Molecular identification based on the sequence of internal transcribed spacer (ITS) of the ribosomal nuclear DNA (rDNA) of pathogenic fungus Pythium aphanidermatum (Edson) Fitzp. isolated from soil and its morphology

M. George, P. Ramteke
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Abstract

Soil-borne fungi Pythium aphanidermatum causes damping-off of cucumber, coriander and other economically important plants in India and presumably in many other countries. The objective of the present investigation was to characterize the pathogen morphologically in vitro and to confirm its molecular identity by the sequence of ITS region of rDNA. P. aphanidermatum was isolated from soil and cultured in PDB broth and PDA solid media. The characteristic of growth was monitored and the reproductive structures were analyzed. Hyphae were grown with a daily increment of 2.5-3.0 cm on PDB. Sporangia were swollen, multinucleate and usually measure 10-50 μm in diameter. Oogonia were found mostly terminal, spherical, 22-27 μm in diameter. Fungus was also characterized using molecular methods based on ITS-PCR. The amplified sequence was compared with the available sequences in the NCBI GenBank. The sequence showed 99% similarity with other species of Pythium. Based on the morphological and molecular characters isolate was confirmed as Pythium aphanidermatum. The isolation, morphological characterization and sequencing of ITS region of rDNA will add knowledge to the scientific community for proper identification and in-depth research on the management of this plant pathogen.
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基于致病真菌蛇皮霉(Pythium aphanidermatum, Edson)核糖体核DNA (rDNA)内部转录间隔段(ITS)序列的分子鉴定分离自土壤及其形态
在印度,土壤传播的真菌蛇皮霉(Pythium aphanidermatum)会导致黄瓜、香菜和其他经济上重要的植物枯萎,可能在许多其他国家也是如此。本研究的目的是在体外对病原菌进行形态学表征,并通过rDNA its区序列确定其分子身份。从土壤中分离出假单胞菌,分别在PDB肉汤和PDA固体培养基中培养。对其生长特征进行了监测,并对其生殖结构进行了分析。菌丝在PDB上以每天2.5 ~ 3.0 cm的速度生长。孢子囊肿胀,多核,直径约10 ~ 50 μm。卵原体多为末端球形,直径22 ~ 27 μm。利用ITS-PCR技术对真菌进行了分子鉴定。扩增的序列与NCBI GenBank中可用的序列进行比较。该序列与其他种的相似性达99%。经形态学和分子特征鉴定,该分离物为蛇皮草。rDNA ITS区域的分离、形态鉴定和测序将为科学界正确识别和深入研究该植物病原体的管理提供知识。
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