Bacterial Apical Necrosis Research Articles

Biological role of EPS from Pseudomonas syringae pv. syringae UMAF0158 extracellular matrix, focusing on a Psl-like polysaccharide

Pseudomonas syringae is a phytopathogenic model bacterium that is used worldwide to study plant–bacteria interactions and biofilm formation in association with a plant host. Within this species, the syringae pathovar is the most studied due to its wide host range, affecting both, woody and herbaceous plants. In particular, Pseudomonas syringae pv. syringae (Pss) has been previously described as the causal agent of bacterial apical necrosis on mango trees. Pss exhibits major epiphytic traits and virulence factors that improve its epiphytic survival and pathogenicity in mango trees. The cellulose exopolysaccharide has been described as a key component in the development of the biofilm lifestyle of the P. syringae pv. syringae UMAF0158 strain (PssUMAF0158). PssUMAF0158 contains two additional genomic regions that putatively encode for exopolysaccharides such as alginate and a Psl-like polysaccharide. To date, the Psl polysaccharide has only been studied in Pseudomonas aeruginosa, in which it plays an important role during biofilm development. However, its function in plant-associated bacteria is still unknown. To understand how these exopolysaccharides contribute to the biofilm matrix of PssUMAF0158, knockout mutants of genes encoding these putative exopolysaccharides were constructed. Flow-cell chamber experiments revealed that cellulose and the Psl-like polysaccharide constitute a basic scaffold for biofilm architecture in this bacterium. Curiously, the Psl-like polysaccharide of PssUMAF0158 plays a role in virulence similar to what has been described for cellulose. Finally, the impaired swarming motility of the Psl-like exopolysaccharide mutant suggests that this exopolysaccharide may play a role in the motility of PssUMAF0158 over the mango plant surface.

Pseudomonas syringae pv. syringae Associated With Mango Trees, a Particular Pathogen Within the "Hodgepodge" of the Pseudomonas syringae Complex.

The Pseudomonas syringae complex comprises different genetic groups that include strains from both agricultural and environmental habitats. This complex group has been used for decades as a “hodgepodge,” including many taxonomically related species. More than 60 pathovars of P. syringae have been described based on distinct host ranges and disease symptoms they cause. These pathovars cause disease relying on an array of virulence mechanisms. However, P. syringae pv. syringae (Pss) is the most polyphagous bacterium in the P. syringae complex, based on its wide host range, that primarily affects woody and herbaceous host plants. In early 1990s, bacterial apical necrosis (BAN) of mango trees, a critical disease elicited by Pss in Southern Spain was described for the first time. Pss exhibits important epiphytic traits and virulence factors, which may promote its survival and pathogenicity in mango trees and in other plant hosts. Over more than two decades, Pss strains isolated from mango trees have been comprehensively investigated to elucidate the mechanisms that governs their epiphytic and pathogenic lifestyles. In particular, the vast majority of Pss strains isolated from mango trees produce an antimetabolite toxin, called mangotoxin, whose leading role in virulence has been clearly demonstrated. Moreover, phenotypic, genetic and phylogenetic approaches support that Pss strains producers of BAN symptoms on mango trees all belong to a single phylotype within phylogroup 2, are adapted to the mango host, and produce mangotoxin. Remarkably, a genome sequencing project of the Pss model strain UMAF0158 revealed the presence of other factors that may play major roles in its different lifestyles, such as the presence of two different type III secretion systems, two type VI secretion systems and an operon for cellulose biosynthesis. The role of cellulose in increasing mango leaf colonization and biofilm formation, and impairing virulence of Pss, suggests that cellulose may play a pivotal role with regards to the balance of its different lifestyles. In addition, 62-kb plasmids belonging to the pPT23A-family of plasmids (PFPs) have been strongly associated with Pss strains that inhabit mango trees. Further, complete sequence and comparative genomic analyses revealed major roles of PFPs in detoxification of copper compounds and ultraviolet radiation resistance, both improving the epiphytic lifestyle of Pss on mango surfaces. Hence, in this review we summarize the research that has been conducted on Pss by our research group to elucidate the molecular mechanisms that underpin the epiphytic and pathogenic lifestyle on mango trees. Finally, future directions in this particular plant–pathogen story are discussed.

