Marco Scortichini

The Kiwifruit Emerging Pathogen Pseudomonas syringae pv. actinidiae Does Not Produce AHLs but Possesses Three LuxR Solos

Pseudomonas syringae pv. actinidiae (Psa) is an emerging phytopathogen causing bacterial canker disease in kiwifruit plants worldwide. Quorum sensing (QS) gene regulation plays important roles in many different bacterial plant pathogens. In this study we analyzed the presence and possible role of N-acyl homoserine lactone (AHL) quorum sensing in Psa. It was established that Psa does not produce AHLs and that a typical complete LuxI/R QS system is absent in Psa strains. Psa however possesses three putative luxR solos designated here as PsaR1, PsaR2 and PsaR3. PsaR2 belongs to the sub-family of LuxR solos present in many plant associated bacteria (PAB) that binds and responds to yet unknown plant signal molecules. PsaR1 and PsaR3 are highly similar to LuxRs which bind AHLs and are part of the canonical LuxI/R AHL QS systems. Mutation in all the three luxR solos of Psa showed reduction of in planta survival and also showed additive effect if more than one solo was inactivated in double mutants. Gene promoter analysis revealed that the three solos are not auto-regulated and investigated their possible role in several bacterial phenotypes.

Field efficacy of chitosan to control Pseudomonas syringae pv. actinidiae, the causal agent of kiwifruit bacterial canker

In order to provide an alternative for controlling bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae, in case of the appearance of copper/antibiotic-resistant strains of the pathogen, the field efficacy of a chitosan-based compound was compared with a copper compound on Actinidia deliciosa cv. Hayward. The 3-year trials were carried out in central Italy, in an area heavily affected by the disease. Both compounds were sprayed on the same day according to the following schedule: every 14xa0days, from early April to early June (a total of seven treatments), and once a month, from mid November to mid February (a total of four treatments). Chitosan did not incite any phytotoxic effect on plant and fruit and showed an overall higher level of performance than the copper compound in reducing disease symptoms throughout the 3-year trials. Chitosan significantly reduced also the presence of exudates on trunk and leader recorded at the end of winter.

FIELD EFFICACY OF A ZINC-COPPER-HYDRACID OF CITRIC ACID BIOCOMPLEX COMPOUND TO REDUCE OOZING FROM WINTER CANKERS CAUSED BY PSEUDOMONAS SYRINGAE pv. ACTINIDIAE TO ACTINIDIA spp.

Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of bacterial canker of green-fleshed (Actinidia deliciosa) and yellow-fleshed (A. chinensis) kiwifruit and is causing severe economic losses worldwide. In Italy, a combined strategy that takes into consideration timely spray treatments to the plant canopy, a correct pruning management including the disinfection of the tools and the protection of the pruning cuts, the avoidance of excessive nitrogen fertilization and water stress, and training systems favoring a better air circulation, are giving satisfactory results in controlling the pathogen severity in the field. However, despite these efforts, the dormant phase of the plant still represents a main problem, especially in the areas where winter frosts regularly occur. During winter 2013-14 and 2014-15 in four sites, spanning from Northern to Southern Italy, the field efficacy of a zinc (4.7%)-copper (2.6%)-hydracid of citric acid (21.4%) biocomplex compound has been tested on Actinidia deliciosa and A. chinensis orchards. The biocomplex compound was applied three times, at a dose of 1.0%, to reduce the occurrence of bacterial exudates oozing out from the main trunk and leaders in early spring. During the trial, winter frosts were recorded in all sites. The biocomplex compound was more effective than a 1% Bordeaux mixture used under the same conditions. This biocomplex formulation could be used to effectively reduce the amount of exudates caused by Psa in areas where winter frost occurs regularly.

Xanthomonas arboricola pv. fragariae: a confirmation of the pathogenicity of the pathotype strain

Xanthomonas arboricola pv. fragariae is the causal agent of strawberry bacterial leaf blight, and, so far, has been reported in Italy and Turkey. Some years later the description of this new pathovar, and its pathogenicity, was questioned based upon artificial inoculations performed with some strains of the pathogen, including the pathotype strain; it was concluded that it is a non-pathogenic xanthomonad. This notion has also been accepted in other studies aiming at elucidating the molecular characteristics of X. arboricola strains. In the present investigation, we performed again pathogenicity tests using the pathotype strain of X. a. pv. fragariae, namely PD 2780xa0=xa0LMG 19145xa0=xa0CFBP 6771, by a spray leaf inoculation technique on strawberry cultivars Candonga, Sabrina and Murano. Similarly, it was also inoculated onto pot-cultivated walnut, hazelnut, peach, apple and kiwifruit plants. All typical symptoms of bacterial leaf blight of strawberry were observed within 25xa0days after the artificial inoculation on all tested strawberry cultivars. None of the other plant species showed symptoms of disease. We conclude again that the pathotype strain of X. a. pv. fragariae is a pathogenic bacterium. This technique also allowed us to observe typical symptoms of strawberry bacterial blight in a shorter time than that obtained previously by puncturing the major vein of the three leaflets of the leaf.

