Dissemination of Pseudomonas syringae pv. actinidiae through pollen and its epiphytic life on leaves and fruit

The role of pollen in disseminating Pseudomonas syringae pv. actinidiae (Psa) in kiwifruit orchards was investigated and the survival of the pathogen as an epiphyte on leaves and fruits was followed, from pollination time until the pre-harvest season. Pollen with natural inoculum was obtained from an infected orchard and pollen harvested without any contamination by Psa was experimentally inoculated at approximately the same contamination level. Two pollination techniques were used in glasshouse experiments: dusting and spraying. In parallel, field trials in commercial orchards were carried out: two plots were designed in two orchards, where bacterial canker was present at low incidence. From petal fall to 3 weeks before harvesting, leaf and fruit samples were taken and analysed for the presence of Psa, using two different PCR protocols and direct isolation. Results confirmed the dissemination of Psa through pollen, especially when using the aqueous suspensions. Both in glasshouse experiments and in the orchards Psa was found as an epiphyte for several weeks after pollination. Pathogen populations on leaves were, initially, 10 to 100 times less than on fruitlets. As the summer continued, the epiphytic contamination levels of fruits decreased constantly, being no more detectable from early August, whereas Psa was present at detectable levels on leaves until early October, approx. 20 days before harvesting time. Our results confirmed the role of pollen in disseminating Psa, the long epiphytic survival of the pathogen on kiwifruit leaves and the increasing unsuitability of fruits to harbour detectable, epiphytic populations of the bacterium through the summer season in commercial orchards with low disease incidence, when they are reaching the final development stage. Thus, kiwifruit surfaces do not appear to be a suitable niche for a long term survival of Psa as an epiphyte and, therefore, kiwifruits should not represent a pathway for Psa dissemination and pose a negligible risk for the introduction of the pathogen into new areas

Pollen as a possible pathway for the dissemination of Pseudomonas syringae pv. actinidiae and bacterial canker of kiwifruit

Pollen collected in a kiwifruit orchard with symptoms of bacterial canker and naturally contaminated by Pseudomonas syringae pv. actinidiae (Psa), was used to pollinate an experimental orchard, in order to confirm its role, under commercial orchard conditions, in disseminating the pathogen and, possibly, contributing to disease spread. A pollen lot, certified free from Psa, was used with the same methods as a control. Two pollination techniques were used: dusting (dry pollen) and spraying (pollen suspension in water). The orchard was monitored during 2 years from experimental pollination, with regular sampling of flowers, fruits, leaves, and vines, to check for Psa as an epiphyte or endophyte, and for bacterial canker symptoms. Psa was recovered from flowers, fruitlets and leaves during the first season, mainly in plots where contaminated pollen had been sprayed in water suspension. From early August until harvesting time (mid-October), Psa detection was possible only on leaves. No symptoms developed during the first season after pollination. No endophytic Psa was detected in pruned vines in the following winter. During the second season, detection and isolation of Psa was erratic, but direct isolation was achieved from four plots. During the second season after pollination, typical leaf symptoms were observed on a few vines, and Psa was isolated and identified. Our results suggest that Psa could be disseminated via contaminated kiwifruit pollen as a pathway for spread of bacterial canker. However, further pollination experiments are needed to establish, beyond any doubt, whether contaminated pollen may contribute to possible disease outbreaks

Plant Growth Promoting and Biocontrol Activity of Streptomyces spp. as Endophytes

There has been many recent studies on the use of microbial antagonists to control diseases incited by soilborne and airborne plant pathogenic bacteria and fungi, in an attempt to replace existing methods of chemical control and avoid extensive use of fungicides, which often lead to resistance in plant pathogens. In agriculture, plant growth-promoting and biocontrol microorganisms have emerged as safe alternatives to chemical pesticides. Streptomyces spp. and their metabolites may have great potential as excellent agents for controlling various fungal and bacterial phytopathogens. Streptomycetes belong to the rhizosoil microbial communities and are efficient colonizers of plant tissues, from roots to the aerial parts. They are active producers of antibiotics and volatile organic compounds, both in soil and in planta, and this feature is helpful for identifying active antagonists of plant pathogens and can be used in several cropping systems as biocontrol agents. Additionally, their ability to promote plant growth has been demonstrated in a number of crops, thus inspiring the wide application of streptomycetes as biofertilizers to increase plant productivity. The present review highlights Streptomyces spp.-mediated functional traits, such as enhancement of plant growth and biocontrol of phytopathogens

