Phenotypic and genetic characterization of Pseudomonas syringae strains associated with the recent citrus bacterial blast and bacterial black pit epidemics in Tunisia

In the spring of 2012, symptoms of a disease resembling citrus blast and citrus black pit were observed in some orchards in Tunisia. The epidemic spread rapidly in the following years. Twenty‐four commercial citrus orchards from four Tunisian regions showing characteristic symptoms of bacterial diseases were surveyed during a 3‐year study. Eighty‐eight Pseudomonas‐like bacterial isolates were successfully obtained from the northeast and west of Tunisia. No isolates were recovered from the central region. Overall, 46 isolates were identified as Pseudomonas syringae pv. syringae and most of them showed similar phenotypic and genetic profiles. The virulence of three selected isolates differed from one plant cultivar to another as well as from the type of plant organ used for the inoculation. In a bioassay test, all isolates produced syringomycin, which was confirmed by molecular detection based on the syrB and syrD genes. Only EC122 possessed syrD but not syrB. DNA fingerprints, based on repetitive sequence‐based polymerase chain reaction (rep‐PCR) and PCR melting profile (PCR MP), were used to determine the potential genetic diversity among strains. Clustering of PCR MP fingerprinting data matched with rep‐PCR fingerprinting data. The generated distribution tree showed that Tunisian isolates were closely related to the citrus reference strain LMG5496. In contrast, EC112, isolated from citrus, and the almond isolate EC122 were distantly related to the type strain LMG1247^T isolated from lilac. Such studies have not been reported until now for P. syringae from citrus.     

Correction to: Phenotypic and marker-assisted characterization of new apple genotypes with high resistance to fire blight

European Journal of Plant Pathology - A Correction to this paper has been published: https://doi.org/10.1007/s10658-021-02315-7     

BTH-mediated antioxidant system responses in apple leaf tissues

BTH (S-methylbenzo-1,2,3-thiadiazole-7-carbothiate), an active compound of the commercial preparation Bion, has been studied as an elicitor of resistance to fire blight (Erwinia amylovora) in apple. However, the biochemical mechanisms of its action are not fully elucidated. Our study indicated that BTH at the best time of its protection activity (2–14 days after application) induced changes in prooxidant–antioxidant balance in the leaves of apple trees, but in different ways in the enzymatic and nonenzymatic antioxidants. Glutathione as low molecular antioxidant as well as superoxide anion radical and lipid peroxides as oxidants exhibited changes at the early phase of BTH action. Glutathione-dependent enzymes were strongly affected by the elicitor used. On the 2nd day glutathione transferase (GST) and glutathione peroxidase (GSH-Px) activities increased by about 70% and 30% above the control, respectively. GST activity normalized about the 14th day but GSH-Px at the same time showed 27% of the control value. Among enzymes utilising hydrogen peroxide only catalase showed increase (37%) at the early phase of experiment. Compared with the control, BTH-treated plants did not show changes in ascorbate peroxidase and phenylalanine ammonia-lyase activities. Tocopherol (TOC) level diminished starting from the 7th day after BTH treatment and on the 14th day it was only 28% of the control. It is proposed that extinguishing of BTH-mediated signal resulted from TOC and glutathione action. The diminished ascorbate level at all examined times may play a crucial role in BTH-mediated cell growth regulation. The direct influence of BTH on lipid metabolism should be also taken into consideration.     

Evaluation of growth response of phytopathogens Alternaria alternata,Diaporthe nobilis and Phytophthora plurivora to inhibitory potential of three essential oils of Monarda didyma genotypes

Journal of Plant Diseases and Protection - The growth response of three phytopathogens to inhibitory activity of essential oils (EOs), derived from three monarda (Monarda didyma L.) genotypes, was...     