Pantoea agglomerans as a New Etiological Agent of a Bacterial Necrotic Disease of Mango Trees.

Bacterial apical necrosis of mango trees, a disease elicited by Pseudomonas syringae pv. syringae, is a primary limiting factor of mango crop production in the Mediterranean region. In this study, a collection of bacterial isolates associated with necrotic symptoms in mango trees similar to those produced by bacterial apical necrosis disease were isolated over five consecutive years in orchards from the Canary Islands. The bacterial isolates were characterized and identified as Pantoea agglomerans. Pathogenicity tests conducted on onion bulbs and mango plants confirmed that P. agglomerans strains isolated from mango trees are a new etiological agent of a bacterial necrotic disease in the Canary Islands. Pathogenicity plasmids of the pPATH family have been previously reported in P. agglomerans. The majority of putatively pathogenic (n = 23) and pathogenic (n = 4) P. agglomerans strains isolated from mango trees harbored four plasmids, one of which was close in size to the 135-kb pPATH pathogenicity plasmid. The analysis of the presence of two major genes in pPATH plasmids (repA and hrpJ) was undertaken in P. agglomerans strains isolated from mango trees. The hrpJ gene was detected in the 140-kb plasmid of pathogenic P. agglomerans strains isolated from mango trees but it showed differences in nucleotide sequences compared with other pathogenic strains. In contrast, the repA gene was not detected in any of the putatively pathogenic and pathogenic P. agglomerans strains isolated from mango trees. Finally, genetic characterization and phylogenetic analysis using the hrpJ gene and the housekeeping genes gyrB and rpoB showed that almost all P. agglomerans strains that were putatively pathogenic and pathogenic on mango trees clustered together, forming a differentiated phylogroup with respect to the other pathogenic P. agglomerans strains described from other hosts.

First Report of Bacterial Dry Rot of Mango Caused by Sphingomonas sanguinis in China

First Report of Bacterial Dry Rot of Mango Caused by <i>Sphingomonas sanguinis</i> in China

First Report of Bacterial Apical Necrosis of Mango Caused by Pseudomonas syringae pv. syringae in Greece

The mango, Mangifera indica L., native to India and Southeast Asia, is grown throughout the tropics and subtropics and marketed as fresh or processed fruit contributing greatly to those countries’ income. According to FAO database, from 2007 to 2012 the mango production in Greece reached the 438 tonnes, an increase of 338% indicating a strong economic potential for the cultivation. During spring of 2014 and 2015, symptoms similar to that of the bacterial apical necrosis disease caused by Pseudomonas syringae pv. syringae (Pss) were observed on commercial mango orchards planted with cv Irwin throughout two growing areas in Chania distinct, Crete, Greece. The disease was characterized by rapidly expanding necrotic lesions on leaves, buds, stems and floral panicles, whereas fruits were seemed to be un-affected. Lesions on leaves started as interveinal, angular, water soaked spots which may coalesce, becoming black and slightly raised (Figure 1). Isolations were made from affected buds, leaf petioles, and nec...

CHARACTERIZATION OF PSEUDOMONAS SYRINGAE pv. SYRINGAE ISOLATED FROM MANGO IN SICILY AND OCCURRENCE OF COPPER-RESISTANT STRAINS

Mango (Mangifera indica) is grown throughout a wide range of frost-free climates and is one of the world’s most important fruit crops. During 2010-2014, severe symptoms of bacterial apical necrosis (BAN) caused by Pseudomonas syringae pv. syringae (Pss) were observed on mango plants throughout all mango-growing areas in Sicily (southern Italy). The causal agent was identified based on phenotypic, genotypic and pathogenicity tests. The genetic variability of strains obtained from different areas and cultivars were assessed by rep- PCR and allowed clear differentiation of the Pss strains isolated from mango from other representative strains of the pathovar. In addition, the respective production of syringomycin, syringopeptin and mangotoxin was checked by in vitro tests and PCR detection. All tested strains showed the presence of mgoA and mgoB of the mangotoxin operon. Some copper- resistant Pss strains showing cusCBA genes were also found in some orchards. This feature could explain the failure of the disease control using copper compounds. Among 71 strains tested using in vitro assay, no copper-sensitive strains were detected, whereas 44 strains (62%) had high resistance, 16 strains (22.5%) were resistant and 11 strains (15.5%) had low resistance to copper sulfate. To our knowledge, this is the first report of copper resistance among Pss strains causing BAN on mango in Sicily.

