Epidemiological studies on Pseudomonas syringae pv. actinidiae causal agent of actinidia bacterial canker

Il cancro batterico dell’actinidia causato da Pseudomonas syringae pv.actinidiae (Psa) suscita grande interesse a livello globale a partire dal 2008. La malattia è comparsa in Giappone e in due anni ha avuto una diffusione epidemica in tutte le aree di coltivazione mondiale di actinidia. Gravi perdite economiche hanno attirato l’attenzione internazionale su questa problematica e grandi sforzi sono stati rivolti allo studio di questo patosistema ancora poco conosciuto. E’ emerso infatti che il patogeno può rimanere in fase latente per lunghi periodi senza causare sintomi caratteristici nelle piante infette, e che dalla comparsa dei sintomi la pianta muore nell’arco di un paio d’anni. Il monitoraggio ed il controllo della situazione è perciò di fondamentale importanza ed è ancora più importante prevenire la comparsa di nuovi focolai di infezione. A questo proposito sarebbe opportuno l’impiego di materiale vegetale di propagazione non infetto, ma in molti casi questo diventa difficile, dal momento che il materiale impiegato è generalmente quello asintomatico, non analizzato precedentemente per la presenza del patogeno. Negli ultimi anni sono state perciò messe a punto molte tecniche molecolari per l’identificazione di Psa direttamente da materiale vegetale. L’obiettivo di questo lavoro è stato quello di studiare l’epidemiologia di Psa in piante adulte infette e di verificare l’efficacia di metodi di diagnosi precoce per prevenire la malattia. A tale scopo il lavoro sperimentale è stato suddiviso in diverse fasi: i) studio della localizzazione, traslocazione e sopravvivenza di Psa nelle piante, a seguito di inoculazione in piante adulte di actinidia di ceppi marcati Psa::gfp; ii) studio della capacità di Psa di essere mantenuto in germogli di actinidia attraverso sette generazioni di micropropagazione dopo l’inoculazione delle piante madri con lo stesso ceppo marcato Psa::gfp; iii) studio ed applicazioni di un nuovo metodo di diagnosi precoce di Psa basato sull’analisi molecolare del “pianto”.Bacterial canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) involved all global interest since 2008. The disease started from Japan and in two year it was causing epidemic outbreaks around the world, in every country that has an actinidia’s cultivations. Huge amount of economical losses brought the international attention on this problem and on strong efforts were devoted study this relatively unknown pathosystem. It appears in fact that pathogen can be maintained in latent form for long periods, without show any characteristic symptom on the affected plants, when it suddenly induce symptoms the plant die in one or two years. Monitor and control the real situation on symptoms are fundamental, but more important is to prevent the appearance of new infection events. This could be supported by the use of pathogen free propagation materials, but in several cases this is only theoretically achieved since materials employed are just asymptomatic but not tested to be pathogen-free. In the recent years a lot of molecular techniques were developed for Psa detection and diagnosis, directly from plant material. The objectives of this work were to clarify the spreading of Psa into the infected adult plant, and to verify the effectiveness of Psa early detection methods on disease prevention. Toward these aims the following experimental steps were carried out: i) study the localization, movement and survival ability of Psa into the plant after inoculation with a reference marked strains of Psa::gfp several actinidia adult plants; ii) study the Psa ability to be maintained in shoots during seven micropropagated generations after inoculation with the same marked strain of Psa::gfp mother plants; iii) study and application of a novel Psa detection method based on bleeding sap molecular testing

The Italian inter-laboratory study on the detection of Pseudomonas syringae pv. actinide

A severe form of bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae (Psa), has been detected in all the main areas of cultivation of kiwifruit (Actinidia deliciosa and A. chinensis). Since 2010 several research groups have been assessing methods and procedures to detect and identify Psa, both from symptomatic and symptomless host material. In 2011, a study to compare Psa diagnostic methods was performed with reference to Psa strains and related pathovars, and with plant extracts or DNA obtained from healthy and naturally infected leaves, pollen or wood. The study revealed the strengths and the weaknesses of the assessed methods. The procedure included screening tests for Psa detection and for identification of Psa colonies. The methods assessed were bacterial isolation on generic and semi-selective media, PCR analysis (single, duplex and rep-PCR assay, the latter for identification only). The results highlighted the best performance of semi-selective with respect the generic media; the usefulness of the direct-PCR as screening tests for Psa detection; and the greater specificity of duplex-PCR and sensitivity of simple-PCR. The use of semi-selective medium for isolation and of two PCR-based methods - in parallel - for Psa detection are suggested. Both rep-PCR and duplex-PCR, were found to be specific, and are recommended as an identification test for this pathogen

Endophytic distribution of Pseudomonas syringae pv. actinidiae after a five-year latency into Actinidia chinensis var. chinensis plants: a real-time-PCR analysis

The ability of phytopathogenic bacteria to survive for long time within asymptomatic host plants represents one of the main critical factors to control outbreaks. The epidemiological role of the bacterial latency phase is very important since the control strategies are based on preventive chemical treatments by spraying on plant surfaces. In seven-year-old plants of Actinidia chinensis var. chinensis ‘Hort16A’ inoculated five years before with a virulent Pseudomonas syringae pv. actinidiae gfp-expressing/rifampicin-resistant strain (Psagfp-Rifres) at low inoculum dose, the dangerous latency phase of Psagfp-Rifres was studied dissecting the whole plants by cutting, from the apex to the root collar, the shoots/stems in segments of 20 cm (approx. 10-15/plant). In this study, to better clarify the endophyte distribution in asymptomatic plants and the preference of the pathogen for certain portions of the plant stem, the data previously obtained from microbiological (direct isolation on selective media, DI), biological (pathogenicity/HR assay) and PCR (Bio- and Nested) analyses were compared with those from Real-time PCR (RT-PCR) analysis. In the pathosystem Psa–Actinidia, the pathogen reached a high degree of pathoadaptation which is highlighted by the pluriannual latency period of Psa. The RT-PCR of the DNA extracted and quantified by the different segments of each entire plant confirmed the results previously obtained from microbiological and Bio/Nested PCR analyses and allowed to detect Psagfp-Rifres in segments of the stem that in Bio/Nested PCR analyses were Psagfp-Rifres-negative. Direct isolation revealed the Psagfp-Rifres presence in accordance with the RT-PCR data. The long time required for DI of Psagfp-Rifres from plant samples five years after the plant inoculation was largely compatible with the low concentrations of Psagfp-Rifres detected by RT-PCR

Draft whole genome sequence analyses on Pseudomonas syringae 1 pv. actinidiae HR negative strains detected from kiwifruit bleeding sap samples

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 IPVBO 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

Draft whole genome sequence analyses on pseudomonas syringae pv. actinidiae hypersensitive response negative strains detected from kiwifruit bleeding sap samples

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