Phellinus noxius: molecular diversity among isolates from Taiwan and its phylogenetic relationship with other species of Phellinus based on sequences of the ITS region

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.


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 1995Chang , 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 Open Access *Correspondence: kowh@nchu.edu.tw 3 Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan Full list of author information is available at the end of the article P. noxius with other species of Phellinus. Details of the study are reported herein.

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 (Zymeset, Taiwan) was subjected to thermal cycling in a Perkin-Elmer Thermal Cycler 9700 (Perkin-Elmer Applied Biosystem, USA). Cycling conditions for amplification were an initial denaturation at 94 °C for 3 min, followed by 35 cycles at 94 °C for 45 s, 50 °C for 45 s, 72 °C for 45 s, and a final elongation at 72 °C for 7 min. The PCR products were electrophoresed on a 1.5% agarose gel. Direct sequencing of the PCR products was performed by the Seeing Bioscience Company (Taipei, Taiwan), using ITS4, ITS5 (White et al. 1990), PN-5.8S-1 (5′-GCA GCG AAA TGC GAT AAG TA-3′), or PN-5.8S-2 (5′-CAT GAC ACT CAA ACA GGC AT-3′) as the primer.
The sequences of ITS region obtained from the sequencing process were assembled, trimmed and edited using the Vector NT1 software v. 10.0 (InforMax Inc., USA). The sequence of ITS tail was determined using the ITS 2 annotation tool (Keller et al. 2009). The polymorphic portions were marked by IUPAC ambiguity codes. The ITS sequences of 36 isolates of P. noxius, representing all ITS types found in Taiwan, were submitted to NCBI (National Center for Biotechnology Information; http:// www.ncbi.nlm.mih.gov).

Phylogenetic analysis
The ITS sequences of 91 isolates of P. noxius from Taiwan were analyzed in order to understand the phylogenetic relationship among these isolates. Multiple alignments and minor adjustments of the sequences of these isolates were performed using clustal X 1.81 (Thompson et al. 1977) followed by BioEditor software. Sequence alignment was deposited at TreeBase (http://purl.org/phylo/ treebase/phylows/study/TB2:S16384). Phylogenetic relationships were analyzed using the Philip 3.67 software (Phylogeny Inference Package, Version 3.67) and the neighbor joining program with 1000 bootstrap replicates. The program of Treeview was used to view phylogenetic trees.
In order to study the phylogenetic relationship between isolates of P. noxius from Taiwan and other countries and other Phellinus species, the ITS sequences of all Phellinus species in the GenBank were retrieved. A total of 58 isolates belonging to 39 species of Phellinus was obtained and used for phylogenetic analysis ( Table 1). The ITS types L, M and S divided based on ITS length were used as local strains for analysis. The method described above was used for phylogenetic relationship analysis.

Phylogenetic relationship among Taiwanese isolates of P. noxius
A total of 91 isolates of P. noxius was obtained from 46 species of plants distributed in different geographic locations in Taiwan from 1991 to 2009 ( Table 2). Analysis of the phylogenetic relationship of these Taiwanese isolates did not show distinct grouping based on ITS sequences. The bootstrap values on the branches were very low and were all below 50% (data not shown) with accession number JN836346-JQ003229 (Tables 1, 2).

Nucleotide variation in ITS region among Taiwanese isolates of P. noxius
The examination of ITS nucleotide variation revealed the existence of 20 different kinds of variants, designated     as V1 to V20 in ITS1 and ITS2 but not 5.8S in the 91 Taiwanese isolates of P. noxius obtained in this study (Table 3) . The variation included insertion, deletion and single nucleotide polymorphism. Some isolates showed single nucleotide polymorphism among chromosomes in the same isolate.

Grouping based on ITS sequence length
The examination of ITS nucleotide variation also revealed the possible division into three distinct groups based on sequence length among the 91 isolates of P. noxius from Taiwan (Table 4). Isolates with long sequence of 613 bp were termed type L. Only two isolates belonged to this type. Isolates with median sequence length of 606-609 bp were termed type M. The majority of the Taiwanese isolates with a total of 72 isolates belonged to this type. Type M was further divided into 33 subtypes based on single nucleotide polymorphisms, single nucleotide deletion (V12), double nucleotide deletion (V19) and single nucleotide insertion (V20) (Tables 3, 4) . Isolates with short sequence of 601 bp were termed type S. Type S was further divided into two subtypes as a result of a single nucleotide polymorphism at position 114. Seventeen isolates belonged to this type.
Isolates of P. noxius from GenBank fitted or nearly fitted the M or S ITS types (in Taiwan; Table 1). Isolate CBS170.32 of unknown origin belonged to type S, so was the Japanese isolate. The isolate from India belonged to type M. Among the six isolates from Malaysia, isolate FRIM154 fitted the type S and isolate FRIM 638 nearly fitted the type M with 1 bp more than the Taiwanese type M. Isolates FRIM 618, FRIM 613 and FRIM 551 nearly fitted type S with 1-2 bp more than the Taiwanese type S, while isolates FRIM 147 was also close to type S with 2 bp less than the Taiwanese type S. However no isolates from other countries were founded to fit the Taiwanese type L in this study.

