Bacterium Pseudomonas Savastanoi Pv Research Articles

Varietal Susceptibility of Olive to Pseudomonas savastanoi pv. savastanoi and the Antibacterial Potential of Plant-Based Agents.

Olive knot disease, caused by the bacterium Pseudomonas savastanoi pv. savastanoi, causes great damage in olive orchards. While control measures of P. savastanoi pv. savastanoi in olive orchards primarily rely on pruning and copper-based treatments, the use of antibiotics as bactericidal preparations in agriculture is limited and highly restricted. However, plants are naturally endowed with protective molecules, such as phenolic compounds, which defend them against herbivores, insects, and microorganisms. This research aimed to test the virulence of five strains of P. savastanoi pv. savastanoi isolated from different growing regions and olive varieties, and to examine whether there is a difference in plant susceptibility based on the variety. An additional goal was to test the antimicrobial activity of olive mill wastewater, known for its high content of phenolic compounds, and aqueous garlic hydrolysate, as well as to compare them with a commercial copper-based product, pure hydroxytyrosol, and a standard antibiotic as references. Analysis of knot characteristics showed variations in the virulence of the P. savastanoi pv. savastanoi strains, with the highest virulence being observed for the strain I7L and the lowest virulence for the strain B45C-PR. The olive cultivar Rosinjola displayed higher susceptibility compared to Frantoio, Buža, and Leccino, while cv. Istarska bjelica exhibited the least susceptibility compared to the other investigated olive cultivars. In an attempt to explore alternative solutions for disease control, in vitro tests revealed that the phenol HTyr, GE, and the wastewater with the highest total phenolic content (cv. Istarska bjelica) possess the highest antibacterial activity. This supports the role of polyphenols in host defense, aligning with previous field observations of lower susceptibility of cv. Istarska bjelica to olive knot disease. These findings highlight the complex nature of olive knot interactions with bacterial strains and olive cultivars, simultaneously accentuating and underscoring the importance of considering the host's defenses as well as bacterial virulence in disease management strategies.

Cultivar Susceptibility to Olive Knot Disease and Association with Endophytic Microbiota Community

Olive knot disease (OKD) induced by the bacterium Pseudomonas savastanoi pv. savastanoi seriously affects olive production in the Mediterranean basin. Nowadays, the only strategies to control the disease are pruning and the application of cupric products. An essential strategy to enhance protection is represented by the identification of resistant cultivars, which represents a crucial opportunity for future investments and breeding. We undertook a three-year-long survey at the International Olive Germplasm Collection of “Villa Zagaria” (Sicily, Italy) on thirty-six Sicilian cultivars that were monitored for symptom development. Cultivars with different levels of susceptibility were divided into five clusters. Moreover, in order to investigate possible interactions with endophytic microbial communities, two cultivars with contrasting susceptibilities, Zaituna (highly resistant) and Giarraffa (highly susceptible), were selected for an amplicon-based metagenomic analysis. Distinct endophytic communities colonized the two cultivars, suggesting an interaction between the resident bacterial community and the pathogen. Significantly higher bacterial richness was detected in the shoots of the susceptible cv. Giarraffa, although it had lower diversity. The opposite trend was observed for fungal communities. Among the microbes resulted to be enriched in cv. Giarraffa, it is important to underline the presence of Pseudomonas among the bacterial genera, and Alternaria, Neofusicoccum, Epicoccum, Ascochyta, and Elsinoe among the fungal genera, which include many species often described as plant pathogens and biocontrol agents. Starting from this basic information, new strategies of control, which include breeding for resistance and integrated disease management, can be envisaged.

Evaluating molecular diagnostic techniques for seed detection of Pseudomonas savastanoi pv. phaseolicola, causal agent of halo blight disease in mungbean (Vigna radiata)

Halo blight of mungbean (Vigna radiata var. radiata) is caused by the bacterium Pseudomonas savastanoi pv. phaseolicola. This pathogen is transmitted via infected seed, facilitating the spread of the disease into new cultivated areas. Prospective mungbean seed crops are currently subjected to visual inspection as a means of determining disease status, however, this is a poor method that relies on visible symptoms and does not account for latent infections. A range of molecular diagnostics targeting P. savastanoi pv. phaseolicola have been developed, but these have not been deployed on seeds. Quantitative PCR (qPCR) SYBR assay, hydrolysis probe, and conventional PCR, using the same primers were optimised against a plate-truthed dilution series of P. savastanoi pv. phaseolicola. The detection limit of the conventional PCR assay was approximately 9,000 CFU µl-1, while both qPCR assays could detect 9 CFU µl-1. These tests were then used to screen DNA extracted from 200 g allotments of 38 seed lots comprising six mungbean cultivars representing the primary Australian production area, and two seed lots of known infection status. Of these, the pathogen was detected in six seed lots by conventional PCR. The SYBR assay and hydrolysis probe methods detected 20 and 24 infected seed lots respectively. This shows that the hydrolysis probe method was the most effective at diagnosing the presence of P. savastanoi pv. phaseolicola in mungbean seed, providing a valuable molecular diagnostic to aid in integrated disease management and seed certification, substantially mitigating losses to halo blight disease.

