Medfly Ceratitis capitata as Potential Vector for Fire Blight Pathogen Erwinia amylovora: Survival and Transmission.

Monitoring the ability of bacterial plant pathogens to survive in insects is required for elucidating unknown aspects of their epidemiology and for designing appropriate control strategies. Erwinia amylovora is a plant pathogenic bacterium that causes fire blight, a devastating disease in apple and pear commercial orchards. Studies on fire blight spread by insects have mainly focused on pollinating agents, such as honeybees. However, the Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae), one of the most damaging fruit pests worldwide, is also common in pome fruit orchards. The main objective of the study was to investigate whether E. amylovora can survive and be transmitted by the medfly. Our experimental results show: i) E. amylovora can survive for at least 8 days inside the digestive tract of the medfly and until 28 days on its external surface, and ii) medflies are able to transmit the bacteria from inoculated apples to both detached shoots and pear plants, being the pathogen recovered from lesions in both cases. This is the first report on E. amylovora internalization and survival in/on C. capitata, as well as the experimental transmission of the fire blight pathogen by this insect. Our results suggest that medfly can act as a potential vector for E. amylovora, and expand our knowledge on the possible role of these and other insects in its life cycle.

Marinomonas alcarazii sp. nov., M. rhizomae sp. nov., M. foliarum sp. nov., M. posidonica sp. nov. and M. aquiplantarum sp. nov., isolated from the microbiota of the seagrass Posidonia oceanica.

Five novel Gram-reaction-negative aerobic marine bacterial strains with DNA G+C contents <50 mol% were isolated from the seagrass Posidonia oceanica. 16S rRNA sequence analysis indicated that they belonged to the genus Marinomonas. Major fatty acid compositions, comprising C10:0 3-OH, C16:0, C16:1ω7c and C18:1ω7c, supported the affiliation of these strains to the genus Marinomonas. Strains IVIA-Po-14b(T), IVIA-Po-145(T) and IVIA-Po-155(T) were closely related to Marinomonas pontica 46-16(T), according to phylogenetic analysis. However, DNA-DNA hybridization values <35 % among these strains revealed that they represented different species. Further differences in the phenotypes and minor fatty acid compositions were also found among the strains. Another two strains, designated IVIA-Po-181(T) and IVIA-Po-159(T), were found to be closely related to M. dokdonensis DSW10-10(T) but DNA-DNA relatedness levels <40 % in pairwise comparisons, as well as some additional differences in phenotypes and fatty acid compositions supported the creation of two novel species. Accordingly, strains IVIA-Po-14b(T )( = CECT 7730(T)  = NCIMB 14671(T)), IVIA-Po-145(T) ( = CECT 7377(T)  = NCIMB 14431(T)), IVIA-Po-155(T) ( = CECT 7731(T)  = NCIMB 14672(T)), IVIA-Po-181(T) ( = CECT 7376(T)  = NCIMB 14433(T)) and IVIA-Po-159(T) ( = CECT 7732(T)  = NCIMB 14673(T)) represent novel species, for which the names Marinomonas alcarazii sp. nov., Marinomonas rhizomae sp. nov., Marinomonas foliarum sp. nov., Marinomonas posidonica sp. nov. and Marinomonas aquiplantarum sp. nov. are proposed, respectively.

Exopolysaccharides favor the survival of Erwinia amylovora under copper stress through different strategies.

Erwinia amylovora causes fire blight, a destructive disease of rosaceous plants very difficult to control. We demonstrated that copper, employed to control plant diseases, induces the "viable-but-nonculturable" (VBNC) state in E. amylovora. Moreover, it was previously reported that copper increases production of its main exopolysaccharide (EPS), amylovoran. In this work, the copper-complexing ability of amylovoran and levan, other major EPS of E. amylovora, was demonstrated. Following this, EPS-deficient mutants were used to determine the role of these EPSs in survival of this bacterium in AB mineral medium with copper, compared to their wild type strain and AB without copper. Total, viable and culturable counts of all strains were monitored for six months. With copper, a larger fraction of the viable population of EPS mutants entered into the VBNC state, and earlier than their wild type strain, showing the contribution of both EPSs to long-term survival in a culturable state. Further, we demonstrated that both EPSs can be used as carbon source by E. amylovora under deprivation conditions. Overall, these previously unreported functions of amylovoran and levan provide survival advantages for E. amylovora, which could contribute to its enhanced persistence in nature.

Taxonomic study of Marinomonas strains isolated from the seagrass Posidonia oceanica, with descriptions of Marinomonas balearica sp. nov. and Marinomonas pollencensis sp. nov.

Novel aerobic, Gram-negative bacteria with DNA G+C contents below 50 mol% were isolated from the culturable microbiota associated with the Mediterranean seagrass Posidonia oceanica. 16S rRNA gene sequence analyses revealed that they belong to the genus Marinomonas. Strain IVIA-Po-186 is a strain of the species Marinomonas mediterranea, showing 99.77 % 16S rRNA gene sequence similarity with the type strain, MMB-1(T), and sharing all phenotypic characteristics studied. This is the first description of this species forming part of the microbiota of a marine plant. A second strain, designated IVIA-Po-101(T), was closely related to M. mediterranea based on phylogenetic studies. However, it differed in characteristics such as melanin synthesis and tyrosinase, laccase and antimicrobial activities. In addition, strain IVIA-Po-101(T) was auxotrophic and unable to use acetate. IVIA-Po-101(T) shared 97.86 % 16S rRNA gene sequence similarity with M. mediterranea MMB-1(T), but the level of DNA-DNA relatedness between the two strains was only 10.3 %. On the basis of these data, strain IVIA-Po-101(T) is considered to represent a novel species of the genus Marinomonas, for which the name Marinomonas balearica sp. nov. is proposed. The type strain is IVIA-Po-101(T) (=CECT 7378(T) =NCIMB 14432(T)). A third novel strain, IVIA-Po-185(T), was phylogenetically distant from all recognized Marinomonas species. It shared the highest 16S rRNA gene sequence similarity (97.4 %) with the type strain of Marinomonas pontica, but the level of DNA-DNA relatedness between the two strains was only 14.5 %. A differential chemotaxonomic marker of this strain in the genus Marinomonas is the presence of the fatty acid C(17 : 0) cyclo. Strain IVIA-Po-185(T) is thus considered to represent a second novel species of the genus, for which the name Marinomonas pollencensis sp. nov. is proposed. The type strain is IVIA-Po-185(T) (=CECT 7375(T) =NCIMB 14435(T)). An emended description of the genus Marinomonas is given based on the description of these two novel species, as well as other Marinomonas species described after the original description of the genus.

Survival strategy of Erwinia amylovora against copper: induction of the viable-but-nonculturable state.

Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu(2+) was inoculated with 10(7) CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper.