Botanical Studies

An International Journal

Impact Factor 1.452

Botanical Studies Cover Image
Open Access

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 Tsai1,
  • Pao-Jen Ann1,
  • Ruey-Fen Liou2,
  • Wen-Hsui Hsieh3 and
  • Wen-Hsiung Ko3Email author
Botanical StudiesAn International Journal201758:9

https://doi.org/10.1186/s40529-017-0162-1

Received: 29 August 2016

Accepted: 8 January 2017

Published: 16 January 2017

Abstract

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.

Keywords

Deletion Insertion ITS sequence Nucleotide variation Sequence length Single nucleotide polymorphism

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 (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.
Table 1

List of taxa of Phellinus noxius and related species from GenBank used for phylogenetic analysis

Species

Geographic origina

Strain no.b

ITS sequence length (bp)c

Accession no.

1. Phellinus alni

 

TW322

610

AY340041

2. P. badius

 

CBS 449.76

663

AY558609

3. P. baumii

 

MPNU 7006

711

AF200231

4. P. bicuspidatus

 

KCTC 6651

621

AY558610

5. P. calcitratus

  

584

JF894115

6. P. chrysoloma

  

644

AF055370

7. P. cinereus

 

05-37

620

AM931248

8. P. conchatus

 

CBS 167.29

708

AY558614

9. P. ferrugineovelutinus

 

CBS 218.48

542

AY558618

10. P. gilvus

 

ATCC26729

613

AF250932

11. P. hartigii

 

CBS 162.30

692

AY558621

12. P. h ippophaeicola

 

CBS 252.50

705

AY558622

13. P. igniarius

 

CFMR 5698

609

AY558623

 P. igniarius

 

KCTC6228

598

AF056192

14. P. igniarius var. trivialis

 

CBS 512.63

596

AY558624

15. P. johnsonianus

 

ATCC60051

702

AF250931

16. P. laevigatus

 

CFMR 5640

588

AY558626

17. P. linteus

 

MPNU 7002

670

AF200228

18. P. lundellii

 

CBS 540.72

605

AY558630

19. P. merrillii

 

PM950703-1

707

EU035310

20. P. nigricans

 

CBS 213.48

611

AY558631

 P. nigricans

 

H6002112

621

GQ383726

21. P. noxius

Taiwan

PN72.1

613-L

JQ003239

 P. noxius

Taiwan

PNP1.2

609-M

JN836341

 P. noxius

Taiwan

PN29.1

609-M

JN836344

 P. noxius

Taiwan

PNA4.1

609-M

JN836346

 P. noxius

Taiwan

PN5.2

608-M

JQ003233

 P. noxius

Taiwan

PNP4.2

607-M

JQ029276

 P. noxius

Taiwan

PN22.1

601-S

EF065630

 P. noxius

 

CBS170.32

601-S

EF065631

 P. noxius,

Japan

Tf566

601-S

JQ003238

 P. noxius

Malaysia

FRIM638

610-M

HQ400698

 P. noxius

Malaysia

FRIM618

602-S

HQ400699

 P. noxius

Malaysia

FRIM613

602-S

HQ400700

 P. noxius

Malaysia

FRIM551

603-S

HQ400702

 P. noxius

Malaysia

FRIM154

601-S

HQ400703

 P. noxius

Malaysia

FRIM147

599-S

HQ400704

 P. noxius

India

608-M

AB639022

22. P. occidentalis

 

CBS 196.55

706

AY558634

23. P. pachyphloeus = Inonotus pachphloeus

 

CBS 193.37

571

AY558635

24. P. pini

 

ATCC12240

635

AF250930

25. P. pini var. cancriformans

 

IMSNU 32031

636

AF200242

26. P. pomaceus

 

25

599

FR686572

27. P. populicola

 

CBS 638.75

599

AY558638

28. P. punctatus

 

CBS 386.66

649

AY558640

29. P. repandus

 

CBS 616.89

658

AF534076

30. P. rhabarbarinus

 

CBS 282.77

714

AY558642

31. P. ribis f. ulicis

 

CBS 579.50

653

AY558644

32. P. rimosus

 

MDJCBS86

608

DQ103885

33. P. robustus

 

KCTC 6657

679

AY558645

34. P. senex

 

CBS 442.76

578

AY558647

35. P. spiculosus

 

KTCC 6658

641

AY558648

36. P. tremulae

 

CBS 123.40

595

AY558650

37. P. tropicalis

 

CBS 617.89

636

AF534077

38. P. tuberculosus

 

CBS 171.32

600

AY558652

39. P. weirii

 