Halo Blight of Mungbean in Australia

Halo blight, one of the major diseases of mungbean, is caused by the bacterium Pseudomonas savastanoi pv. phaseolicola. The pathogen infects the foliar parts of the plant, causing water-soaked spots that eventually develop surrounding yellow margins. The disease is particularly destructive under moderate temperature and high humidity, especially when it occurs during late vegetative through to early reproductive stage. In such conditions, severely infected crops could experience a yield loss up to 70%. Halo blight can be widespread on mungbeans grown in Southern Queensland and Northern New South Wales. However, due to its seedborne and cryptic nature of transmission, the disease is likely to be under-reported. This report addresses major aspects of halo blight symptomology, pathology and epidemiology.

A MATE Transporter is Involved in Pathogenicity and IAA Homeostasis in the Hyperplastic Plant Pathogen Pseudomonas savastanoi pv. nerii.

During the last years, many evidences have been accumulating about the phytohormone indole-3-acetic acid (IAA) as a multifaceted compound in the microbial world, with IAA playing a role as a bacterial intra and intercellular signaling molecule or as an effector during pathogenic or beneficial plant–bacteria interactions. However, pretty much nothing is known on the mechanisms that bacteria use to modulate IAA homeostasis, in particular on IAA active transport systems. Here, by an approach combining in silico three-dimensional (3D) structural modeling and docking, mutagenesis, quantitative gene expression analysis, and HPLC FLD auxin quantitative detection, for the first time a bacterial multidrug and toxic compound extrusion (MATE) transporter was demonstrated to be involved in the efflux of IAA, as well as of its conjugate IAA–Lysine, in the plant pathogenic hyperplastic bacterium Pseudomonas savastanoi pv. nerii strain Psn23. Furthermore, according to the role proved to be played by Psn23 MatE in the development of plant disease, and to the presence of Psn23 MatE homologs in all the genomospecies of the P. syringae complex, this membrane transporter could likely represent a promising target for the design of novel and selective anti-infective molecules for plant disease control.

Quorum Sensing in Pseudomonas savastanoi pv. savastanoi and Erwinia toletana: Role in Virulence and Interspecies Interactions in the Olive Knot.

The olive knot disease (Olea europea L.) is caused by the bacterium Pseudomonas savastanoi pv. savastanoi. P. savastanoi pv. savastanoi in the olive knot undergoes interspecies interactions with the harmless endophyte Erwinia toletana; P. savastanoi pv. savastanoi and E. toletana colocalize and form a stable community, resulting in a more aggressive disease. P. savastanoi pv. savastanoi and Etoletana produce the same type of the N-acylhomoserine lactone (AHL) quorum sensing (QS) signal, and they share AHLs in planta In this work, we have further studied the AHL QS systems of P. savastanoi pv. savastanoi and Etoletana in order to determine possible molecular mechanism(s) involved in this bacterial interspecies interaction/cooperation. The AHL QS regulons of P. savastanoi pv. savastanoi and Etoletana were determined, allowing the identification of several QS-regulated genes. Surprisingly, the P. savastanoi pv. savastanoi QS regulon consisted of only a few loci whereas in Etoletana many putative metabolic genes were regulated by QS, among which are several involved in carbohydrate metabolism. One of these loci was the aldolase-encoding gene garL, which was found to be essential for both colocalization of P. savastanoi pv. savastanoi and Etoletana cells inside olive knots as well as knot development. This study further highlighted that pathogens can cooperate with commensal members of the plant microbiome.IMPORTANCE This is a report on studies of the quorum sensing (QS) systems of the olive knot pathogen Pseudomonas savastanoi pv. savastanoi and olive knot cooperator Erwinia toletana These two bacterial species form a stable community in the olive knot, share QS signals, and cooperate, resulting in a more aggressive disease. In this work we further studied the QS systems by determining their regulons as well as by studying QS-regulated genes which might play a role in this cooperation. This represents a unique in vivo interspecies bacterial virulence model and highlights the importance of bacterial interspecies interaction in disease.