Cellulose production in Pseudomonas syringae pv. syringae: a compromise between epiphytic and pathogenic lifestyles.

Genome sequencing and annotation have revealed a putative cellulose biosynthetic operon in the strain Pseudomonas syringae pv. syringae UMAF0158, the causal agent of bacterial apical necrosis. Bioinformatics analyses and experimental methods were used to confirm the functionality of the cellulose biosynthetic operon. In addition, the results showed the contribution of the cellulose operon to important aspects of P. syringae pv. syringae biology, such as the formation of biofilms and adhesion to the leaf surface of mango, suggesting that this operon increases epiphytic fitness. However, based on the incidence and severity of the symptoms observed in tomato leaflets, cellulose expression reduces virulence, as cellulose-deficient mutants increased the area of necrosis, whereas the cellulose-overproducing strain decreased the area of necrosis compared with the wild type. In conclusion, the results of this study show that the epiphytic and pathogenic stages of the P. syringae pv. syringae UMAF0158 lifestyle are intimately affected by cellulose production.

A Pseudomonas syringae Diversity Survey Reveals a Differentiated Phylotype of the Pathovar syringae Associated with the Mango Host and Mangotoxin Production

Pseudomonas syringae pv. syringae, the causal agent of bacterial apical necrosis (BAN) in mango crops, has been isolated in different mango-producing areas worldwide. An extensive collection of 87 P. syringae pv. syringae strains isolated from mango trees affected by BAN from different countries, but mainly from Southern Spain, were initially examined by repetitive sequence-based polymerase chain reaction (rep-PCR) to analyze the genetic diversity with an epidemiological aim. rep-PCR was powerful in assessing intrapathovar distribution and also allowing clustering of the P. syringae pv. syringae strains isolated from mango, depending on the isolation area. A clear pattern of clustering was observed for all the P. syringae pv. syringae strains isolated from mango distinct from strains from other hosts, including strains for the same geographical regions as the mango isolates. For this reason, a representative group of 51 P. syringae pv. syringae strains isolated from mango and other hosts, as well as some P. syringae strains from other pathovars, were further characterized to determine their possible genetic, phenotypic, and phylogenetic relationships. Similar to the rep-PCR results, the randomly amplified polymorphic DNA PCR (RAPD-PCR) and catabolic diversity analysis using the Biolog GN2 profile grouped 90% of the mango isolates together in a unique cluster. Interestingly, the majority of P. syringae pv. syringae strains isolated from mango produced mangotoxin. The analysis of the phylogenetic distribution using the multilocus sequence typing analysis strongly supports the existence of a differentiated phylotype of the pathovar syringae mainly associated with the mango host and characterized by the mangotoxin production.

Characterisation of the mgo operon in Pseudomonas syringae pv. syringae UMAF0158 that is required for mangotoxin production.

