ผลงานวิจัยนี้แบ่งออกเป็น 3 เรื่องใหญ่ๆ ดังนี้

เรื่องที่หนึ่ง เป็นการเตรียมโปรโตพลาสของเชื้อรา Fusarium oxysporum เพื่อใช้ในการเตรียม Chromosome ของเชื้อรา วิธีการเตรียมโปรโตพลาสของเชื้อราดังกล่าว

Production and reversion of protoplasts of Fusarium oxysporum

A procedure for efficient isolation and cell wall regeneration of protoplasts from Fusarium oxysporum f.sp. lycopersici is described. Protoplasts were obtained from the mycelia cultured in Czapek liquid medium containing 0.1 M acetate salt (potassium of sodium acetate). A large quantity of protoplasts were released from young mycelia (1 or 2-day old) by treatment with NovoZyme 234 (NZ). The optimal conditions for isolation were summarized as follows; i) 1.3 M KCl was at the best among 0.6-1.5 M of different stabilizers (Mannitol, Sorbitol, MgSO4, and KCI), ii) 30 mg/ml NovoZyme gave the highest yield in the concentration range of 20 and 50 mg/ml, iii) the optimum pH was around 5.0, iv) 1 or 2-day old mycelia, and v) incubation at 30°C. This conditions also suitable for isolation of the protoplasts form other formae speciales, cucumerinum, fragariae, melonis, and spinaciae. Reversion of renerated cell was found the best when the protoplasts were cultured on SN medium containing 1% agar suppllemented with 25% sucrose as osmotic stabilizer.

เรื่องที่สอง เป็นการหาวิธีที่เหมาะสมในการสกัด DNA จากเชื้อรา Fusarium เพื่อนำมาวิเคราะห์หาความสัมพันธ์ในระดับโมเลกุล

Rapid Extraction of Nucleic Acids from Fusarium oxysporum for PCR Amplification

An efficient, relatively quick, inexpensive and convenient method is presented for the isolation of megabase-size DNA mycelia of Fusarium oxysporum. The protocol involves (i) disruption of mycelial cells by blending in liquid nitro followed by suspension of cell contents in buffer containing high concentrations of CTAB and EDTA: (ii) deproteinization with chloroform/isoamyl alcohol; (ii) precipitation with isopropanol and (iv) agarose gel electrophoresis to separate and identify DNA. The procedure has been used successfully with several dozens of formae speciales of Fusarium oxysporum and has also been used to prepare DNA from Collectotrichum spp. , Cheatomium cupreum, C. globosum, Trichoderma hamatum and T. harzianum. Average yields of the DNA range from 750 to 1,250 mg/g wet weight of mycelium and was suitable for PCR amplification and degestion with restriction endonucleases. This procedure may prove useful for other difficult where hundreds of samples need to be analyzed in a short period of time.

เรื่องที่สาม เป็นการนำ DNA ของเชื้อราที่สกัดได้มาวิเคราะห์หาความสัมพันธ์ในระดับโมเลกุล โดยใช้เชิ้อราในกลุุ่ม Fusarium oxysporum 9 สายพันธุ์ (formae speciales)

Phylogenetic Relationship Among Formae Speciales of Fusarium oxysporum Inferred from rDNA Sequences

An improved protocol, including DNA extraction, two amplifications with a nested primer set, and DNA purification by electrophoresis, made it possible to analyze nuclear rDNA sequences of nine formae speciales of Fusarium oxysporum. In addition, for comparison, Fusarium solani and Gibberella fujikuroi were used as outer groups. Nucleotide sequence diversity of the nuclear rDNA region containing the two internal transcribed spacers (ITS1 and ITS2) and 5.85 rDNA gene derived from fungal mycelia was investigated and used to develop a phylogenetic tree. The results showed that the nucleotide sequences of the 5.8S rDNA genes of the tested fungi were highly conderved, but those of their ITS regions were variable. Our data indicated that seventeen formae speciales of F. oxysporum can divide into two groups base on nucleotide sequence of the internal transcribed spacers. Thus, the ITS region will be useful for phylogenetic comparison of closely related species or intraspecies.