Global Analysis of Type Three Secretion System and Quorum Sensing Inhibition of Pseudomonas savastanoi by Polyphenols Extracts from Vegetable Residues

Protection of plants against bacterial diseases still mainly relies on the use of chemical pesticides, which in Europe correspond essentially to copper-based compounds. However, recently plant diseases control is oriented towards a rational use of molecules and extracts, generally with natural origin, with lower intrinsic toxicity and a reduced negative environmental impact. In this work, polyphenolic extracts from vegetable no food/feed residues of typical Mediterranean crops, as Olea europaea, Cynara scolymus, and Vitis vinifera were obtained and their inhibitory activity on the Type Three Secretion System (TTSS) and the Quorum Sensing (QS) of the Gram-negative phytopathogenic bacterium Pseudomonas savastanoi pv. nerii strain Psn23 was assessed. Extract from green tea (Camellia sinensis) was used as a positive control. Collectively, the data obtained through gfp-promoter fusion system and real-time PCR show that all the polyphenolic extracts here studied have a high inhibitory activity on both the TTSS and QS of Psn23, without any depressing effect on bacterial viability. Extracts from green tea and grape seeds were shown to be the most active. Such activity was confirmed in planta by a strong reduction in the ability of Psn23 to develop hyperplastic galls on explants from adult oleander plants, as well as to elicit hypersensitive response on tobacco. By using a newly developed Congo red assay and an ELISA test, we demonstrated that the TTSS-targeted activity of these polyphenolic extracts also affects the TTSS pilus assembly. In consideration of the potential application of polyphenolic extracts in plant protection, the absence of any toxicity of these polyphenolic compounds was also assessed. A widely and evolutionary conserved molecular target such as Ca2+-ATPase, essential for the survival of any living organism, was used for the toxicity assessment.

Draft Genome Sequence of Pseudomonas savastanoi pv. savastanoi Strain DAPP-PG 722, Isolated in Italy from an Olive Plant Affected by Knot Disease.

Olive knot disease, caused by the bacterium Pseudomonas savastanoi pv. savastanoi, seriously affects olive trees in the Mediterranean basin. Here, we report the draft genome sequence of P. savastanoi pv. savastanoi DAPP-PG 722, a strain isolated in Italy from an olive plant affected by knot disease.

Draft Genome Sequence of Erwinia toletana, a Bacterium Associated with Olive Knots Caused by Pseudomonas savastanoi pv. Savastanoi

Erwinia toletana was first reported in 2004 as a bacterial species isolated from olive knots caused by the plant bacterium Pseudomonas savastanoi pv. savastanoi. Recent studies have shown that the presence of this bacterium in the olive knot environment increases the virulence of the disease, indicating possible interspecies interactions with P. savastanoi pv. savastanoi. Here, we report the first draft genome sequence of an E. toletana strain.

Sharing of quorum-sensing signals and role of interspecies communities in a bacterial plant disease.

Pathogenic bacteria interact not only with the host organism but most probably also with the resident microbial flora. In the knot disease of the olive tree (Olea europaea), the causative agent is the bacterium Pseudomonas savastanoi pv. savastanoi (Psv). Two bacterial species, namely Pantoea agglomerans and Erwinia toletana, which are not pathogenic and are olive plant epiphytes and endophytes, have been found very often to be associated with the olive knot. We identified the chemical signals that are produced by strains of the three species isolated from olive knot and found that they belong to the N-acyl-homoserine lactone family of QS signals. The luxI/R family genes responsible for the production and response to these signals in all three bacterial species have been identified and characterized. Genomic knockout mutagenesis and in planta experiments showed that virulence of Psv critically depends on QS; however, the lack of signal production can be complemented by wild-type E. toletana or P. agglomerans. It is also apparent that the disease caused by Psv is aggravated by the presence of the two other bacterial species. In this paper we discuss the potential role of QS in establishing a stable consortia leading to a poly-bacterial disease.

