In 2003, a new disease was observed on commercial strawberries (Fragaria × ananassa Duch.) grown in multiple fields in Watsonville, CA. Initial symptoms consisted of brown lesions on the undersides of the sepals of strawberry flowers. The lesions coalesced and spread to upper sepal surfaces and anther bases. No leaf symptoms were observed. Fields affected with this disease appeared to have a greater number of deformed fruit, though incidence data were not collected. A gram-negative, blue-green fluorescent pseudomonad was isolated from lesions on King's medium B agar from both sepals and anthers from 23 of 24 samples from three different fields. All isolates were levan, oxidase, and arginine dihydrolase positive. The strains did rot potato slices but did not induce a hypersensitive reaction in tobacco (Nicotiana tabacum L. cv. Sansun), indicating that the bacteria belonged to Lelliot's LOPAT group IVa, P. marginalis (3). Isolates from strawberry were compared with pathotype strains of Pseudomonas marginalis pv. marginalis, P. marginalis pv. alfalfae, and P. marginalis pv. pastinaceae. The 16S rDNA sequence of type strain of P. marginalis (Z76663) was 97 to 99% similar to the four strawberry isolates sequenced (GQ845121). Identity was further supported by analysis of fatty acid methyl esters (MIS-TSBA version 4.10; MIDI, Inc., Newark, DE). Polymerase chain reaction using BOX-A1R primers (repetitive sequence-based (rep)-PCR [1]) resulted in DNA fragment banding patterns that were identical among strawberry isolates. These banding patterns were different from the three distinct patterns of the P. marginalis pathotypes. Pathogenicity on strawberry (cv. Albion) was confirmed in three experiments using four strawberry isolates originally isolated from plants from three different fields and the P. marginalis pathotype strains. Inoculum was produced by growing bacteria in nutrient broth shake cultures for 48 h (24°C) and washing and suspending the cultures in 0.01 M phosphate buffer (pH 7.0). Three to five attached strawberry flowers on separate plants were dipped in the bacterial suspensions (106 CFU/ml) or sterile buffer for 1 min. To maintain high humidity, flower buds were enclosed in plastic bags for 36 to 48 h and then incubated in the greenhouse (24 to 26°C). After 7 days, approximately half of the flowers inoculated with the strawberry isolates had symptoms on sepals that were identical to symptoms seen in the field. Additionally, reisolates obtained from the symptomatic, inoculated flowers were identical to those used to inoculate the plants as confirmed by LOPAT reactions and rep-PCR, thus completing Koch's postulates. Flowers dipped in phosphate buffer or the P. marginalis pathotype strains did not develop symptoms and no bacteria were reisolated. To our knowledge, this is the first report of blossom blight of strawberry caused by P. marginalis and the first report of P. marginalis on strawberry in California. P. marginalis causes leaf bud rot of strawberry in Japan (2). Further research is needed to determine if the strawberry isolates represent a new or previously described pathovar of P. marginalis.
References: (1) N. A. Cintas et al. Plant Dis. 86:992, 2002. (2) T. Kijima et al. Bull. Tochigi. Agric. Exp. Stn. 36:59, 1989. (3) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.