Phylogenetic,genetic, and phenotypic diversity of Pseudomonas syringae pv. syringae strains isolated from citrus blast and black pit in Tunisia

Citrus blast and black pit caused by Pseudomonas syringae pv. syringae (Pss) is the only bacterial disease reported in Tunisian Citrus orchards. The phylogenetic relationship between Pss strains was studied based on multilocus sequence analysis (MLSA), using partial sequences of housekeeping genes rpoD, rpoB, gyrB, cts, and pfk for 14 representative Pss Citrus strains, including the reference strain LMG5496. The MLSA revealed that the studied Tunisian Citrus strains are closely related to LMG5496 and cluster in phylogroup 02. Based on the cts gene, the majority of Citrus strains clustered in clades “a” and “b”. However, five strains were placed in a newly defined clade “g”. We describe the presence of six different type III secreted effectors (T3SEs). These were found with frequencies of 100% for the effector hopAN1 and the helper hrpK1, 65% for hopT1-2, and 14% for hopN1, hopR1, and hopQ1-2. Investigation of copper resistance showed that 67% of our Pss Citrus strains from Tunisia are resistant to copper sulphate in vitro, and the copper resistance genes copABCDR were detected in 23% of the strains. Our results present new data concerning the genetic diversity and phylogeny, presence of T3SEs, and copper resistance within the Pss populations that affect Citrus in Tunisia.     

The use of PCR melting profile for typing of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> isolates from stone fruit trees

Of thirty fluorescent Pseudomonas isolates originating from symptomatic tissues of sweet (Prunus avium) and sour cherry (Prunus cerasus), plum (Prunus domestica), peach (Prunus persica) and apricot (Prunus armeniaca), 23 were identified as P. syringae using LOPAT tests. Further characterization of those isolates by GATTa and L-lactate utilization tests showed that 10 of them belonged to race 1, six to race 2 of P. syringae pv. morsprunorum (Psm) and six other isolates were identified as pathovar syringae (Pss). One isolate (791) was determined as atypical. Phenotypic determination and genetic analysis of studied isolates for toxin production revealed that isolates of Pss produced syringomycin, 3 Psm race 1 produced coronatine and 6 Psm race 2 produced yersiniabactin. Genetic diversity of all isolates was evaluated with the PCR melting profile (PCR MP) method. A dendrogram constructed with PCR MP patterns showed positive correlation with phenotypically distinguished pathovars. Isolates of Psm races 1 and 2 formed distinct, tight clusters, whereas Pss isolates were more heterogeneous. Isolate 791 was placed within Pss isolates. Bacteria identified as Pss caused more severe symptoms on immature cherry fruits compared to Psm, which corresponded to determined pathovars and races.     

Necrotrophic behaviour of Erwinia amylovora in apple and tobacco leaf tissue

An important issue related to the epidemiology of fire blight, a devastating disease of apples and pears, is how its causal agent, the bacterium Erwinia amylovora, survives and disseminates in the environment. Almost no information is available on the possibility of this pathogen overwintering as a necrotroph. In this study, bacterial survival in dead apple and tobacco (a non‐host) leaf tissues was addressed. In necrotized leaves collected 5, 6, 7 and 8 months following shoot inoculation of apple trees, viable E. amylovora cells were present in over 50% of samples from the midrib and in over 10% of samples from lateral veins, but were never found in parenchyma. Using a PCR‐based method, pathogen DNA was detected in more than 50% of samples that were found to be free of viable cells by conventional plating out. However, PCR analysis was insufficient to distinguish between the DNA of viable and dead bacteria. Sugars appropriate for bacterial growth were found in dead apple leaves. In spot‐inoculated attached apple and tobacco leaves, a remarkable increase in the bacterial population was observed in lesions that developed as a hypersensitive response (HR). As in other necrotrophic interactions, bacterial proliferation was associated with massive hydrogen peroxide production and progression toward plant cell death. The results indicate that E. amylovora has an ability to survive as a semi‐necrotroph or necrotroph, which allows for overwintering in dead apple leaves.