Investigation of the Antibacterial Mechanism of Aflatoxins in the BioArena System

The influence of monomethylated basic amino acids [NG-monomethyl-L-arginine (MMA) and Nepsilon-monomethyl-L-lysine (MML)] and ozone capturers (indigo carmine, d-limonene) on the antibacterial effect of the mycotoxins aflatoxins B1, B2, G1 and G2 was studied in BioArena, which is a complex bioautographic system especially suitable for investigating biochemical interactions. In the presence of the formaldehyde precursors MMA or MML, the antibacterial-toxic activity of all the aflatoxins against the phytopathogenic bacterium Pseudomonas savastanoi pv. phaseolicola was enhanced dose-dependently. Indigo carmine and d-limonene, in appropriate concentrations, decreased the inhibition zones of aflatoxins. These results support the original idea that HCHO and its derivative 03 may be involved in the antibacterial activity of aflatoxins and so, potentially, in their known toxic effect.

OLEANDER KNOT CAUSED BY PSEUDOMONAS SAVASTANOI pv. NERII IN TURKEY

A knot disease was detected on some oleander plants in two park since 2007 and in one home gardens in 2008 in the Sanliurfa province (south-east Turkey). Although galls were excised in the first year, they developed again with a bigger size on the lower parts of the main stems. Knots on newly infected plants were common on the upper parts, particularly on twigs, flowers and pistils or at the pistil base. Almost all infected plants belonged to cultivars with pink or red flowers. Biochemical and pathogenicity tests identified the plant pathogenic bacterium Pseudomonas savastanoi pv. nerii as the causal agent of the disease. Although the presence of knots on oleander has long been known, to our knowledge, Pseudomonas savastanoi pv. nerii had never been previously reported on Nerium oleander in Turkey.

Systemic spread of Pseudomonas savastanoi pv. savastanoi in olive explants

The manner in which the bacterium Pseudomonas savastanoi pv. savastanoi (Pss), the causal agent of knot disease, infects olive plants is erratic and has not been fully documented. To investigate the process of Pss invasion, olive explants were inoculated in vitro and examined visually and by light microscopy at 2‐weekly intervals for 10 weeks. In all host genotypes tested, interaction with the pathogen resulted in: (i) a progressive collapse of the stem, originating at the inoculation site at the apex of the explant, and proceeding downwards; and (ii), localized outgrowths on the stem located at various distances from the inoculation site. Histological analysis revealed that the anatomy of the outgrowths closely resembled that of knots formed in vivo; they showed that Ps. savastanoi also diffused within the olive explants through the xylem vessels, and that the olive host reacted to pathogen invasion, possibly by producing substances of polysaccharidic and/or phenolic nature.

Potential role of formaldehyde in the mechanism of action of ascorbigens on the basis of BioArena studies

The effect of ascorbigen and 1'-methylascorbigen as a model compound pair was studied on the phytopathogenic bacterium Pseudomonas savastanoi pv. phaseolicola in the BioArena experimental system after overpressured layer chromatography. Results showed a characteristic, strong antibacterial effect of 1'-methylascorbigen and weak effect of ascorbigen present on the adsorbent layer as chromatographic spot. Addition of formaldehyde capture compounds (L-arginine, glutathione, dimedone) partially or totally reduced the antibacterial effect of 1'-methylascorbigen and ascorbigen. On adding Cu(II) ions--which mobilize and coordinate formaldehyde--to the culture medium, the antibacterial effect of both compounds became stronger. It is supposed that the weak antibacterial effect of ascorbigen may have originated from the 1'-methylascorbigen formed in situ on the adsorbent layer by partial enzymatic methylation of ascorbigen.

First report of Pseudomonas savastanoi pv. nerii on oleander in the Czech Republic

The bacterium Pseudomonas savastanoi pv. nerii was identified as the causal agent of parenchymatous galls on leaves of potted oleander plants grown at Brno, Moravia, in 2004. The plants had originated from cuttings made from firm shoots of a supposedly asymptomatic plant grown in and introduced from the Mediterranean region. The Biolog GN microplate system was used to identify the isolated bacterial strains. Successful inoculation of Nerium oleander seedlings proved the pathogenicity of the isolates. This is the first record of P. savastanoi pv. nerii in the Czech Republic. :

Multiplex Nested Reverse Transcription-Polymerase Chain Reaction in a Single Tube for Sensitive and Simultaneous Detection of Four RNA Viruses and Pseudomonas savastanoi pv. savastanoi in Olive Trees