CNU 6017

620

AF251438

aThe country where P. noxius was isolated

b CBS Centraalbureau voor Schimmelcultures, NPMU National Programme Management Unit, KCTC Korean Collection for Type Cultures, ATCC American Type Culture Collection, CFMR Colegiul Fizicienilor Medicali din România, FRIM Forest Research Institute Malaysia, IMSNU Institute of Microbiology, Seoul National University, CNU Collection of Newcastle University

cL: ITS type L; M: ITS type M; S: ITS type S

Results

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).
Table 2

List of hosts, locations, ITS information, GenBank accession no. of strains of Phellinus noxius from Taiwan used in the study

Scientific name (common name)

Isolate

Location

Year of isolation

GenBank accession no.

Sequence ITS1/5.8S/ITS2 (bp)

ITS type

1. Annona squamosa (custard apple)

PNA4.1

Taitung County

1996

JN836346

609

M6

2. Araucaria cunninghamii (hook pine)

PN29.1

Taichung City

2004

JN836344

609

M4

 A. cunninghamii

PN30.1

Taichung City

2004

 

609

M4

3. Averrhoa carambola (carambola)

PNS1.1

Tainan City

1992

 

607

M32

4. Bauhinia × hybrid (butterfly tree)

PN40.2

Changhua County

2005

JQ003235

606

M33

5. Bauhinia variegata (orchid tree)

PN7.1

Nantou County

1996

 

609

M6

 B. variegata

PN35.1

Hualian County

2005

JN836349

609

M9

 B. variegata

PN35.2

Hualian County

2005

 

609

M9

6. Calocedrus formosana (Taiwan incense cedar)

PN70.2

Taichung City

2009

JQ003232

608

M23

7. Casuarina equisetifolia (ironwood tree)

PN22.1

Nantou County

1998

EF065630.1

601

S1

8. Cinnamomum kotoensis (botel tobago cinnamon tree)

PN74.2

Taitung County

2009

 

608

M24

 C. kotoensis

PN74.1

Taitung County

2009

JQ029271

608

M27

9. C. osmophloeum (Taiwan cinnamon)

PN50.1

Nantou County

2006

 

601

S1

 C. osmophloeum

PN51.1

Nantou County

2006

 

608

M24

10. Cinnamomun camphora (camphor)

PN32.1

Taichung City

2005

 

608

M24

 C. camphora

PN32.2

Taichung City

2005

 

608

M24

 C. camphora

PN94001.1

Nantou County

2005

 

607

M32

 C. camphora

94001.2

Nantou County

2005

 

607

M32

11. Citrus limon (lemon)

PNC1.1

Tainan City

2003

 

609

M1

 C. limon

PNC1.2

Tainan City

2003

 

609

M1

 C. limon

PNC4.1

Chiayi County

2006

 

607

M32

12. Delonix regia (flame tree)

PN37.1

Hualian County

2005

 

601

S1

 D. regia

PN37.2

Hualian County

2005

JQ029275

607

M31

 D. regia

PN42.1

Hualian County

2005

 

601

S1

13. Dimocarpus. longan (longan)

PNLn5.1

Tainan City

1992

JQ003236

601

S1

 D. longan

PNLn9.2

Changhua County

1998

JQ003226

608

M17

 D. longan

PNLn10.1

Tainan City

2003

 

609

M6

 D. longan

PNLn10.2

Tainan City

2003

JQ003222

609

M13

 D. longan

PNLn14.2

Changhua County

2006

 

601

S1

14. Diospyros kaki (persimmon)

PNPe1.1

Chiayi County

1991

 

609

M1

15. Duranta repens (creeping sky flower)

PN3.1

Nantou County

1996

JQ003231

608

M22

16. Eriobotrya japonica (loquat)

PNLo3.1

Taitung County

1997

 

601

S1

 E. japonica

PNLo5.1

Taitung County

2009

 

609

M1

17. Eucalyptus citriodora (lemon gum eucalyptus)

PN6.1

Nantou County

1996

 

608

M22

18. Ficus microcarpa (small-leafed banyan)

PN21.1

Miaoli County

2003

 

608

M24

 F. microcarpa

PN21.2

Miaoli County

2003

 

608

M24

 F. microcarpa

PN12.1

Taichung City

1996

JQ029274

607

M30

 F. microcarpa

PN26

Nantou County

2003

 

608

M24

 F. microcarpa

PN28.2

Taichung City

2004

 

608

M19

 F. microcarpa

PN49.2

Taichung City

2005

JQ003227

608

M18

 F. microcarpa

PN57.1

Taichung City

2005

 