Dissemination of Pseudomonas syringae pv. actinidiae through pollen and its epiphytic life on leaves and fruits

The role of pollen in disseminating Pseudomonas syringae pv. actinidiae (Psa ) in kiwifruit orchards was investigated and the survival of the pathogen as an epiphyte on leaves and fruits was followed, from pollination time until the pre-harvest season. Pollen with natural inoculum was obtained from an infected orchard and pollen harvested without any contamination by Psa was experimentally inoculated at approximately the same contamination level. Two pollination techniques were used in glasshouse experiments: dusting and spraying. In parallel, field trials in commercial orchards were carried out: two plots were designed in two orchards, where bacterial canker was present at low incidence. From petal fall to 3 weeks before harvesting, leaf and fruit samples were taken and analysed for the presence of Psa, using two different PCR protocols and direct isolation. Results confirmed the dissemination of Psa through pollen, especially when using the aqueous suspensions. Both in glasshouse experiments and in the orchards Psa was found as an epiphyte for several weeks after pollination. Pathogen populations on leaves were, initially, 10 to 100 times less than on fruitlets. As the summer continued, the epiphytic contamination levels of fruits decreased constantly, being no more detectable from early August, whereas Psa was present at detectable levels on leaves until early October, approx. 20 days before harvesting time. Our results confirmed the role of pollen in disseminating Psa, the long epiphytic survival of the pathogen on kiwifruit leaves and the increasing unsuit­ability of fruits to harbour detectable, epiphytic populations of the bacterium through the summer season in commercial orchards with low disease incidence, when they are reaching the final development stage. Thus, kiwifruit surfaces do not appear to be a suitable niche for a long term survival of Psa as an epiphyte and, therefore, kiwifruits should not represent a pathway for Psa dissemination and pose a negligible risk for the introduction of the pathogen into new areas

In vitro characterization of plant growth promoting and biocontrol activity of beneficial microorganisms

Plant roots are associated with numerous and diverse types of beneficial and pathogenic microorganisms. Among them, plant growth\u2013promoting (rhizo)bacteria (PGPB or PGPR) are isolated from plants crops worldwide, and many of them are used as agricultural inoculants. Agricultural biofertilization and biocontrol of pathogens are eco-friendly alternatives to chemical usage and have less energy, environmental, and economic costs. PGPB isolation and evaluation are essentials steps for determining bacteria that could improve plant development and productivity. In the present study three Streptomyces sp. strains SB14, SA51 & SL81, two Pseudomonas sp. strains PT65 & PN53, an Agrobacterium sp. strain AR39 and an internal control (IC) Pseudomonas synxantha were evaluated in vitro for different plant growth promoting and biocontrol activities. The results were aimed to identify possible antagonists able to inhibit different plant bacterial (Xanthomonas vesicatoria, Clavibacter michiganensis subsp. michiganensis, Clavibacter michiganensis subsp. sepedonicus, Acidovorax citrulli and Ralstonia solanacearum) and fungal (Rhizoctonia solani, Sclerotium sp., Fusarium oxysporum, Alternaria solani and Monilia laxa) pathogens. All the strains were screened for biocontrol activity on three different media\u2019s and AIA (average inhibition area) was calculated. Among the isolates, each strain showed different ability to inhibit the pathogens: Streptomyces sp. strain SA51 was found to be most active. The most prospective strains SA51, AR39 and DLS65 were further evaluated in the field, as possible biocontrol agents for the tomato spot disease (X. vesicatoria), singularly and as a consortium. Results will improve our understanding on the use of such microbial biocontrol agents and will implement innovative biocontrol strategies to bacterial diseases