ABSTRACT A multiplex nested reverse transcription-polymerase chain reaction (RT-PCR) in a single closed tube was developed for the simultaneous detection of four RNA viruses: Cucumber mosaic virus, Cherry leaf roll virus, Strawberry latent ringspot virus, and Arabis mosaic virus, and the bacterium Pseudomonas savastanoi pv. savastanoi. The method enabled, for the first time, the sensitive and simultaneous detection of RNA and DNA targets from plant viruses and a bacterium, saving time, decreasing risks of contamination, and reducing costs compared with conventional monospecific nested amplifications. The method was successfully coupled with colorimetric detection of amplicons using specific oligoprobes to simplify routine detection. Two hundred forty-five olive trees from 15 different cultivars were analyzed by multiplex RT-nested PCR coupled with colorimetric detection. Multiplex nested RT-PCR for viral detection increased the identification of positive trees by 8.1%. An uneven distribution of the viruses was observed in the infected trees. The bacterium was detected in 28.7% of the analyzed trees by the developed multiplex nested method and by a nested PCR previously developed. This powerful methodology could be applied to other models for the detection of several pathogens in a single assay.

Root promoting compounds detected in olive knot extract in high quantities as a response to infection by the bacterium Pseudomonas savastanoi pv. savastanoi

Olive knot crude extract has been found to promote rooting in vitro of Koroneiki olive explants. The analysis of this extract revealed the presence of the naturally occurring auxins indole-3-acetic acid (IAA) and indole-3-acetonitrile (IAN) in high quantities. Both auxins were found in higher amounts in the knots, formed in olive shoots after the infection by the bacterium Pseudomonas savastanoi pv. savastanoi, than in healthy shoots. Further research showed that the knots were rich in phenolic compounds and o-diphenols, as well as that they contained higher amounts of oleuropein (an o-diphenol) and another unidentified compound in contrast to healthy shoots. The analysis of polyamines showed higher concentrations of putrescine in the knots than in the healthy shoots, while it was not possible to detect any traces of spermidine or spermine in the olive knot extract. The results suggested a possible role and collaboration of auxins, phenolic compounds and polyamines, such as putrescine, in the root induction procedure of olive explants in vitro . Furthermore, a possible defense mechanism of olive tree against the bacterium was postulated, through the increased production of possible anticipins, like IAN and the unknown phenolic compound.

In vitro propagation of olive (Olea europaea L.) cv. Koroneiki

Single node explants of 'Koroneiki' olive trees werecultured for one month on a modified Driver-Kuniyuki for Walnut medium, lackinggrowth regulators. The explants were subcultured once a month on a mediumsupplemented with zeatin riboside, 6-(γ-γ-dimethylallylamino)purine,6-benzyladenine or thidiazuron. Zeatin riboside proved to be superior to othercytokinins in inducing shoot proliferation. The combination of olive knotextract at 25 or 50 mg l−1 with cytokininssuppressed shoot proliferation. After two months at the proliferation stage,theexplants were cultured for one week in the dark in 1 ml liquidWoody Plant Medium supplemented with IBA, α-NAA or IBA+α-NAA. Theexplants were then transferred to the same solid medium lacking growthregulators, with a small layer of perlite on the surface. The combination ofthetwo auxins at 1+1 mg l−1 resulted in almost 76%rooting. The combination of olive knot extract at 50 mgl−1 with auxins increased the rooting percentage up toalmost 87%. Artificial infection of explants with the bacteriumPseudomonas savastanoi pv. savastanoiinhibited rhizogenesis, even in the presence of auxins. Rooted explants weresuccessfully acclimatised under a mist system, with the survival rate reachingalmost 75%.

The structure of the exopolysaccharide fraction from Pseudomonas savastanoi strain ITM519 and the defence-response it induces in non-hosts plants

The main exopolysaccharide (EPS) obtained from the phytopathogenic bacterium Pseudomonas savastanoi pv. nerii, strain ITM519, has a very complex highly branched structure consisting of fucose, galactose, Nacetylgalactosamine and N-acetylglucosamine. EPS triggers a defence response in non-host plant cells. This capability could be a consequence of the complex and heterogeneous structure of the molecule, part of which might mimic elicitors produced in the plant–pathogen interaction.

The structure of the exopolysaccharide fraction from Pseudomonas savastanoi strain ITM519 and the defence-response it induces in non-hosts plants

The main exopolysaccharide (EPS) obtained from the phytopathogenic bacterium Pseudomonas savastanoi pv. nerii, strain ITM519, has a very complex highly branched structure consisting of fucose, galactose, Nacetylgalactosamine and N-acetylglucosamine. EPS triggers a defence response in non-host plant cells. This capability could be a consequence of the complex and heterogeneous structure of the molecule, part of which might mimic elicitors produced in the plant–pathogen interaction.