BackgroundMangotoxin is an antimetabolite toxin that is produced by strains of Pseudomonas syringae pv. syringae; mangotoxin-producing strains are primarily isolated from mango tissues with symptoms of bacterial apical necrosis. The toxin is an oligopeptide that inhibits ornithine N-acetyl transferase (OAT), a key enzyme in the biosynthetic pathway of the essential amino acids ornithine and arginine. The involvement of a putative nonribosomal peptide synthetase gene (mgoA) in mangotoxin production and virulence has been reported.ResultsIn the present study, we performed a RT-PCR analysis, insertional inactivation mutagenesis, a promoter expression analysis and terminator localisation to study the gene cluster containing the mgoA gene. Additionally, we evaluated the importance of mgoC, mgoA and mgoD in mangotoxin production. A sequence analysis revealed an operon-like organisation. A promoter sequence was located upstream of the mgoB gene and was found to drive lacZ transcription. Two terminators were located downstream of the mgoD gene. RT-PCR experiments indicated that the four genes (mgoBCAD) constitute a transcriptional unit. This operon is similar in genetic organisation to those in the three other P. syringae pathovars for which complete genomes are available (P. syringae pv. syringae B728a, P. syringae pv. tomato DC3000 and P. syringae pv. phaseolicola 1448A). Interestingly, none of these three reference strains is capable of producing mangotoxin. Additionally, extract complementation resulted in a recovery of mangotoxin production when the defective mutant was complemented with wild-type extracts.ConclusionsThe results of this study confirm that mgoB, mgoC, mgoA and mgoD function as a transcriptional unit and operon. While this operon is composed of four genes, only the last three are directly involved in mangotoxin production.

Environmentally friendly treatment alternatives to Bordeaux mixture for controlling bacterial apical necrosis (BAN) of mango

Bacterial apical necrosis (BAN), caused by the bacterium Pseudomonas syringae pv. syringae (Pss), is currently the most limiting disease affecting the mango crop in the Mediterranean area. The copper‐based compound Bordeaux mixture (BM) is considered to be the conventional treatment against BAN, but it does not act as a bactericide. Alternative experimental treatments to BM that are compatible with organic farming were tested for their ability to control BAN disease. Field trials were conducted over six seasons in different mango orchards with natural infestation of Pss. The experimental treatments included applications of Silicon gel (a commercial formulation of aqueous potassium silicate), dicalcium phosphate, Kaolinite, and Ulmasud B® (bentonite, powder); BM was applied as the conventional treatment. During the first two seasons (small‐scale trial, 2002–2004), all these experimental compounds were applied in order to select those treatments providing the greatest reduction of BAN symptoms. In the next three seasons (2005–2008), a semi‐commercial scale trial was carried out with the best‐performing treatments, resulting in the selection of Silicon gel. Finally, Silicon gel was tested in a commercial scale trial during the last season (2008–2009). Trees treated with Silicon gel showed significantly fewer necrotic buds and leaves, reaching disease levels very similar to those using the conventional treatment with BM. However, minor differences in P. syringae‐like population levels were observed, as has been described in previous studies. The possible mode of action of the Silicon gel is discussed. Currently, the Silicon gel compound is undergoing the registration process for its commercial use in mango management in Spain.

A Nonribosomal Peptide Synthetase Gene (mgoA) of Pseudomonas syringae pv. syringae Is Involved in Mangotoxin Biosynthesis and Is Required for Full Virulence

Pseudomonas syringae pv. syringae, which causes the bacterial apical necrosis of mango, produces the antimetabolite mangotoxin. We report here the cloning, sequencing, and identity analysis of a chromosomal region of 11.1 kb from strain P syringae pv. syringae UMAF0158, which is involved in mangotoxin biosynthesis. This chromosomal region contains six complete open reading frames (ORFs), including a large gene (ORF5) with a modular architecture characteristic of nonribosomal peptide synthetases (NRPS) named mgoA. A Tn5 mutant disrupted in mgoA was defective in mangotoxin production, revealing the involvement of the putative NRPS gene in the biosynthesis of mangotoxin. This derivative strain impaired in mangotoxin production also showed a reduction in virulence as measured by necrotic symptoms on tomato leaflets. Mangotoxin production and virulence were restored fully in the NRPS mutant by complementation with plasmid pCG2-6, which contains an 11,103-bp chromosomal region cloned from the wild-type strain P syringae pv. syringae UMAF0158 that includes the putative NPRS gene (mgoA). The results demonstrate that mgoA has a role in the virulence of P. syringae pv. syringae. The involvement of an NRPS in the production of an antimetabolite toxin from P. syringae inhibiting ornithine acetyltransferase activity is proposed.

Pseudomonas syringae Diseases of Fruit Trees: Progress Toward Understanding and Control.

Pseudomonas syringae Diseases of Fruit Trees: Progress Toward Understanding and Control.