Relation between ITS types and hosts and locations from where P. noxius was found in Taiwan
Type L was detected only in Taichung City (Fig. 1). Type M was found in three cities and seven counties, while type S was found in two cities and eight counties. P. noxius was not found in Yilan County, Taoyuan County, Hsinchu Tounty and Pingtung County during this study.
Subtype S1 was found on 12 plant species located in three cities and seven counties, while subtype S2 was found only on flame gold-rain tree in Taichung City (Table 2). Other isolates found on flame gold-rain tree in  on custard apple in Taitung County and on orchid tree in Nantou County.

Phylogenetic analysis based on ITS sequences among Phellinus species
The ITS sequences of 58 isolates belonging to 39 species of Phellinus retrieved from GenBank and seven P. noxius isolates representing type L, type M and type S of ITS sequences from Taiwan were used in the analysis of the phylogenetic relationship among Phellinus species. The result showed that all the isolates of P. noxius including isolates from Taiwan and other countries were in the same clade with 100% bootstrap support (Fig. 2). The sequence similarity between P. noxius and other Phellinus species was less than 85%. The species most closely related to P. noxius was P. pachphloeus with 83% similarity, whereas the most distant species was P. badius with only 67% similarity.

Discussion
Results from this study showed that the isolates of P. noxius from Taiwan can be divided into type L, type M and type S based on ITS sequence length. From 1991 to 2009, 2 type L isolates, 82 type M isolates and 17 type S isolates were found on 46 plant species in Taiwan (Table 2). To our best knowledge, this is the first report of division of isolate from the same fungal species into different groups based on ITS length. P. noxius was reported from Taiwan as early as 1928 (Sawada 1928). It is conceivable that type M and type S may have existed in Taiwan for a very long period of time and that type M may have evolved in Taiwan earlier and became the predominant type. Only two isolates of type L was obtained from apricot at Taichung City. It is possible that type L may be a recent mutation from subtype M25 through an 6 bp insertion at position 116-117 (V5), and deletion at position 600-601 (V20) (Tables 3, 4). However, the possibility that it may be due to host specificity of type L has not been ruled out. The results also suggested the possibility that type S may originate from type M through an 8 bp deletion at the position between 135 and 142 (V9) (Tables 3, 4). After the deletion, the ITS sequences seem to become stable because there were only two subtypes among 17 isolates of type S obtained in this study. Moreover, the difference between subtype S1 and subtype S2 was the occurrence of a single nucleotide polymorphism at sequence position 114 (V4) in the latter.
Phellius noxius is one of the plant pathogens with a very wide host range. Among the more than 200 plant species representing 59 families 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). This is compatible with the discovery of great nucleotide variation in ITS region among isolates of P. noxius found in Taiwan in this study. The variation included 15 kinds of single nucleotide polymorphisms, three kinds of deletions and two kinds of insertions (Table 3).
Analysis of the ITS sequences of the Taiwanese isolates of P. noxius revealed that the 5.8 S region was identical in all isolates, while significant sequence variation was observed in ITS regions. This is in agreement with those reported with powdery mildews (Hirata and Takamatsu 1996) and Fusarium species (Naqvi et al. 2013). Our studies showed that the ITS1 was more variable than ITS 2 ( Table 3). The former contained 10 single nucleotide polymorphisms, one 8 bp deletion, one 1 bp deletion and one 6 bp insertion, while the latter consisted of only five single nucleotide polymorphisms, one 2 bp deletion and one 1 bp insertion.
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 (Fig. 2). Phylogenetic relationship among Phellinus species based on ITS sequences has been reported previously (Shin 2001;Wagner and Fischer 2002;Jeong et al. 2005;Decock et al. 2006). However, none of them  Fig. 2 The (unrooted) distance tree of phylogenic relationship of 17 Phellinus noxius isolates and other 38 species in Phellinus spp. based on the internal transcribed spacer (ITS1-5.8Sr DNA-ITS2) region of nuclear ribosomal DNA. Branch lengths and boot strap (1000 replicates) were displayed in the distance tree by using neighbor-joining and bootstrap methods of Philip 3.67 software. The distance bar is corresponded to 10 substitutions per 100 nucleotide sites has included P. noxius in their studies. P. noxius has been transferred to Phellinidium noxium (Corner) Bondartseva & S. Herrera in 1992 (Bondartseva et al. 1992). However, Phellinidium noxium was distinctly separated phylogenetically from other Phellinidium species (Dai 2010), indicating that more study is needed in the future.
During this study, P. noxius was not found in the counties of Yilan, Taoyuan, Hsinchu and Pingtung (Fig. 1). This does not mean that the fungus was not present in those areas because detection of P. noxius in those counties had been reported previously (Ann et al. 2002).

Conclusion
The 91 isolates of Phellinus noxius obtained from 46 plant species in Taiwan showed 20 different kinds of variation including single nucleotide polymorphisms, deletion, insertion in ITS1 and ITS2, but none in 5.8S. 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. Phylogenetic analysis of ITS sequence among Phellinus species showed the isolate of P. noxius were in the same clade distinctly separated from other Phellinus species.