Molecular and biochemical characterization of an emerging pathogen in walnut (Juglans regia cv chandler)

CARRASCO, FRANCA; BALBI, FERNANDO; CALIGIORE GEI, PABLO; PUCCI, GRACIELA; SIMONE, IVANA; MAGRIS, GINA; DOMÍNGUEZ, ANA LUZ; MOYANO, PRISCILA; PELLEGRINO, MATÍAS; ORTIZ, MARIA ISABEL; PRINCIPE, ANALÍA

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research (SAIB); 2022

The walnut represents one of the most valuable plants with an important role in the economy. However, different diseases can cause an important reduction of quality and production of walnut. In Argentina, cracks were observed in the bark of walnut trees (Juglans regia cv Chandler) in the province of Catamarca (27°53´57.30"S, 65°50´37.80´´W) during the summer of 2020-2021.Thecracks, found both on trunks and branches, were accompanied by a soft rot with exudate. Intense leaf and shoot blight was also observed on the affected branches, which eventually died. On the trunks, the lesions reached different depths but managed to affect the plants’ vascular system. A bacterial strain was isolated from the symptomatic tissues and named S2-FC. In the present study,the pathogenicity of this strain was investigated by injecting bacterial suspensions (108 CFU/ml from a 28 h LB culture) into the mesocarp of disinfected walnut fruits and under the bark of walnut branches. Non-inoculated fruits and branches served as the control. All the treatments were covered with polyethylene bags for 5 days to maintain high humidity, and kept under greenhouseconditions at temperatures ranging from 25 to 35ºC. Necrosis and reddish brown exudate started to appear at the sites inoculated with the strain as early as 3 days after inoculation. No symptoms were detected in the controls. Reinfection tests (following Koch’s postulates) were carried out on immature fruits and on branches, and confirmed the strain’s pathogenicity. S2-FC was classified asa facultatively anaerobic gram-negative rod, nonfluorescent on King´s B medium. It elicited a hypersensitive response in geranium and formed white, mucoid colonies with well-defined edges on Tryptic Soy Agar medium (TSA). It tested negative for the hydrolysis of gelatin and starch, as well as for production of indole, urease, and H2S. It was able to use glucose, sucrose, mannitol,lactose, raffinose, cellobiose and citrate as sources of carbon, and proved to have oxidative and fermentative metabolism. Furthermore, it could partially grow in media containing xylose, maltose and glycerol as sources of carbon, both under aerobiosis and micro-anaerobiosis. Pathogenicity and pectolytic enzymes activity tests were carried out on carrot and potato discs. They showed that S2-FC is highly virulent: maceration areas were visible at the infection sites 24 hours after initial infection. For genetic identification, a 1509-bp fragment of the strain’s 16S rRNA gene was sequenced. The obtained sequence was compared to the reference sequences retrieved from GenBank. The results, which indicated a 99.7% similarity to Pectobacterium carotovorumsubsp. carotovorum strain JR1.1 (CP034237.1), were later confirmed through their fatty acid profiles and bio-typed by MALDITOF. Additional epidemiological studies are being carried out to gain further knowledge about its behaviour in the areas where symptoms have been observed.