Biological Characteristics of Phellinus noxius and its Molecular Methods for Diagnosis

• Main Authors: Jyh-Nong Tsai, 蔡志濃
• Other Authors: 謝文瑞
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/26536330203172316529

博士 === 國立中興大學 === 植物病理學系所 === 96 === Brown root rot caused by Phellinus noxius is one of the most important root diseases of woody trees in Taiwan. During the investigation from 2002 through 2007, a total of 19 species of new recorded hosts of the pathogen were found and studied. The morphology of the fungus was studied by culturing the purified cultures of several isolates on PDA and saw dust media as well as by diagnosing the diseased spaceman collected from nature. The fungal colonies on PDA were white in the beginning and turned irregular brown gradually. Clamp connection was not found. Mycelia produced hyaline arthrospores and dark brown trichocyst on PDA, whereas only trichocyst but no arthrospores were found in natural specimens. All tested isolates present multinuclei in hyphae. Fruiting bodies brown and 0.5-1 cm thick; basidiospores smooth, hyaline, and ovoid to broadly ellipsoid, measuring 3.6-6.2 x 1.8-5.2 μm; contextual setal hyphae reddish brown, measuring 340-680 μm. The temperatures for mycelial growth on PDA were 12-36℃ with optimum growth at 24-28 ℃. On artificial media amended with adequate substrates, all the 5 tested isolates collected from different territorial hosts were able to produce amylase, cellulase, lipase, pectinase and protease. Nucleotide sequences of ITS1, 5.8S and ITS2 of 80 strains of Phellinus noxius obtained from 33 tree species at different geographic locations in Taiwan from 1991 to 2006 were analyzed. These strains were divided into two ITS types based on the nucleotide length, type L with 614 bp and types S with 606 bp. Majority of the strains belonged to type L. Both type L and type S were present at most counties of Taiwan. In order to develop a pair of specific primers for rapid and accurate diagnosis of the disease associated with the pathogen in the fields, the ribosomal DNA (rDNA) sequences in internal transcript spacer (ITS, including ITS1/5.8S/ITS2) regions from P. noxius and several other soil-borne fungi were studied. Based on the ITS sequences, a set of primers PN-1F/PN-2R was designed. The forward primer PN-1F sequence is “5’-agtttgcgctcatccatctc-3’ ” and the reverse primer PN-2R is “5’-agccgacttacgccagcag-3’ ”. Using PN-1F/PN-2R primers for PCR amplification under a setup condition and the fungal genomic DNA as templates, a distinct 414 bp or 422 bp fragment were amplified form P. noxius, whereas no PCR products can be amplified from other soil-borne fungi tested. The PCR could amplify the fragments recognizable within 3-4 hr when concentration of purified DNA was higher or equal to 0.01 ng. Meanwhile, similar amplified fragments were obtained when the diseased root tissues of Dimocarpus longana and Acasia confusa from P. noxius infested fields were used as templates. Results presented here suggest that the primer pairs PN-1F/PN-2R can be efficiency in auxiliary identifying P. noxius and accurately and rapidly detecting disease caused by the pathogen in the field. In order to search of effective control measures, potato dextrose agar amended with individual chemical was used to evaluate the effects of different concentrations of synthetic chemicals on suppression of P. noxius. Among the 46 chemicals tested against P. noxius, 25% propiconazole EC was most effective, completely inhibiting the mycelial growth at the active ingredient dosage of 10 ppm. Bordeaux mixtures were also very effective in inhibiting mycelial growth. Another study was conducted to determine survival of P. noxius in infected stems of loquat buried in soil amended with fungicides, fertilizers, or by flooding. Results showed that treatment of soil with urea at 2.0 g/L soil or flooding of soil for 10 days completely eliminated the pathogen in the stems. A field trial was carried out on 3-5 year-old grapes in a field naturally infested with P. noxius in Shuili, Nantou. Results showed that soil drenching every 3 months with the solution containing 10 g 5% triadimefon+10 g urea+10 g CaCO3 in 1 L of water was the most effective treatment with no infected grapevines developed in all the 60 plants after treating for 2.5 years. In contrast, 6 of 42 (14.3%) tested plants in the untreated control were killed by P. noxius in 2.5 years.