First Report of Elm Yellows Subgroup 16SrV-B Phytoplasma as the Cause of Rose Balsam Phyllody in China.

Abstract

Rose balsam (Impatiens balsamina L.) is an ornamental species frequently cultivated in China and the red flower is often used as nail polish in rural regions. The phytoplasmas previously reported with rose balsam phyllody in China have been classified as aster yellows group (16SrI) (1). In August 2012, some rose balsams were observed with typical phytoplasma symptoms in Handan City, Hebei Province, China, with an incidence of about 70% in the fields. The flowers turned green and petals fascicled. The new leaves wrinkled and deformed and internodes shortened. Infected plants were stunted, matured prematurely, and failed to produce seeds. To confirm phytoplasma infection, 100 mg of plant tissue (leaves, petals) was collected from five symptomatic and four asymptomatic plants and total DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method (2). The 16S rDNA gene was amplified by nested PCR using primer pair P1/P7 followed by R16F2n/R16R2 (3). No amplicons were generated with DNA from asymptomatic samples, but amplicons of approximately 1.2 kb were obtained with DNA from five symptomatic samples. The amplified products were purified with aTIANgel midi purification kit (Tiangen, Beijing) and sequenced at the Sangon Biotech facility (Shanghai, China). The sequences of the amplicons were 100% identical and deposited in NCBI GenBank (Accession No. KC993832). The 16S rDNA gene sequence from this phytoplasma was 99% similar to Jujube witches broom phytoplasma (JQ675716), Puna chicory flat stem phytoplasma (JN582266), Plum yellows phytoplasma (FJ459914), and other elm yellows group phytoplasmas by BLAST search of the NCBI database. Restriction fragment length polymorphism (RFLP) analyses were carried out by digesting the 1.2-kb R16F2n/R16R2 nested PCR product with restriction enzymes AluI, RsaI, HhaI, HpaI, Eco RI, TaqI, HaeIII, HinfI, and KpnI (Takara, Dalian). The 16S rDNA RFLP patterns matched that of Jujube witches broom phytoplasma (JWB, subgroup 16SrV-B) (4). Nucleotide sequences of rose balsam phyllody were analyzed by iPhyClassifier software, which revealed that it had maximum similarity to the reference pattern of 16Sr group V, subgroup B (AB052876). All samples were detected with transmission electron microscopy. The results showed phytoplasma-like cells in phloem sieve element of symptomatic plants, while no phytoplasma-like cells were observed in healthy phloem tissues. The phytoplasma cells ranged from 230 to 470 nm in diameter and were ellipsoidal or orbicular with visible membranes. Combining the RFLP pattern and sequence analysis by iPhyClassifier, we classified the phytoplasma causing rose balsam phyllody into subgroup 16SrV-B. To our knowledge, this is the first report of 16SrV-B group phytoplasmas infecting rose balsam in China.