ObjectiveCerambycid vectors and fungi are important components in system of pine wilt disease. Illustrating the fungal flora, especially the ophiostomatoid fungi associated with pine wilt disease, to accurately identify the dominant species with combination of their physiological and morphological characteristics and further phylogenetic analysis based on multiple DNA sequences, could provide a solid basis for understanding relationship among the disease, cerambycid vectors and associated fungi.
MethodThe tunnels and pupal chambers of sawyer beetle Monochamus alternatus (the vector of the nematode pathogen, Bursaphelenchus xylophilus) inside the infected Pinus massoniana and P. thunbergii from newly epidemic areas of Shandong Provinces and historical epidemic areas of Zhejiang Provinces were collected to investigate the ophiostomatoid fungal flora. The tissue isolation method was used to obtain the associated fungi. Based on cultural characteristics and morphological identification, the representative strains were selected according to their geographical and host distribution to conduct a phylogenetic analysis based on the rDNA-ITS and β-tubulin sequences.
ResultTwo hundred and fifty-four ophiostomatoid fungal strains were isolated from 98 samples, accounting for 20.6% of the fungal flora, indicating the dominant status of the group. According to the cultural and morphological characteristics, 235 strains among them were identified as Ophiostoma ips, which accounted for 92.5% of the ophiostomatoid fungi, giving rise to its predominant status. The species exhibited obvious internal differentiations on colony and micro-morphological characteristics. The temperature dependent growths and phylogenetic analyses based on rDNA-ITS and β-tubulin sequences of representative strains further support the phenotype status of the species.
ConclusionThis is the first report confirmed by the comprehensive identification that O. ips as the predominantly species extensively and intensively associated with the pine wilt disease involved by both the sawyer beetle and the pine wood nematode in China. The studies on the association mechanism between O. ips and the pine wilt disease, and on the fungus roles reacting on the nematode population dynamics are further necessary, which can lead to new strategies for the effective prevention and control of the disease.