Giorgio Mariano Balestra

Pseudomonas syringae pv. actinidiae (PSA) isolates from recent bacterial canker of kiwifruit outbreaks belong to the same genetic lineage.

Intercontinental spread of emerging plant diseases is one of the most serious threats to world agriculture. One emerging disease is bacterial canker of kiwi fruit (Actinidia deliciosa and A. chinensis) caused by Pseudomonas syringae pv. actinidiae (PSA). The disease first occurred in China and Japan in the 1980s and in Korea and Italy in the 1990s. A more severe form of the disease broke out in Italy in 2008 and in additional countries in 2010 and 2011 threatening the viability of the global kiwi fruit industry. To start investigating the source and routes of international transmission of PSA, genomes of strains from China (the country of origin of the genus Actinidia), Japan, Korea, Italy and Portugal have been sequenced. Strains from China, Italy, and Portugal have been found to belong to the same clonal lineage with only 6 single nucleotide polymorphisms (SNPs) in 3,453,192 bp and one genomic island distinguishing the Chinese strains from the European strains. Not more than two SNPs distinguish each of the Italian and Portuguese strains from each other. The Japanese and Korean strains belong to a separate genetic lineage as previously reported. Analysis of additional European isolates and of New Zealand isolates exploiting genome-derived markers showed that these strains belong to the same lineage as the Italian and Chinese strains. Interestingly, the analyzed New Zealand strains are identical to European strains at the tested SNP loci but test positive for the genomic island present in the sequenced Chinese strains and negative for the genomic island present in the European strains. Results are interpreted in regard to the possible direction of movement of the pathogen between countries and suggest a possible Chinese origin of the European and New Zealand outbreaks.

A Multiplex PCR Assay for Detection of Pseudomonas syringae pv. actinidiae and Differentiation of Populations with Different Geographic Origin

Pseudomonas syringae pv. actinidiae is responsible for severe outbreaks of bacterial canker of kiwifruit currently occurring around the world. Although molecular detection methods have been reported, none provide complete selectivity for this pathovar or discriminate among pathogen haplotypes. Therefore, a new multiplex polymerase chain reaction (PCR) assay was developed and validated. The assay was tested on 32 P. syringae pv. actinidiae isolates and 15 non-P. syringae pv. actinidiae strains and correctly assigned P. syringae pv. actinidiae strains to three different haplotypes: a Japanese/Korean group, a European group, and a Chinese group. Two P. syringae pv. actinidiae isolates from New Zealand were found to belong to the Chinese group whereas two other isolates from New Zealand, which were isolated from kiwifruit plants but which do not cause bacterial canker, tested negative. The described PCR assays has a limit of detection of approximately 5 to 50 pg of purified DNA or of 5 × 102 bacteria/PCR and were shown to work with both artificially and naturally infected plant tissues. Thus, the described method represents a suitable tool for detection of P. syringae pv. actinidiae and haplotype attribution, in particular, when testing a high number of samples during surveillance and prevention activities.

A framework to gauge the epidemic potential of plant pathogens in environmental reservoirs: the example of kiwifruit canker

New economically important diseases on crops and forest trees emerge recurrently. An understanding of where new pathogenic lines come from and how they evolve is fundamental for the deployment of accurate surveillance methods. We used kiwifruit bacterial canker as a model to assess the importance of potential reservoirs of new pathogenic lineages. The current kiwifruit canker epidemic is at least the fourth outbreak of the disease on kiwifruit caused by Pseudomonas syringae in the mere 50 years in which this crop has been cultivated worldwide, with each outbreak being caused by different genetic lines of the bacterium. Here, we ask whether strains in natural (non-agricultural) environments could cause future epidemics of canker on kiwifruit. To answer this question, we evaluated the pathogenicity, endophytic colonization capacity and competitiveness on kiwifruit of P. syringae strains genetically similar to epidemic strains and originally isolated from aquatic and subalpine habitats. All environmental strains possessing an operon involved in the degradation of aromatic compounds via the catechol pathway grew endophytically and caused symptoms in kiwifruit vascular tissue. Environmental and epidemic strains showed a wide host range, revealing their potential as future pathogens of a variety of hosts. Environmental strains co-existed endophytically with CFBP 7286, an epidemic strain, and shared about 20 virulence genes, but were missing six virulence genes found in all epidemic strains. By identifying the specific gene content in genetic backgrounds similar to known epidemic strains, we developed criteria to assess the epidemic potential and to survey for such strains as a means of forecasting and managing disease emergence.

