of CsGSTU promotes tolerance to the herbicide alachlor and resistance to Pseudomonas syringae pv. tabaci in transgenic tobacco

Glutathione transferases (GSTs) mainly catalyze the nucleophilic addition of glutathione to a large variety of hydrophobic molecules participating to the vacuole compartmentalization of many toxic compounds. In this work, the putative tolerance of transgenic tobacco plants over-expressing CsGSTU genes towards the chloroacetanilide herbicide alachlor was investigated. Our results show that the treatment with 0.0075 mg cm-3 of alachlor strongly affects the growth of both wild type and transformed tobacco seedlings with the sole exception of the transgenic lines overexpressing CsGSTU2 isoform that are barely influenced by herbicide treatment. In order to correlate the in planta studies with en…

First report of Pleurotus fuscosquamulosus (Pleurotaceae, Basidiomycota) in Italy naturally occurring on new tropical hosts

Basidiomata of a mushroom macroscopically recognised as Pleurotus cystidosus sensu latu were collected on the trunks of three tropical ornamental trees such as Broussonetia papyrifera, Yucca elephantipes, and Paulownia tomentosa in the Parco d’Orleans, an urban park in the town of Palermo (Sicily, Italy). All the plants showed cavities and white rot symptoms at the base of the trunks. Macro- and microscopic observations on both collected basidiomata and isolated colonies, as well as molecular analysis, allowed identifying the collected basidiomata as Pleurotus fuscosquamulosus and its anamorph as Antromycopsis fuscosquamulosus. These species belong to the subgenus Coremiopleurotus that incl…

Transcriptional analysis of pha genes in pseudomonas mediterranea CFBP 5447 grown on glycerol

We analysed the draft genome sequence of Pseudomonas mediterranea CFBP 5447 in order to identify firstly the central metabolic pathways that convert fatty acids or carbohydrate intermediates into mcl-PHA and secondly the genes involved in glycerol metabolism (glpF, glpK, glpD, glpR). Absence of the glpF gene, which codifies for the "glycerol uptake facilitator protein", was highlighted. In order to understand the expression of the pha gene cluster, we investigated the promoter activity of phaC1, phaC2, phaZ, phaD and phaI genes. When glycerol was present as the carbon source, PI was found to be the most active promoter. Expression analysis of the knock-out mutant of the phaD gene, which is …