AN INSIGHT IN SOME POPULATION FEATURES OF XANTHOMONAS ARBORICOLA pv. JUGLANDIS

Xanthomonas arboricola pv. juglandis (Xaj) is the causal agent of bacterial blight of walnut, an emerging disease, which has the potential to severely affect walnut orchards. An Italian strain collection of Xaj, obtained during the past 3 years from affected orchards in Romagna, was first assayed with conventional PCR with XajF/XajR primer pair developed to confirm strain identity. The population structure of the collection of Xaj isolates, confirms the presence of different genetic groups identified by rep-PCR (using Italian collection are currently being analysed by MLSA (multi locus sequence analysis), using 7 primers for 7 different housekeeping genes, with the purpose to better characterise the Italian isolates for phylotyping. The study of copper resistance on a wide collection of over 150 Xaj strains frequently showed high resistance (up to 500 ppm Cu++): two strains have been further studied confirming the presence of chromosomal genes copA and copB involved in the general copABCD copper resistance structure, as described for Pseudomonas syringae. Sequencing and comparing with other Xanthomonads were done. The elucidation of Xaj population structure may help to deeper investigate some additional aspects of the molecular epidemiology of the disease, thus allowing a better control strategy in the field. the REP, BOX and ERIC primers) and by multilocus sequence typing (MLST) and multilocus variable number analysis of tandem repeat (MLVA). Xaj and Xaj-like bacterial isolates from th

Factors influencing the detection of Acidovorax citrulli in naturally contaminated cucurbitaceous seeds by PCR-based assays.

The success of Acidovorax citrulli detection by seed health testing of commercial cucurbitaceous seeds depends on the efficiency of pathogen extraction. In the present study, different extraction techniques were compared to identify factors that influence A. citrulli detection in naturally contaminated cucurbit seeds. Embryo-infected watermelon seeds, produced by pistil inoculation, were mixed with non-infected seeds to generate samples (n = 1000 seeds) with low infection levels (approximately 104 cells per sample). Additionally, two naturally infested melon seed lots were tested. A. citrulli was extracted from seeds by soaking or crushing, followed by one or two centrifugation steps. Samples extracted by soaking seeds yielded better amplification efficiency (103%) compared with crushing (93%), as determined by quantitative real-time PCR analysis. This was most likely due to a reduction of the concentration of inhibitors present in the DNA samples. PCR assays using three different A. citrulli-specific primer sets highlighted that soaking followed by two centrifugation steps enhanced pathogen detection (100% of the samples) and the mean cycle threshold (Ct) value was significantly lower than those observed for the other pathogen extraction techniques. These results indicate that the optimised extraction protocol combined with PCR analysis can improve routine seed health testing for A. citrulli

Pollen as a possible pathway for the dissemination of Pseudomonas syringae pv. actinide and bacterial canker of kiwifruit

Pollen collected in a kiwifruit orchard with symptoms of bacterial canker and naturally contaminated by Pseudomonas syringae pv. actinidiae (Psa), was used to pollinate an experimental orchard, in order to confirm its role, under commercial orchard conditions, in disseminating the pathogen and, possibly, contributing to disease spread. A pollen lot, certified free from Psa, was used with the same methods as a control. Two pollination techniques were used: dusting (dry pollen) and spraying (pollen suspension in water). The orchard was monitored during 2 years from experimental pollination, with regular sampling of flowers, fruits, leaves, and vines, to check for Psa as an epiphyte or endophyte, and for bacterial canker symptoms. Psa was recovered from flowers, fruitlets and leaves during the first season, mainly in plots where contaminated pollen had been sprayed in water suspension. From early August until harvesting time (mid-October), Psa detection was possible only on leaves. No symptoms developed during the first season after pollination. No endophytic Psa was detected in pruned vines in the following winter. During the second season, detection and isolation of Psa was erratic, but direct isolation was achieved from four plots. During the second season after pollination, typical leaf symptoms were observed on a few vines, and Psa was isolated and identified. Our results suggest that Psa could be disseminated via contaminated kiwifruit pollen as a pathway for spread of bacterial canker. However, further pollination experiments are needed to establish, beyond any doubt, whether contaminated pollen may contribute to possible disease outbreaks

Virulence - associated genes in Avian Pathogenic Escherichia coli of turkey

50 Escherichia coli (APEC-Avian Pathogenic Escherichia coli) strains and 15 E. coli (AFEC-Avian Faecal Escherichia coli) from turkeys affected by colibacillosis and from healthy turkeys were tested for the presence of eight different virulence-associated genes. Besides, APEC were serotyped. O78 has been the most detected serotyped. The presence of the tested virulence genes was prevalently related to the APEC isolates. With reference to serogroup, all the tested O78 resulted iss and irp2 positive. Besides, tsh e cva/cvi were respectively present in 88.9 and 83.3% of O78. Nevertheless, the finding of a not typeable strains equipped with all the eight tested virulence genes among the APEC isolates suggest the importance of a careful and complete characterisation of the isolate to evaluate the real potential pathogenic attitude of the bacterium