Bacterial Populations Related to Gerbera ("Gerbera jamesonii" L.) Stem Break

Bacterial distribution, both external (epiphytic) and internal (endophytic), on Gerbera jamesonii L. cv. Provence and its relationship to gerbera stem break and ethylene production were investigated. The greatest number of epiphytic bacteria was found at capitulum level and 20 cm below. Three genera of bacteria were identified: Acinetobacter, Bacillus and Pantoea. A silver-nitrate solution greatly reduced ethylene production in cut flowers. The use of acid fuchsin solution revealed an occlusion of the xylem vessels, probably due to bacterial cells. The bacteria Acinetobacter, Pantoea and Bacillus appeared to be involved in stem break once their populations reached 105 cfu g-1 of stem tissue.

Comparison and utilization of different PCR-based approaches for molecular typing of Pseudomonas syringae pv. actinidiae strains from Italy

Abstract Over the last few years, a severe bacterial disease affecting kiwifruit has been reported in Italy. Its causal agent was identified as Pseudomonas syringae pv. actinidiae. For epidemiological purposes, we evaluated the discriminatory power of various PCR-based fingerprinting approaches. Profiles were generated by rep-PCR ((GTG)5-PCR), IS50-PCR and RAPD, from a wide collection of Italian, Korean and Japanese strains. The genetic relationships between strains of pv. actinidiae and strains from related pathovars were investigated. No differences between Italian strains were identified. However, some differences were identified between Italian strains and strains from Japan and Korea. The other pathovars clustered separately, and pv. theae was always more closely related to pv. actinidiae than the others. In general, the results obtained with the different methods were in good agreement, with RAPD showing the highest values of divergence between groups.

The Pseudomonas viridiflava phylogroups in the P. syringae species complex are characterized by genetic variability and phenotypic plasticity of pathogenicity-related traits.

As a species complex, Pseudomonas syringae exists in both agriculture and natural aquatic habitats. P.viridiflava, a member of this complex, has been reported to be phenotypically largely homogenous. We characterized strains from different habitats, selected based on their genetic similarity to previously described P.viridiflava strains. We revealed two distinct phylogroups and two different kinds of variability in phenotypic traits and genomic content. The strains exhibited phase variation in phenotypes including pathogenicity and soft rot on potato. We showed that the presence of two configurations of the Type III Secretion System [single (S-PAI) and tripartite (T-PAI) pathogenicity islands] are not correlated with pathogenicity or with the capacity to induce soft rot in contrast to previous reports. The presence/absence of the avrE effector gene was the only trait we found to be correlated with pathogenicity of P.viridiflava. Other Type III secretion effector genes were not correlated with pathogenicity. A genomic region resembling an exchangeable effector locus (EEL) was found in S-PAI strains, and a probable recombination between the two PAIs is described. The ensemble of the variability observed in these phylogroups of P.syringae likely contributes to their adaptability to alternating opportunities for pathogenicity or saprophytic survival.

Effect of cellulose and lignin on disintegration, antimicrobial and antioxidant properties of PLA active films

This study reports the effects on antimicrobial, antioxidant, migration and disintegrability activities of ternary nanocomposite films based on poly(lactic acid) incorporating two biobased nanofillers, (cellulose nanocrystals (CNC) and lignin nanoparticles (LNP)), in two different amounts (1 and 3% wt.). Results from antimicrobial tests revealed a capacity to inhibit the Gram negative bacterial growth of Xanthomonas axonopodis pv. vesicatoria and Xanthomonas arboricola pv. pruni along the time, offering innovative opportunities against dangerous bacterial plant pathogens. LNP proved to be highly efficient in antioxidation activity, based on the disappearance of the absorption band at 517nm of the free radical, 2,2-diphenyl-1-picrylhydrazyl (DPPH) upon reduction by an antiradical compound; moreover the combination of LNP and CNC generates a synergistic positive effect in the antioxidation response of PLA ternary films. Furthermore, all the studied formulations showed a disintegrability value up to 90% after 15days of incubation in composting conditions. Migration results showed that the films can be considered suitable for application in food packaging field.