Field evaluation of treatments for the control of the bacterial apical necrosis of mango (Mangifera indica) caused by Pseudomonas syringae pv. syringae

Bacterial apical necrosis is a critical disease in the main production area of mango in Europe. It is caused by Pseudomonas syringae pv. syringae, and produces necrotic lesions on mango buds and leaves, causing severe yield losses due to a decrease of flowering and fruit set. A field study to evaluate control treatments against bacterial apical necrosis was carried out during three seasons on mango trees cv. Tommy Atkins in Huelva (Spain). Experimental treatments included Bordeaux mixture, fosetyl-Al, acibenzolar-s-methyl, gibberelic acid, silicon gel, a mixture between acibenzolar-S-methyl and Bordeaux mixture, and combined applications of fosetyl-Al with Bordeaux mixture or silicon gel. The treatments which caused a consistent reduction in bacterial apical necrosis symptoms at similar levels to the conventional treatment with Bordeaux mixture, were the plant resistance activator acibenzolar-S-methyl and the phosphonate derivative fosetyl-Al applied singly or in combination with other compounds, which could be alternative treatments. These treatments showed a significant decrease in the necrotic buds and/or leaves numbers; however, minor differences in P. syringae-like population levels were observed. The analysis of the inhibitory and bactericidal concentrations of cupric compounds against P. syringae strains isolated from mango tissues suggests that the commercial copper-based treatments with Bordeaux mixture used in the management of mango crops do not work in a bactericidal mode of action.

Mangotoxin: a novel antimetabolite toxin produced by Pseudomonas syringae inhibiting ornithine/arginine biosynthesis

Pseudomonas syringae pv. syringae is a common inhabitant of a large variety of plants and the causal agent of bacterial apical necrosis of mango. In this work, we describe production and preliminary characterization of a novel antimetabolite toxin, designated mangotoxin, by P. syringae pv. syringae strains mainly isolated from mango trees. The toxin was detected by the Escherichia coli growth inhibition assay, since growth inhibition was reversed by l-ornithine and not by N-acetyl ornithine, suggesting that mangotoxin could interfere with this step of ornithine/arginine biosynthesis. Cell-free culture filtrates from different mangotoxin-producing strains strongly decreased, around 75%, the activity of ornithine N-acetyltransferase (OAT; EC 2.3.1.35) from tomato leaf protein extracts, confirming this enzyme as a target of the novel toxin. Biochemical data suggest that mangotoxin is a hydrophylic oligopeptide (<3 kDa) without a complex secondary structure (sensitive to proteases, but stable at high temperatures and extreme pH), as other antimetabolite toxins, such as phaseolotoxin or tabtoxin. HPLC analyses of cell-free culture filtrates allowed us to associate mangotoxin activity to a single peak obtained from a mangotoxin-producer strain but not present in filtrates from a derivative mutant defective in the toxic activity.

Copper Resistance in Pseudomonas syringae Strains Isolated from Mango Is Encoded Mainly by Plasmids

ABSTRACT Bacterial apical necrosis of mango, elicited by Pseudomonas syringae pv. syringae, limits fruit production in southern Spain and Portugal. Examination of a collection of P. syringae pv. syringae isolates for copper resistance showed that 59% were resistant to cupric sulfate. The survey of a mango orchard revealed an increase in frequencies of copper-resistant bacteria after repeated treatments with Bordeaux mixture. These data suggest that selection of copper-resistant strains could be a major reason for control failures following management with copper bactericides. Most copper-resistant isolates harbored plasmids, although the majority of them contained a 62-kb plasmid that also was present in copper-sensitive strains. The 62-kb plasmids were differentiated by restriction enzyme analysis and hybridization to copABCD DNA. The most frequently found copper-resistant plasmid type (62.1) was transferable by conjugation. Southern blot hybridizations showed that genetic determinants partially homologous to copABCD were present in all the copper-resistant strains examined, and usually were associated with plasmids; these determinants were not detected in copper-sensitive strains. The selective pressure exerted by copper bactericide sprays on the diversity of copper resistance determinants in bacterial populations of mango is discussed.