609

M7

 F. microcarpa

PN75.1

Taichung City

2009

 

607

M30

 F. microcarpa

PN76.1

Taichung City

2009

 

609

M4

19.  Ficus pumila var. awkeotsang (jellyfig)

PN10.1

Chiayi County

1991

JQ029272

608

M28

20.  F. religiosa (botree fig)

PN90.1

Taichung

2009

JN836342

609

M2

21. Juniperus chinensis var. kaizuka (dragon juniper).

PN65.1

Nantou County

2007

 

609

M4

22. Kigelia pinnata (sausage tree)

PN14.1

Nantou County

1998

JN836348

609

M8

23. Koelreuteria henryi (flame gold-rain tree)

PN94.1

Taichung City

2009

JQ003237

601

S2

K. henryi

PN33.2

Hualian County

2005

JQ003223

609

M14

K. henryi

PN41.1

Hualian County

2005

 

609

M14

K. henryi

PN41.2

Hualian County

2005

 

609

M14

24. Liquidambar formosana (maple)

R9218

New Taipei city

1992

 

608

M15

25. Litchi chinensis (litchi)

PNL2.1

Chiayi County

1992

JN836347

609

M7

L. chinensis

PNL2.2

Chiayi County

1992

 

601

S1

L. chinensis

PNL5.1

Kaohsiung City

2003

JQ029273

607

M29

L. chinensis

PNL5.2

Kaohsiung City

2003

 

607

M29

26. Mangifera indica (mango)

PNM4.1

Changhua County

2009

 

609

M6

27. Melaleuca bracteata ‘Revolution Gold’ (white cloud tree)

PN73.2

Taichung City

2009

 

609

M6

M. bracteata ‘Revolution Gold’

PN73.1

Taichung City

2009

 

609

M6

28. Murraya paniculata (orange jasmine)

PN5.1

Nantou County

1996

 

608

M24

M. paniculata

PN5.2

Nantou County

1996

JQ003233

608

M24

M. paniculata

PN25.1

New Taipei city

2004

 

601

S1

M. paniculata

PN25.2

New Taipei city

2004

 

601

S1

29. Oncidium Gower Ramsey

PN44

Yungling County

2005

 

601

S1

30. Osmanthus fragrans (osmanthus)

PN140.1

Changhua County

2009

JQ003221

609

M12

31. Psidium guajava (guava)

PN98007

Kaohsiung City

2009

JQ029270

608

M26

32. Podocarpus macrophyllus (long-leaved podocarpus)

PN98.3

Taichung City

2009

 

601

S1

33. Prunus armeniaca (apricot)

PN72.1

Taichung City

2009

JQ003239

613

L1

P. armeniaca

PN72.2

Taichung City

2009

 

613

L1

34. Prunus campanulata (Taiwan cherry)

PN71.1

Taichung City

2009

JN386350

609

M10

P. campanulata

PN71.2

Taichung City

2009

 

609

M10

35. Prunus mume (plum)

PNP1.2

Kaohsiung City

1991

JN836341

609

M1

P. mume

PNP2.1

Nantou County

1996

JN836345

609

M5

36. Prunus persica (peach)

PNP5.1

Nantou County

1999

JQ003228

608

M19

P. persica

PNP10.1

Changhua County

2005

JQ003225

608

M16

37. Pterocarpus indicus (rose wood)

PN104.1

Taichung City

2009

JQ003220

609

M11

38. Pyrus pyrifolia (pear)

PNP4.1

Miaoli County

2003

 

601

S1

P. pyrifolia

PNP4.2

Taichung City

1998

JQ029276

607

M32

P. pyrifolia

PNP9.1

Nantou County

2004

JN836343

609

M3

39. Schinus terebinthifolius (Brazilian peppertree)

PN48.1

Taichung City

2005

 

601

S1

S. terebinthifolinus

PN48.2

Taichung City

2005

 

601

S1

40. Spathodea campanulata (African tulip tree)

PN147

Changhua County

2009

 

601

S1

41. Sterculia nobilis (ping-pong)

PN17.1

Nantou County

1999

JQ003224

608

M15

S. nobilis

PN84.1

Taichung City

2009

 

607

M32

S. nobilis

PN124.1

Taichung City

2009

 

609

M6

42. Syzygium samarangense (wax apple)

PNW1.1

Chiayi County

1991

JQ003234

608

M25

43. Terminalia catappa (Indian almond)

PN2.1

Chiayi County

1996

 

609

M4

44. T. catappa

PN63.1

Changhua County

2007

 