Cellulose nanocrystals from Actinidia deliciosa pruning residues combined with carvacrol in PVA_CH films with antioxidant/antimicrobial properties for packaging applications

Kiwi Actinidia deliciosa pruning residues were here used for the first time as precursors for the extraction of high performing cellulose nanocrystals (CNC) by applying a bleaching treatment followed by an acidic hydrolysis. The resultant cellulosic nanostructures, obtained by an optimize extraction procedure (0.7% wt/v two times of sodium chlorite NaClO2) followed by an hydrolysis step, were then used as reinforcements phases in poly(vinyl alcohol) (PVA) blended with natural chitosan (CH) based films and also combined, for the first time, with carvacrol used here as active agent. Morphological and optical characteristics, mechanical response, thermal and migration properties, moisture content and antioxidant and antimicrobial assays were conducted. The morphological, optical and colorimetric results underlined that no particular alterations were induced on the transparency and color of PVA and PVA_CH blend by the presence of CNC and carvacrol, while they were able to modulate the mechanical responses, to induce antioxidant activities maintaining the migration levels below the permitted limits and suggesting the possible application in industrial sectors. Finally, inhibitions on bacterial development were detected for multifunctional systems, suggesting their protective function against microorganisms contamination.

Development of a Multiple Loci Variable Number of Tandem Repeats Analysis (MLVA) to Unravel the Intra-Pathovar Structure of Pseudomonas syringae pv. actinidiae Populations Worldwide

The bacterial canker of kiwifruit by Pseudomonas syringae pv. actinidiae is an emblematic example of a catastrophic disease of fruit crops. In 2008 a new, extremely virulent form of the pathogen emerged and rapidly devastated many Actinidia spp. orchards all over the world. In order to understand differences in populations within this pathovar and to elucidate their diffusion and movements on world scale, it is necessary to be able to quickly and on a routine basis compare new isolates with previous records. In this report a worldwide collection of 142 strains was analyzed by MLVA, chosen as investigative technique for its efficacy, reproducibility, simplicity and low cost. A panel of 13 Variable Number of Tandem Repeats (VNTR) loci was identified and used to describe the pathogen population. The MLVA clustering is highly congruent with the population structure as previously established by other molecular approaches including whole genome sequencing and correlates with geographic origin, time of isolation and virulence. For convenience, we divided the VNTR loci in two panels. Panel 1 assay, using six loci, recognizes 23 different haplotypes, clustered into ten complexes with highest congruence with previous classifications. Panel 2, with seven VNTR loci, provides discriminatory power. Using the total set of 13 VNTR loci, 58 haplotypes can be distinguished. The recent hypervirulent type shows very limited diversity and includes, beside the strains from Europe, New Zealand and Chile, a few strains from Shaanxi, China. A broad genetic variability is observed in China, but different types are also retrievable in Japan and Korea. The low virulent strains cluster together and are very different from the other MLVA genotypes. Data were used to generate a public database in MLVAbank. MLVA represents a very promising first-line assay for large-scale routine genotyping, prior to whole genome sequencing of only the most relevant samples.

Influence of Nitrogen Fertilization on the Colonization of Olive Phylloplane by Pseudomonas syringae subsp. savastanoi.

P. s. subsp. savastanoi (Smith) Young et al. is the causai agent of one of the most common disease of oiive (Olea europea L.) and oieander (Nerium oleander L.) plants. On olive plant the disease is known as “knot” or “tubercle”. Populations of the pathogen were tested to verify the influence of calcium nitrate on their ability to colonize olive phyiloplane. Olive plants, one-year young, cv. Nocellara del Belice, have been utilized in pot in greenhouse. Two different levels of nitrate were used: 0,65 g/l (low concentration) and 1,3 g/l (high concentration); as control, olive plants have been utilized without fertilization. The inoculum (108 cfu/ml) has been spreaded for each thesis, as follow; (a) 48 h after fertilization; (b) 48 h before fertilization; (c) at the same time with fertilization; (d) 7 days after fertilization. Nitrogen fertilization show a positive effect on the colonization of olive phylloplane by P. s. subsp. savastanoi. Indipendently by doses, calcium nitrate distributed before the inoculum is the most effective on colonization of olive phylioplane by P. s. subsp. savastanoi. The trend of inoculum without nitrogen fertilization, following an initial increment shows a decrease.