First Report of Endophytic Candida ipomoeae Isolated from Ovules of Upland Cotton in Mississippi

Cotton is grown on approximately 34.5 million ha worldwide to provide fiber, food oil, and animal feed. To our knowledge, this report is the first of Candida ipomoeae on cotton, and this yeast was found on ovules of the most commercially important cotton species in a major cotton-growing region. The yeast was isolated from ovules of upland cotton grown in vitro. A culture (NRRL Y-48065) was sent to Microbial ID Inc. (Newark, DE) where a partial 176-bp sequence for the D2 domain of the large subunit rDNA was obtained. A BLAST search on the GenBank database ( www.ncbi.nih.gov/Genbank/index.html ) found a 100% match between our sequence and accessions from two strains of C. ipomoeae (Accession Nos. AF050148 and AF050149). In addition, the distinctive colony morphology (white pseudomycelium with a raised stellate to lobate edge) was consistent with previous descriptions of C. ipomoeae (1). No growth was observed at 37°C for the current and previously described isolates. C. ipomoeae is a recently described asexual species (1) that has been isolated from morning glory (Ipomoea spp.) flowers and their insect visitors in Hawaii and the Americas (2). C. ipomoeae has also been found on insects that have visited flowers of the indigenous wild Hawaiian cotton species, Gossypium tomentosum (2) but it has not been isolated previously from cotton per se. Endogenous microbes are common in field-grown upland cotton and can be an impediment to obtaining aseptic plant tissue cultures. During August and September 2005, as part of an effort to rescue interspecific cotton hybrids, ovules were cultured in vitro for 4 days after pollination from plants grown in a field at Stoneville, MS. Fruit were washed in soap and water, surface sterilized in a laminar flow hood by immersion in an aqueous solution of 2.6% sodium hypochlorite and 0.1% Tween 20 for 10 min with intermittent shaking, followed by immersion in ethanol for 10 min, and then allowed to air dry. This surface sterilization protocol is >99% effective on greenhouse-grown fruit. For each fruit, ovules were placed on a single 100 × 25-mm petri dish containing 25 ml of modified Murashige and Skoog media with Gambourg's B5 vitamins (M0404; Sigma-Aldrich, St. Louis, MO) plus 1.9 g l-1 KNO3, 0.5 g l-1 asparagine, 1.0 g l-1 glutamine, 20.0 g l-1 glucose, 0.25 g l-1 cefotaxime, and 2.2 g l-1 gelrite, with a pH of 5.8. Plated ovules were incubated at 30°C with 12 h of fluorescent light each day. C. ipomoeae was first observed on ovules of the cv. Deltapine 90 crossed with G. arboreum; other fungal contaminants were also observed but all of these contaminants originated from ovules within 2 weeks of culture, indicating that the contaminants were endogenous. Subsequently, ovules from the self-pollination of cv. FiberMax 832 were grown on media containing 50 mg l-1 benomyl. On the benomyl-containing plates, the only fungal contaminant observed was C. ipomoeae and it was found on 22 of 120 plates. On plates with or without benomyl, C. ipomoeae grew slowly but caused the infected ovules to become necrotic and die, in contrast to uninfected ovules. Over time, the cultured ovules were completely overrun by the C. ipomoeae colonies. By identifying the contaminant as C. ipomoeae, pursuit of a targeted strategy for controlling it in cotton tissue cultures will now be possible. References: (1) M. A. Lachance et al. Can J. Microbiol. 44:718, 1998. (2) M. A. Lachance et al. FEMS (Fed. Eur. Microbiol. Soc.) Yeast Res. 1:1, 2001.