607

M32

45. Toona sinensis, Cedrela sinensis (Chinese cedar)

PN64.1

Taichung City

2007

JQ003230

608

M21

46. Vitis vinifera (grape)

PNG1.1

Nantou County

1999

 

609

M6

Zizyphus mauritiana (Indian jujube)

PNZ2.1

Kaohsiung City

2001

JQ003229

608

M20

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.
Table 3

Nucleotide variation in ITS detected among isolates of Phellinus noxius in Taiwan

Kind of variant

Sequence position

Nucleotide variation

Single nucleotide polymorphism

Deletion

Insertion

ITS1

    

 V1/V1*

19

G, A/R

  

 V2

31

T, C

  

 V3/V3*

32

G, C/S

  

 V4

114

G, C

  

 V5

116–117

  

GGAGAG

 V6

117–118

TG, AT

  

 V7

125–126

TC, AT

  

 V8/V8*

129

T, A/C

  

 V9

135–142

 

ATTTATTC

 

 V10

152

A, G

  

 V11

168

C, T

  

 V12

193

 

A

 

 V13

197

T, C

  

ITS2

    

 V14/V14*

420

T, C/Y

  

 V15/V15*

442

G, A/R

  

 V16/V16*

469

A, G/R

  

 V17/V17*

546

A, G/R

  

 V18/V18*

593

C, G/S

  

 V19

594–595

 

AC

 

 V20

600–601

  

C

*The variants with an asterisk symbol represent isolates with single nucleotide polymorphisms 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.
Table 4

ITS types and subtypes among Phellinus noxius isolates from Taiwan

ITS type and subtype

ITS sequence length (bp)

Nucleotide variation

Representative isolate (total no.)

Type L

   

 L1

613

V1, V3, V5, V15, V16, V19

PN72.1 (2)

Type M

   

 M1

609

 

PNP1.2 (5)

 M2

609

V16

PN90.1 (1)

 M3

609

V17

PNP9.1 (1)

 M4

609

V18

PN29.1 (5)

 M5

609

V1, V3

PNP2.1 (1)

 M6

609

V14, V15

PNA4.1 (8)

 M7

609

V15, V16

PNL2.1 (2)

 M8

609

V3, V14, V15

PN14.1 (1)

 M9

609

V1, V3, V16, V17

PN35.1 (2)

 M10

609

V1, V3, V11, V15, V17

PN71.1 (2)

 M11

609

V15*, V18*

PN104.1 (1)

 M12

609

V1, V15*, V16

PN140.1 (1)

 M13

609

V14*, V15, V17*

PNLn10.2 (1)

 M14

609

V1*, V3*, V15*, V16*

PN33.2 (3)

 M15

608

V3, V12

PN17.1 (2)

 M16

608

V3, V12, V17

PNP10.1 (1)

 M17

608

V10, V12, V15, V16

PNLn9.2 (1)

 M18

608

V1, V3, V11, V12

PN49.2 (1)

 M19

608

V1, V3, V8, V11, V12, V16

PNP5.1 (2)

 M20

608

V1, V3, V6, V11, V12, V15

PNZ2.1 (1)

 M21

608

V1, V2, V3, V11, V12, V15, V16

PN64.1 (1)

 M22

608

V1, V2, V3, V8, V11, V12, V13, V16

PN3.1 (2)

 M23

608

V1, V2, V3, V6, V7, V11, V12, V15, V16

PN70.2 (1)

 M24

608

V15, V16, V19, V20

PN5.2 (9)

 M25

608

V1, V3, V15, V16, V19, V20

PNW1.1 (1)

 M26

608

V3, V12, V15*, V16*

PN98007 (1)

 M27

608

V15, V16*, V19, V20

PN74.1 (1)

 M28

608

V1, V3*, V15, V19, V20

PN10.1 (1)

 M29

607

V19

PNL5.1 (2)

 M30

607

V16, V19

PN12.1 (2)

 M31

607

V15, V16, V19

PN37.2 (1)

 M32

607

V1, V3, V15, V16, V19

PNP4.2 (7)

 M33

606

V1, V2, V3, V6, V7, V11, V12, V15, V16, V19

PN40.2 (1)

Type S

   

 S1

601

V9

PNLn5.1 (16)

 S2

601

V4, V9

PN94.1 (1)

*The variants with an asterisk symbol represent isolates with single nucleotide polymorphisms among chromosomes in the same isolate

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.
Fig. 1

Distribution of different ITS types of Phellinus noxius from Taiwan. Number referred to 1 New Taipei City, 2 Taoyuan County, 3 Hsinchu County, 4 Miaoli County, 5 Taichung City, 6 Nantou County, 7 Changhua County, 8 Yunlin County, 9 Chiayi County, 10 Tainan City, 11 Kaohsiung City, 12 Pingtung County, 13 Yilan County, 14 Hualian County, 15 Taitung County