Evaluation of different training systems on Annurca apple fruits revealed by agronomical, qualitative and NMR-based metabolomic approaches

Nine different training systems for Annurca Rossa del Sud apple fruits, including oblique palmette, free palmette, V-shaped, Tatura trellis, Bibaum®, modified Bibaum®, triple leader, slender spindle and Solaxe, were evaluated based on agronomic, qualitative and metabolomic traits. Fruits were analysed at harvest and after the reddening process. The slender spindle training system showed the highest cumulative efficiency yield compared to the others. Furthermore, an increase in the content of bioactive compounds in flesh and fruit peels was observed after the reddening process and was influenced by the different training systems. The metabolic variations in apple peel were measured and analysed. Changes in the metabolome highlight the influence of different training systems on apple quality. This multidisciplinary study expands our knowledge of the influence of training systems on a typical Italian apple cultivar.

Transcriptome reprogramming of resistant and susceptible peach genotypes during Xanthomonas arboricola pv. pruni early leaf infection

Bacterial spot caused by Xanthomonas arboricola pv. pruni (Xap) is a major threat to Prunus species worldwide. The molecular mechanisms of peach resistance to Xap during early leaf infection were investigated by RNA-Seq analysis of two Prunus persica cultivars, ‘Redkist’ (resistant), and ‘JH Hale’ (susceptible) at 30 minutes, 1 and 3 hours-post-infection (hpi). Both cultivars exhibited extensive modulation of gene expression at 30 mpi, which reduced significantly at 1 hpi, increasing again at 3 hpi. Overall, 714 differentially expressed genes (DEGs) were detected in ‘Redkist’ (12% at 30 mpi and 1 hpi and 88% at 3 hpi). In ‘JH Hale’, 821 DEGs were identified (47% at 30 mpi and 1 hpi and 53% at 3 hpi). Highly up-regulated genes (fold change > 100) at 3 hpi exhibited higher fold change values in ‘Redkist’ than in ‘JH Hale’. RNA-Seq bioinformatics analyses were validated by RT-qPCR. In both cultivars, DEGs included genes with putative roles in perception, signal transduction, secondary metabolism, and transcription regulation, and there were defense responses in both cultivars, with enrichment for the gene ontology terms, ‘immune system process’, ‘defense response’, and ‘cell death’. There were particular differences between the cultivars in the intensity and kinetics of modulation of expression of genes with putative roles in transcriptional activity, secondary metabolism, photosynthesis, and receptor and signaling processes. Analysis of differential exon usage (DEU) revealed that both cultivars initiated remodeling their transcriptomes at 30 mpi; however, ‘Redkist’ exhibited alternative exon usage for a greater number of genes at every time point compared with ‘JH Hale’. Candidate resistance genes (WRKY-like, CRK-like, Copper amine oxidase-like, and TIR-NBS-LRR-like) are of interest for further functional characterization with the aim of elucidating their role in Prunus spp. resistance to Xap.

Methyl jasmonate and ozone affect the antioxidant system and the quality of wine grape during postharvest partial dehydration

Postharvest partial dehydration is a technique used in the production of important dry and sweet wines in Italy. An accurate management of the dehydration environmental parameters allows for the modulation of berry metabolism and the maintenance/improvement of the enochemical quality of grapes. As it is known that water loss induces oxidative processes in berries, our hypothesis was that methyl jasmonate (MeJA) and ozone (O3), as postharvest treatments before partial dehydration, might be beneficial for grape berry quality. Grape bunches were postharvest treated with 10 or 100u202fμM MeJA at 20u202f°C or with ozone gas at 10u202f°C, in 70% relative humidity (RH) and air flow, for 12u202fh; the control bunches were untreated and kept at 20u202f°C for 12u202fh. Subsequently, partial dehydration was performed at 10u202f°C until a 30% weight loss (w.l.) was reached. MeJA hastened grape berry water loss. Polyphenol and flavonoid contents at the end of the partial dehydration were lower in the MeJA-treated berries than in the control and ozone samples. Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (GPX) activity rates increased in the treated samples. In contrast, lipoxygenase (LOX) and polyphenoloxidase (PPO) had lower activities in the MeJA-treated samples than in the controls. It would seem that MeJA accelerates grape water loss but at the same time activates the antioxidant system. Ozone does not accelerate grape water loss but induces the antioxidant system and increases polyphenol content.

