Calendula officinalis: A New Natural Host of Pseudomonas viridiflava in Italy

Abstract

In November 2010, small necrotic spots surrounded by chlorotic halos, which sometimes enlarged and coalesced to form large dead areas, were observed on leaves of marigold (Calendula officinalis L.) plants grown in the Medieval Garden at the Agricultural Faculty of Perugia (central Italy). Cream-colored bacterial colonies were consistently isolated on nutrient agar (NA) from the diseased leaf tissues. Four representative selected strains, which were gram negative, fluorescent on King's medium B, and had oxidative but not fermentative metabolism, were subjected to a pathogenicity test by inoculating 1-month-old marigold plants. To prepare the inoculum, the bacterial strains were grown on NA at 27°C for 24 h, suspended in sterile deionized water, and adjusted to 1 × 106 CFU/ml. Sterile water was used for control plants. Marigold leaves were infiltrated with a glass atomizer at high pressure, and plants were kept in a growth chamber at 22 to 24°C, 70 μE·m-2·s-1 illumination and 12-h light period, and 80% relative humidity. Small, water-soaked necrotic spots were observed 10 days after inoculation, and the bacterium with the same cultural features of the original strains was reisolated from inoculated plants. For bacterial identification, the four original strains and two reisolates were subjected to LOPAT tests. They were levan negative, oxidase negative, potato rot positive, arginine dihydrolase negative, and tobacco hypersensitive response positive. These results were similar to those obtained with the type strain LMG 2352T of Pseudomonas viridiflava (Burkholder) Dowson. When 16S rDNA was amplified with the universal primers, P0 (6-27f Escherichia coli) and P6 (1515-1495r E. coli), and digested with the endonucleases, SacI and HinfI as previously reported (2), an identical restriction profile was obtained for marigold strains and reisolates and P. viridiflava strains, LMG 2352T, LMG 2353, LMG 5397, and NCPPB 1382. A completely different profile was obtained for P. syringae pv. syringae LMG 1247T. The 16S rDNA (1,364 bp) and the gyrB (570 bp) sequences of two selected marigold strains (GenBank Accession Nos. JN406504 and JN406505; JN406506 and JN406507), amplified by using universal and previously reported PCR primers (3), respectively, shared 100% sequence identity with P. viridiflava (GenBank Accession Nos. HM190229 and AY606763) for 16S rDNA and gyrB gene, respectively. On the basis of biochemical, physiological, molecular, and pathogenicity tests, it was concluded that the bacteria isolated from marigold leaves are P. viridiflava. To our knowledge, this is the first report of C. officinalis as a natural host of P. viridiflava. The plant was previously reported as a host of the bacterium by artificial inoculation (1).