First Report of Phaeoacremonium mortoniae Causing Petri Disease of Grapevine in Spain.

In May 2006, symptoms of grapevine decline were observed on 4-year-old grapevines (cv. Cabernet Sauvignon) grafted onto 110 R rootstock in Daimiel (Ciudad Real Province, central Spain). Affected vines had low vigor, reduced foliage, and chlorotic leaves. Cross or longitudinal sections of the rootstock trunk showed black spots and dark streaking of the xylem vessels. Five symptomatic plants were collected and analyzed for fungal isolation. Sections (10 cm long) were cut from the basal end of the rootstocks, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally and small pieces of discolored tissues were plated onto malt extract agar (MEA) supplemented with 0.5 g L of streptomycin sulfate. Plates were incubated at 25 to 26°C in the dark for 14 to 21 days and all colonies were transferred to potato dextrose agar (PDA). A Phaeoacremonium sp. was consistently isolated from necrotic tissues. Single conidial isolates were obtained and grown on PDA and MEA in the dark at 25°C for 2 to 3 weeks until colonies produced spores (3). Colonies were yellowish white on PDA and white-to-pale gray on MEA. Conidiophores were short and unbranched, 12.5 to 37.5 (20.5) μm long, and often consisting of a single subcylindrical phialide. Conidia were hyaline, oblong to ellipsoidal or reniform, 2.5 to 7.5 (4.6) μm long, and 1.2 to 1.9 (1.6) μm wide. On the basis of these characteristics, the isolates were identified as Phaeoacremonium mortoniae (2,3). Identity of isolate Pmo-1 was confirmed by PCR-restriction fragment length polymorphism of the internal transcribed spacer region (Phaeoacremonium-specific primers Pm1-Pm2) with the restriction enzymes BssKI, EcoO109I, and HhaI (1). Additionally, the β-tubulin gene fragment (primers T1 and Bt2b) of this isolate was sequenced (GenBank Accession No. EF517921). The sequence was identical to the sequence of P. mortoniae (GenBank Accession No. DQ173109). Pathogenicity tests were conducted on 2-month-old grapevine seedlings (cv. Tempranillo) using two isolates, Pmo-1 and a reference isolate of P. mortoniae (CBS-101585) obtained from the Centraalbureau voor Schimmelcultures (Utrecht, the Netherlands). Seedlings were inoculated when two to three leaves had emerged by watering the roots with 25 mL of a conidial suspension (10 conidia mL) harvested from 21-day-old cultures grown on PDA. Controls were inoculated with sterile distilled water. There were 20 replicates for each isolate with an equal number of uninoculated plants. Seedlings were maintained in a greenhouse at 23 to 25°C. Within 2 months after inoculation, symptoms developed as reduced growth, chlorotic leaves, severe defoliation, and finally wilting. Control plants did not show any of these symptoms. The fungus was reisolated from internal tissues of the crown area and the stems of all inoculated seedlings, completing Koch's postulates. To our knowledge, this is the first report of P. mortoniae causing young grapevine decline in Spain. References: (1) A. Aroca and R. Raposo. Appl. Environ. Microbiol. 73:2911, 2007. (2) M. Groenewald et al. Mycol. Res. 105:651, 2001. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006.