TITLE:
PCR-Mediated Detection of Endophytic and Phytopathogenic Fungi from Needles of the Japanese Black Pine, Pinus thunbergii
AUTHORS:
Junichi Kihara, Makoto Ueno, Sakae Arase
KEYWORDS:
Phytopathogenic Fungi, Endophytic Fungi, Pinus thunbergii, Japanese Black Pine, PCR-Mediated Detection
JOURNAL NAME:
Open Journal of Forestry,
Vol.5 No.4,
April
17,
2015
ABSTRACT: A specific
and sensitive polymerase chain reaction (PCR) assay based on the internal transcribed spacer (ITS) region of rDNA sequences
was developed to detect endophytic and phytopathogenic fungi from needles of the
Japanese black pine, Pinus thunbergii.
Sequences of the ITS regions of Lophodermium
conigenum, Lecanosticta acicola, Pestalotiopsis neglecta, Rhizosphaera kalkhoffii, and Septorioides pini-thunbergii were compared,
and each specific primer pair for these species was designed. First, the designed
primer pairs were tested for their specificity to detect each species. A PCR product
was amplified only each combination of species and its specific primer pair, confirming
the specificity of the designed primer pairs. These primer pairs were also tested
on DNA extracted from the needles of P. thunbergii. The PCR products were amplified not only in needles with lesions but also in healthy
needles without symptoms. Furthermore, several endophytic and phytopathogenic fungi
could be simultaneously detected from the same region in a needle. The PCR-mediated
detection method developed in this study will be a valuable tool for the detection
of the endophytic and phytopathogenic fungi, not only as a rapid diagnostic tool
for early detection but also for monitoring variations in both the quality and quantity
of the endophytic and phytopathogenic fungi in needles in Japanese black pines.