Phellinus noxius: molecular diversity among isolates from Taiwan and its phylogenetic relationship with other species of Phellinus based on sequences of the ITS region
Tsai et al. Bot Stud
Phellinus noxius: molecular diversity among isolates from Taiwan and its phylogenetic relationship with other species of Phellinus based on sequences of the ITS region
Jyh‑Nong Tsai 2
PaoJ‑en Ann 2
Ruey‑Fen Liou 1
Wen‑Hsui Hsieh 0
Wen‑Hsiung Ko 0
0 Department of Plant Pathology, National Chung Hsing University , Taichung , Taiwan
1 Department of Plant Pathology and Microbiology, National Taiwan University , Taipei , Taiwan
2 Division of Plant Pathology, Taiwan Agricultural Research Institute , Wufeng, Taichung , Taiwan
Background: Analysis of phylogenetic relationship of 91 isolates of Phellinus noxius obtained from 46 plant species in Taiwan did not show distinct grouping based on ITS sequences. Results: However, the ITS nucleotides showed 20 different kinds of variations including single nucleotide polymorphisms, deletion and insertion in ITS1 and ITS2, but none in 5.8 S. The Taiwanese isolates of P. noxius were dividable into long (type L), median (type M) and short (type S) groups based on ITS sequence length. Two isolates with identical ITS sequence belonged to types L. Type M with 72 isolates was further divided into 33 subtypes, while types S with 17 isolates was further divided into two subtypes. Conclusion: Phylogenetic analysis of ITS sequences among Phellinus species showed that isolates of P. noxius were in the same clade distinctly separated from other Phellinus species.
Deletion; Insertion; ITS sequence; Nucleotide variation; Sequence length; Single nucleotide polymorphism
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Background
Brown root rot caused by Phellinus noxius (Corner) G. H.
Cunn. is widespread among tropical countries in
Southeast Asia, Africa, Oceania, Central America and the
Caribbean (Pegler and Waterston 1968). In China, it has been
reported from the tropical Hainan Island (Tai 1979). In
Japan, it was found on the subtropical island of Okinawa
(Abe et al. 1995). The pathogen attacks more than 120
species of fruit and ornamental trees in both topical and
subtropical districts in Taiwan (Ann et al. 1999; Chang
and Yang 1998). Among the approximately 200 plant
species listed as hosts of P. noxius in the world, about half
of them were reported for the first time from Taiwan
(Ann et al. 2002). Even though the fungus lacks air-borne
spores for efficient dissemination, it is very widespread
and occurs on so many kinds of hosts at very different
geographic locations on the island of Taiwan (Ann et al.
2002). It is, therefore, conceivable that P. noxius may be
an ancient residence of the island where diverse isolates
of this fungus may have existed. There are very few
morphological characters in P. noxius available for testing this
hypothesis because the fungus rarely produces
basidiocarps on diseased trees in the fields (Ann et al. 1999;
Chang 1995, 1996).
In this study, molecular variation in the ITS (ITS1, 5.8S
and ITS2) region among isolates of P. noxius from Taiwan
was investigated and compared with the ITS sequences
reported from other countries available in the GenBank.
We also investigated the ITS phylogenetic relationship of
P. noxius with other species of Phellinus. Details of the
study are reported herein.
Methods
Isolation and storage of the pathogen
Main roots of trees showing quick or slow decline
symptoms (Ann et al. 2002) were exposed and
examined. Those showing typical brown discoloration were
cut and brought back to the laboratory. Small pieces
(5 × 2 × 1 mm) of tissue were obtained from the
advancing margins of the diseased roots, surface-sterilized with
0.5% NaClO for 1 min, plated on potato dextrose agar
(PDA) supplemented with 100 ppm streptomycin sulfate
and 10 ppm benomyl for inhibition of growth of bacteria
and other fungi, and incubated at room temperature (24–
30 °C). Fungal mycelia growing from diseased tissue were
transferred to 2% water agar. Single-hyphal tips obtained
from the fungus growing on water agar were cultured on
PDA and stored in sterile distilled water in test tubes at
room temperature (Boesewinkle 1976; Ko 2003). From
each diseased tree only one isolate was saved for the
study. The cultures were identified as P. noxius based on
the production of brown colonies with irregular dark
brown zone lines on PDA and formation of arthrospores
and trichocysts (Ann and Ko 1992).
DNA extraction, amplification and sequencing
Each isolate of P. noxius was grown on cellophane placed
on PDA (Ko et al. 2011). After incubation at 25 °C for
10 days, mycelia were harvested, lyophilized and stored at
−20 °C until use. About 20 mg lyophilized mycelia were
ground in liquid nitrogen and used for extraction of DNA
using the genomic DNA extraction kit (GenMark
Technology Co., Taichung, Taiwan). The ITS (ITS1-5.8S-ITS2)
region was amplified with primer pair of ITS4 and ITS5
(White et al. 1990). The 25 μl reaction mixture
consisting of 0.2 μg template DNA, 0.2 μM each primer, 200 μM
each dNTP, 2 μl 2X polymerase chain reaction (PCR)
buffer and 1.0 U ZyM Taq DNA polymerase (Zy (...truncated)