dinemasporium

Study Extent

During the years 2012–2013, dead individuals of Sp. littoreus were collected in four locations distributed on sandy sites of western and southern Taiwan, namely beaches of two counties (Taoyuan, 25.047°N, 121.076°E, and Miaoli, 24.622°N, 120.756°E), an artificial plantation 200–300 m above sea level and at a distance of 2 km from the coast (Hengchun Tropical Botanic Garden, Pingtung County, 21.958°N, 120.811°E), and a coastal wet land close to the beach of Haomeiliao (Chiayi County, 23.360°N, 120.129°E). Samples were individually placed in plastic bags, returned to the laboratory and kept at 4°C until further processing.

Sampling

During the years 2012–2013, dead individuals of Sp. littoreus were collected in four locations distributed on sandy sites of western and southern Taiwan, namely beaches of two counties (Taoyuan, 25.047°N, 121.076°E, and Miaoli, 24.622°N, 120.756°E), an artificial plantation 200–300 m above sea level and at a distance of 2 km from the coast (Hengchun Tropical Botanic Garden, Pingtung County, 21.958°N, 120.811°E), and a coastal wet land close to the beach of Haomeiliao (Chiayi County, 23.360°N, 120.129°E). Samples were individually placed in plastic bags, returned to the laboratory and kept at 4°C until further processing.

Method steps

1. Plants and collection sites During the years 2012–2013, dead individuals of Sp. littoreus were collected in four locations distributed on sandy sites of western and southern Taiwan, namely beaches of two counties (Taoyuan, 25.047°N, 121.076°E, and Miaoli, 24.622°N, 120.756°E), an artificial plantation 200–300 m above sea level and at a distance of 2 km from the coast (Hengchun Tropical Botanic Garden, Pingtung County, 21.958°N, 120.811°E), and a coastal wet land close to the beach of Haomeiliao (Chiayi County, 23.360°N, 120.129°E). Samples were individually placed in plastic bags, returned to the laboratory and kept at 4°C until further processing. Isolation and cultivation For the isolation and cultivation of fungi, conidia were transferred to corn meal agar with 0.2% chloramphenicol (CMA, Fluka) with a flamed acupuncture needle from the conidiomata seen under a dissecting microscope. Then the specimen was dried and deposited in the Natural Museum of Natural Science, Taichung, Taiwan (TNM). The culture was deposited in the Bioresource Collection and Research Center, Hsinchu, Taiwan (BCRC). DNA isolation and molecular analysis DNA was extracted from the examined strains with Genomic DNA Spin Kit (Plant), according to the modified manufacturer’s protocol (Bioman Scientific Co., Ltd., Taiwan). The primer pair ITS1F/ITS4 was used for amplification of the internal transcribed spacer region of the ribosomal RNA gene (ITS rDNA) as in Yeh & Kirschner (2014). DNA sequencing was performed by Mission Biotech (Nankang, Taipei) with the same primers used for the PCR. The DNA sequences were edited using CodonCode Aligner version 4.0.1 (CodonCode Corporation, USA). Eleven ITS sequences belonging to six species of Dinemasporium downloaded from GenBank along with our two sequences of D. spinificis were used for phylogenetic analysis including Thozetella nivea as outgroup. Altogether 14 nucleotide sequences were aligned using the default options of MUSCLE implemented in MEGA6 (Tamura et al. 2013). The alignment was not manually manipulated but only trimmed to a block of 490 characters. The phylogenetic tree was inferred by using the Maximum Likelihood method with the default options of MEGA6, based on the Kimura 2-parameter model and 1000 bootstrap replications. Morphological study Description of micromorphology was based on dry leaf lamina samples. The samples were sectioned by hand and mounted in 10% (w/v) KOH solution. Statistical treatment of measurements was based on 30 measurements given as mean value ± standard deviation with outlier values given in brackets.

The anamorphic fungus Stauronema spinificis hitherto known only from the coastal grass Spinifex littoreus in India was collected on dead leaves of Spinifex littoreus in Taiwan. Phylogenetic analysis of the internal transcribed spacer region of the ribosomal RNA genes indicated a placement within Dinemasporium and confirmed that the species is distinct from the morphologically hardly distinguishable D. cruciferum, therefore, the new combination D. spinificis is proposed.

1. Dinemasporium spinificis
   rank: species

The anamorphic fungus Stauronema spinificis hitherto known only from the coastal grass Spinifex littoreus in India was collected on dead leaves of Spinifex littoreus in Taiwan. Phylogenetic analysis of the internal transcribed spacer region of the ribosomal RNA genes indicated a placement within Dinemasporium and confirmed that the species is distinct from the morphologically hardly distinguishable D. cruciferum, therefore, the new combination D. spinificis is proposed.