APPLE TISSUE CULTURE CONTAMINATION BY RHODOTORULA SPP.: IDENTIFICATION AND PREVENTION J. KOLOZSVA ´ RI NAGY 1 *, S. SULE 1 , AND J. P. SAMPAIO 2 1 Plant Protection Institute of the Hungarian Academy of Sciences, 1525 Budapest, P.O. Box 102, Hungary 2 CREM, SABT, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Lisbon, Portugal (Received 13 July 2004; accepted 23 February 2005; editor S. S. Korban) Summary Shoot cultures of apple cv. Pinova were contaminated with faint pink pigmented yeast. Yeast isolates were identified as Rhodotorula slooffiae with standard physiological methods and molecular analysis. Growth of isolated yeasts was tested against different fungicides. The following fungicides inhibited the growth of yeast isolates, and were not phytotoxic to apple shoots at concentrations lower than the minimal phytotoxic concentrations (MPC): ProClin w 300, mancozeb, triforine, myclobutanil, thiabendazole, mancozeb þ zoxamid, and silver nitrate. Some fungicides inhibited growth of yeasts, but were phytotoxic. These included miconazole, PPMe, copper sulfate, potassium sorbate, and cycloheximide. Benomyl was not phytotoxic, but was effective only at high doses. Decontamination of shoots was achieved using a combination of two treatments. Shoots were first soaked in half-strength Murashige and Skoog (MS) liquid medium containing silver nitrate (588 mM) and Silvet 77 (0.01%) for 1–2 h, and then transferred to a solidified MS medium containing both mancozeb (15 mg l 21 ) and thiabendazole (40 mg l 21 ). Key words: isothiazolones; mancozeb; micropropagation; silver nitrate; thiabendazole; yeast. Introduction In vitro contamination of plant tissue cultures by microorganisms is one of the most serious problems in plant micropropagation (Cassells, 1997). Cultures can be infected with a wide range of microorganisms (filamentous fungi, yeasts, bacteria, viruses, and phytoplasmas) (Leifert and Cassells, 2001). Contaminants are either present in the mother explant or introduced during inadequate handling in the laboratory. Contaminating microorganisms may be readily observed immediately or can remain latent for long periods of time. This often makes it difficult to identify the source of contamination. During the establishment of a new culture, plant meristematic tissues are generally utilized as starting material. However, these tissues are frequently colonized by bacteria, filamentous fungi, or yeasts, which may not become visible during the initial stage of culture establishment. The presence of contaminant microorganisms in tissue culture is usually attributed to insufficient surface sterilization or poor sterile techniques (Leifert et al., 1994). Besides increased culture mortality, the presence of contaminants can result in variable growth, tissue necrosis, and reduced shoot proliferation and rooting. Microbial contamination is difficult to eradicate from established cultures. Contaminants of explants grown on rich tissue culture media containing high amounts of sugar multiply in high numbers and invade internal tissues. Sterilization of such highly contaminated tissues is much more difficult than establishment of a new culture. However, in certain circumstances, a valuable and contaminated culture must be saved and cleaned from contaminants. During the course of micropropagation of apple cv. Pinova, which had been maintained through several passages, a pink pigmented bacterium-like contaminant was found. Preliminary observations, based on microscopic examinations, suggested the presence of yeasts. Using several antibiotic treatments and repetition of surface sterilization could not eliminate the contaminants. The present study was undertaken to identify the contaminant microorganism, and to circumvent the problem. Materials and Methods Materials. Chemicals used in tissue culture were from Sigma-Aldrich (Budapest). The fungicides mancozeb, myclobutanil, and mancozeb þ zoxamid were from Dow AgroSciences Hungary (Budapest); triforine from American Cyanamid Company (Princeton, NJ, USA); benomyl from Chinoin (Budapest); ProClin w 300 from Sigma-Aldrich (Budapest), and PPMe Plant Cell Technology Inc. (Washington, DC, USA). Apple tissue culture. Shoot cultures of apple cv. Pinova used in this study were initiated from axillary and actively growing buds. Individual buds were surface-sterilized in 70% ethanol for 1 min, and in 10% sodium hypochlorite containing a drop of Triton X surfactant for 20 min. After rinsing in sterile water, scales and outer leaves were removed. Buds were dipped in 70% ethanol for 5 – 10 s, followed by continuous agitation in 10% sodium hypochlorite for 5 min, and then rinsed twice with sterile distilled water. Meristem tips (1–3 mm) were aseptically excised, and transferred to Petri dishes, each containing 20 ml of the culture medium. After 4 d, contaminant-free meristematic shoot-tips were re-transferred to fresh medium. To confirm that the disinfestation process was successful, randomly selected explants were pressed onto plates with potato dextrose agar (PDA) medium, and plates were evaluated for growth of microorganisms after incubation at 288C for 7 d. Cultures were kept in a culture room at 258C under 16-h cool white illumination (60 mmol m 22 s 21 ). Shoots (5 – 10 mm) were subcultured at monthly intervals. Shoot cultures were maintained on a proliferation medium *Author to whom correspondence should be addressed: Email naju@nki.hu In Vitro Cell. Dev. Biol.—Plant 41:520–524, July – August 2005 DOI: 10.1079/IVP2005647 q 2005 Society for In Vitro Biology 1054-5476/05 $18.00+0.00 520