PCR-Mediated Detection of Endophytic and Phytopathogenic Fungi from Needles of the Japanese Black Pine, &amp;lt;i&amp;gt;Pinus thunbergii&amp;lt;/i&amp;gt;

Junichi Kihara,Makoto Ueno,Sakae Arase

doi:10.4236/ojf.2015.54037

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.

Highlights

The Japanese black pine (Pinus thunbergii Parl.), an evergreen species, is distributed along the seacoasts of Japan and South Korea
Many diseases of the Japanese black pine are known, such as Dothistroma needle blight caused by Dothistroma pini (Ito & Zinno, 1972; Ito et al, 1975), brown spot needle blight caused by Lecanosticta acicola (Suto & Ougi, 1998; Seo et al, 2012), needle cast caused by Lophodermium spp. (Yamamoto et al, 1964; Sakuyama, 1993), Pestalotia disease caused by Pestalotiopsis spp. (Takahashi & Kobayashi, 1998; Takahashi & Kobayashi, 1999), Rhizosphaera needle blight caused by Rhizosphaera kalkhoffii (Tanaka & Chiba, 1971), and sooty mold caused by Septorioides pini-thunbergii (Kaneko et al, 1989; Suto, 2000)
This paper reports the development of specific and rapid detection of endophytic and phytopathogenic fungi from the needles of Japanese black pines using polymerase chain reaction (PCR) assay based on the internal transcribed spacer (ITS) region of rDNA sequences

Summary

Introduction

The Japanese black pine (Pinus thunbergii Parl.), an evergreen species, is distributed along the seacoasts of Japan and South Korea. The identification and detection of both endophytic and phytopathogenic fungi relies upon their culture-based morphological characteristics and on biochemical approaches. These procedures are time-consuming and require extensive knowledge of fungal taxonomy. Species-specific polymerase chain reaction (PCR) has emerged as a powerful tool for the identification and detection of phytopathogenic fungi, such as root rot pathogen Rhizopycnis vagum (Ghignone et al, 2003), collar rot pathogen Sclerotium rolfsii (Pravi et al, 2014), chestnut blight pathogen Cryphonectria parasitica (Popov et al, 2010), and pine needle pathogen Lophodermium spp. This paper reports the development of specific and rapid detection of endophytic and phytopathogenic fungi from the needles of Japanese black pines using PCR assay based on the internal transcribed spacer (ITS) region of rDNA sequences

Methods

Results

Conclusion