In October 2010, 2-year-old papaya (cv. Hawaii) trees with high incidence of stem rot were observed during a survey conducted in Rio Grande do Norte state, northeastern Brazil. Stems showing reddish brown-to-dark brown symptoms were collected and small pieces (4 to 5 mm) of necrotic tissues were surface sterilized for 1 min in 1.5% NaOCl, washed twice with sterile distilled water, and plated onto potato dextrose agar (PDA) amended with 0.5 g liter–1 streptomycin sulfate. Plates were incubated at 25°C with a 12-h photopheriod for 4 days. Pure cultures with white, fluffy aerial mycelia were obtained by subculturing hyphal tips onto PDA. Identification was made using morphological characteristics and DNA based molecular techniques. Colonies grown on PDA and Spezieller Nährstoffarmer agar (SNA) for 10 days at 25°C with a 12-h photoperiod were used for morphological identification (3). The fungus produced cream sporodochia and two types of spores: microconidia were thin-walled, hyaline, ovoid, one-celled, and 6.8 to 14.6 × 2.3 to 4.2 μm; macroconidia were thick walled, hyaline, slightly curved, 3- to 5-celled, and 25.8 to 53.1 × 3.9 to 5.7 μm. Fifty spores of each type were measured. Rounded, thick-walled chlamydospores were produced, with two to four arranged together. On the basis of morphological characteristics (1), three fungal isolates (CMM-3825, CMM-3826, and CMM-3827) were identified as Fusarium solani (Mart.) Sacc. and were deposited in the Culture Collection of Phytopathogenic Fungi of the Universidade Federal Rural de Pernambuco (Recife, Brazil). Single-spore isolates were obtained and genomic DNA of the isolates was extracted and a portion of the translation elongation factor 1-alpha (EF1-α) gene of the isolates was amplified and sequenced (2). When compared with sequences available in the GenBank and Fusarium-ID databases, DNA sequences of the three isolates shared 99 to 100% sequence identity with F. solani species complex (GenBank Accession Nos. JF740784.1, DQ247523.1, and DQ247017.1). Representative sequences of the isolates were deposited in GenBank (Accession Nos. JQ808499, JQ808500, and JQ808501). Pathogenicity tests were conducted with four isolates on 3-month-old papaya (cv. Hawaii) seedlings. Mycelial plugs taken from the margin of actively growing colonies (PDA) of each isolate were applied in shallow wounds (0.4 cm in diameter) on the stem (center) of each plant. Inoculation wounds were wrapped with Parafilm. Control seedlings received sterile PDA plugs. Inoculated and control seedlings (10 each) were kept in a greenhouse at 25 to 30°C. After 2 weeks, all inoculated seedlings showed reddish brown necrotic lesions in the stems. No symptoms were observed in the control plants. The pathogen was successfully reisolated from symptomatic plants to fulfill Koch's postulates. To our knowledge, this is the first report of F. solani species complex causing papaya stem rot in Brazil. Papaya is an important fruit crop in the northeastern Brazil and the occurrence of this disease needs to be taken into account in papaya production. References: (1) C. Booth. Fusarium Laboratory Guide to the Identification of the Major Species. CMI, Kew, England, 1977. (2) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.
Occurrence of the Paracalanus parvus species complex in offshore waters south of Japan and their genetic and morphological identification to species
