Draft Whole Genome Sequence Analyses onPseudomonas syringaepv.actinidiaeHypersensitive Response Negative Strains Detected from Kiwifruit Bleeding Sap Samples

Abstract

Kiwifruit bleeding sap samples, collected in Italian and Chilean orchards from symptomatic and asymptomatic plants, were evaluated for the presence of Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker. The saps were sampled during the spring in both hemispheres, before the bud sprouting, during the optimal time window for the collection of an adequate volume of sample for the early detection of the pathogen, preliminarily by molecular assays, and then through its direct isolation and identification. The results of molecular analyses showed more effectiveness in the P. syringae pv. actinidiae detection when compared with those of microbiological analyses through the pathogen isolation on the nutritive and semiselective media selected. The bleeding sap analyses allowed the isolation and identification of two hypersensitive response (HR) negative and hypovirulent P. syringae pv. actinidiae strains from different regions in Italy. Moreover, multilocus sequence analysis (MLSA) and whole genome sequence (WGS) were carried out on selected Italian and Chilean P. syringae pv. actinidiae virulent strains to verify the presence of genetic variability compared with the HR negative strains and to compare the variability of selected gene clusters between strains isolated in both countries. All the strains showed the lack of argK and coronatine gene clusters as reported for the biovar 3 P. syringae pv. actinidiae strains. Despite the biologic differences obtained in the tobacco bioassays and in pathogenicity assays, the MLSA and WGS analyses did not show significant differences between the WGS of the HR negative and HR positive strains; the difference, on the other hand, between PAC_ICE sequences of Italian and Chilean P. syringae pv. actinidiae strains was confirmed. The inability of the hypovirulent strains IPV-BO 8893 and IPV-BO 9286 to provoke HR in tobacco and the low virulence shown in this host could not be associated with mutations or recombinations in T3SS island.

Genetic and Phenotypic Characterization of Indole-Producing Isolates of Pseudomonas syringae pv. actinidiae Obtained From Chilean Kiwifruit Orchards

“…This suggests that this “hypervirulent” group ( Ciarroni et al, 2015 ) is the only found in Chile, and no other biovar has entered or emerged. The conclusions of this study are consistent with previous research in which the Chilean Psa isolates were classified in the Psa Biovar 3 cluster using different approaches: REP-PCR fingerprinting, MLVA (multiple locus variable number of tandem repeats analysis) assay and MLST ( Ferrante and Scortichini, 2010 , 2015 ; Vanneste et al, 2010 ; Ciarroni et al, 2015 ; Biondi et al, 2017 ). Genomic analyses of the Chilean Psa strains suggest that they originated from China forming a sub-group in biovar 3 ( Butler et al, 2013 ; Ciarroni et al, 2015 ).…”

Bacillus-based products for management of kiwifruit bacterial canker

“…The Psa strains NCPPB 3739 (biovar 1), CRA-FRU 3.1 (biovar 3), CFBP 7286 (biovar 3) and DISTAL (ex-IPV-BO) 9312 (biovar 3; Biondi et al, 2018) were routinely grown at 27°C for 48-72 h, on NSA (Crosse, 1959) or KB (King et al, 1954) media. The mutant strain CRA-FRU 3.1rif r , resistant to rifampicin, was grown at 27°C for 72-96 h on KB medium supplemented with 20 ppm rifampicin.…”

“…The plants were then sealed in polyethylene (PE) bags for 2 d to favour pathogen penetration in the leaves. The greenhouse conditions were set at 16 h light, 23°C and 8 h dark, 17°C, and maintaining the RH% at greater values than 70% (Biondi et al, 2018;Perez et al, 2019) until disease assessments. Streptomycin sulphate (100 ppm) and SDW were used as, respectively, positive and negative experimental controls.…”