FIRST REPORT OF PSEUDOMONAS SYRINGAE pv. ACTINIDIAE ON ACTINIDIA spp. CULTIVATED IN CAMPANIA (SOUTHERN ITALY)

To ascertain the presence of Pseudomonas syringae pv. actinidiae in Campania (southern Italy) field surveys were carried out since 2011 in all areas of kiwifruit (Actinidia deliciosa and A. chinensis) cultivation. In some orchards of Caserta, Benevento, Napoli and Salerno provinces, leaf spot- ting and twig wilting were observed, possibly indicating the occurrence of the pathogen, but no cankers along the trunk and main branches and plant collapse were seen. Gener- ally, the incidence of these symptoms varied from 3% to 20%. Isolation from symptomatic organs and identification procedures were done according to Ferrante and Scortichi- ni (2009, 2010). In parallel, with all samples, duplex PCR (Gallelli et al., 2011) and real-time PCR (Gallelli et al., 2014) were directly applied to plant tissues. The reference P.s. pv. actinidiae CRA-FRU 8.43 strain (Ferrante and Scortichini, 2010) was used as positive control. Pathogenicity tests were carried out on pot-grown, one-year-old plants of A. deliciosa cv. Hayward with representative isolates of P.s. pv. actinidiae obtained from all of the provinces (Ferrante and Scortichini, 2009). Bacterial isolates from all provinces were identified by PCR as P.s. pv. actinidiae. In addition, the successful inocula- tion of cv. Hayward demonstrated the pathogenicity of the representative isolates. The occurrence of the pathogen was also confirmed by the detection techniques applied directly to plant samples. P.s. pv. actinidiae was isolated in Napoli and Benevento provinces from cv. Hayward; in Salerno province from cvs Hayward and G3 Gold, and in Caserta province from cvs. Hayward, JinTao and Soreli.

Emended description of Pseudomonas syringae pv. avellanae, causal agent of European hazelnut (Corylus avellana L.) bacterial canker and decline

Recent studies based on multiple, comparative taxonomic analyses, including MLSA and average nucleotide identity (ANI) approaches, revealed the clear existence of strains associated with bacterial canker and decline of Corylus avellana trees belonging to two different genomospecies: Pseudomonas avellanae and Pseudomonas syringae. Strains of the latter were tentatively named P. s. pv. avellanae. The distinctiveness of the two phytopathogens, both associated with the same symptoms to C. avellana, requires the formal emended description of P. s. pv. avellanae. In fact, the strain BPIC 631u2009=u2009NCPPB 3487, originally described as P. s. pv. avellanae and subsequently elevated to species/genomospecies level, was confirmed as the pathotype strain of P. avellanae. In this paper, the emended description of Pseudomonas syringae pv. avellanae, causal agent of European hazelnut bacterial canker and decline, is provided. P. s. pv. avellanae DSMZ 100307u2009=u2009CRAPAV 013 is designed as the pathotype strain of this pathovar which infect solely C. avellana trees.

Characterisation of the MutS and MutL Proteins from the Pseudomonasavellanae Mismatch Repair (MMR) System

The identification and analysis of the Pseudomonas avellanae mismatch repair system (MMR) were performed via sequencing and cloning the mutS and mutL genes and then analyzing the characteristics of the corresponding proteins studying their function and biological role in an E. coli heterologous system. In these studies, the P. avellanae MutS and MutL proteins were shown to localise at the nucleoid level, in a MutS-dependent manner as far as MutL is concerned, and were also able to complement the defect observed in both the mutS and mutL knockout strains of E. coli. In addition, their ability to form both homo and heterodimers between each other was shown by using the prokaryotic two-hybrid assay. Our results represent a first step to elucidate the MMR mechanism in plant pathogenic pseudomonads since the MMR genes were identified in P. syringae pathovars but there was no evidence on their action as effective repair products.