First Report of Simplicillium lanosoniveum Causing Brown Spot on Salvinia auriculata and S. molesta in Taiwan

Salvinia spp. are small, floating ferns that grow in long chains of two oval leaves and a root-like third leaf. S. natans (L.) All., a native floating fern distributed in paddy fields, ponds, and ditches in Taiwan, has become critically endangered. Another two exotic species, S. auriculata Aublet (eared salvinia) and S. molesta Mitchell (giant salvinia), are sold in increasing frequency at local flower markets and aquarium shops and pose a serious threat when they find their way into the natural environment. Brown spot of S. auriculata was found in a home aquarium in December 2006 in Chiayi, Taiwan. Symptoms of the disease included many, irregular, dark brown spots on both upper and lower leaf surfaces. Lesions on the upper surface of the leaves were covered with white patches of mycelia and abundant conidia. Small pieces (approximately 2 × 2 mm) of diseased leaf tissue from the margin of individual lesions were surface disinfected in 1% sodium hypochlorite solution for 1 min, rinsed in sterile water, plated on water agar, and incubated at 25°C. Six isolates of the fungus were then isolated and transferred to potato dextrose agar (PDA). Isolate Cs0701 was identified morphologically as Simplicillium lanosoniveum (van Beyma) Zare & W. Gams on the basis of morphology of asexual reproduction structures and rDNA sequence analysis (1). In culture, this fungus formed whitish-to-whitish yellow, pulvinate colonies with matted surfaces. The reverse side of cultures was yellow to light brown. Small, ovate to spherical, hyaline conidia, 2.2 to 3.0 × 1.6 to 2.0 μm (average 2.4 × 1.9 μm) were formed. To confirm the identity of the fungus, PCR amplification and DNA sequencing of the internal transcribed spacer (ITS1-5.8S-ITS2 rRNA gene cluster) was conducted on isolates Cs0701 and Cs0702. The sequence of the PCR product was compared with sequences of closely related species listed in the GenBank database. Except for a single nucleotide, the ITS sequence of both isolates (480 bp; GenBank Accession No. EU939525) was identical to the rRNA of Simplicillium lanosoniveum (GenBank Accession No. AJ292396). Koch's postulates were performed to confirm the pathogenicity of the fungus on S. auriculata and S. molesta. After 14 days of growth on PDA, a spore suspension of isolate Cs0701 (10⁶ spores per ml) was sprayed onto approximately 5 and 10 g of healthy S. auriculata and S. molesta plants, respectively, floated in 500-ml beakers filled with 300 ml of tap water. All treatments, including controls misted with sterile water, were replicated three times. The beakers were covered with plastic bags and placed in a growth chamber maintained at 25°C with 12-h fluorescent light cycles. After 2 days, the bags were removed. Symptoms developed on all inoculated plants 4 days after inoculation. In all cases, typical brown spots were observed. Simplicillium lanosoniveum was reisolated from all surface-disinfested infected tissues. Control plants developed no symptoms. Six isolates of the fungus are being maintained at the Department of Microbiology and Immunology, National Chiayi University, Taiwan. To our knowledge, this is the first report of Simplicillium lanosoniveum causing brown spot of S. auriculata and S. molesta in Taiwan. Reference: (1) R. Zare and W. Gams. Nova Hedwigia 73:1, 2001.