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 Hualian County belonged to subtype M14. This study also revealed that isolates of P. noxius obtained from the same plant species in the same location may belong to different subtypes. In Taichung City, isolates of P. noxius found on small-leafed banyan consisted of subtypes M4, M7, M18, M19 and M30. Similarly, isolates obtained from longan in Tainan City contained subtypes S1, M6 and M13. It was also found that isolates obtained from the same host in different locations may belong to the same subtype. For examples, subtype S1 on longan was found in Tainan City and Changhua County, while subtype M24 on small-leafed banyan was found in Miaoli County and Nantou County. Isolates obtained from different hosts in different locations may also belong to the same subtype. For examples, subtype M1 was found on lemon in Tainan City and on persimmon in Chiayi County, while subtype M6 was found 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.
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

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 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.

Declarations

Authors’ contributions

WHK designed the experiments and wrote the manuscript; PJA, RFL and WHH conceived the experiments; JNT performed the experiments. All authors read and approved the final manuscript.

Acknowledgements

The study was supported by the Grants from Council of Agriculture (101N-10.2.1-N-C7(3)) and Ministry of Science and Technology (NSC 101-2321-B-005-004) of Taiwan.

Competing interests

The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors’ Affiliations

(1)
Division of Plant Pathology, Taiwan Agricultural Research Institute
(2)
Department of Plant Pathology and Microbiology, National Taiwan University
(3)
Department of Plant Pathology, National Chung Hsing University

References

  1. Abe Y, Kobayashi T, Onuki M, Hattori T, Tsurumachi M (1995) Brown root rot of trees caused by Phellinus noxius in windbreaks on Ishigaki Island, Japan-incidence of disease, pathogen and artificial inoculation. Ann Phytopathol Soc Jpn 61:425–433View ArticleGoogle Scholar
  2. Ann PJ, Ko WH (1992) Decline of longan trees: association with brown root rot caused by Phellinus noxius. Plant Pathol Bull 1:19–25Google Scholar
  3. Ann PJ, Lee HL, Tsai JN (1999) Survey of brown root disease of fruit and ornamental trees caused by Phellinus noxius in Taiwan. Plant Pathol Bull 8:51–60Google Scholar
  4. Ann PJ, Chang TT, Ko WH (2002) Phellinus noxius brown root rot of fruit and ornamental trees in Taiwan. Plant Dis 86:820–826View ArticleGoogle Scholar
  5. Boesewinkle HJ (1976) Storage of fungal cultures in water. Trans Br Mycol Soc 66:183–185View ArticleGoogle Scholar
  6. Bondartseva MA, Herrera SD, Sandoval FC (1992) Taxonomical problems of the Cuban Hymenochaetaceous fungi. Mikologiia i Fitopatologiia 26:1–13Google Scholar
  7. Chang TT (1995) Decline of nine tree species associated with brown root rot caused by Phellinus noxius in Taiwan. Plant Dis 79:962–965View ArticleGoogle Scholar
  8. Chang TT (1996) Survival of Phellinus noxius in soil and in the roots of dead host plants. Phytopathology 86:272–276View ArticleGoogle Scholar
  9. Chang TT, Yang WW (1998) Phellinus noxius in Taiwan: distribution, host plants and the pH and texture of the rhizosphere soils of infected host. Mycol Res 102:1085–1088View ArticleGoogle Scholar
  10. Dai YC, Zhou LW, Cui BK, Chen YQ, Decock C (2010) Current advances in Phellinus sensu lato: medicinal species, functions, metabolites and mechanisms. Appl Microbial Biotech 87:1587–1593View ArticleGoogle Scholar
  11. Decock C, Figueroa SH, Robledo G, Castillo G (2006) Phellinus caribaeo-quercicolus sp. nov. parasitic on Quercus cubana: taxonomy and preliminary phylogenetic relationships. Mycologia 298:265–274View ArticleGoogle Scholar
  12. Hirata T, Takamatsu S (1996) Nucleotide sequence diversity of rDNA internal transcribed spacers extracted from conidia and cleistothecia of several powdery mildew fungi. Mycoscience 39:283–288View ArticleGoogle Scholar
  13. Jeong WJ, Lim YW, Lee JS, Jung HS (2005) Phylogeny of Phellinus and related genera inferred from combined data of ITS and mitochondrial SSU rDNA sequences. J Microbiol Biotechnol 15:1028–1038Google Scholar
  14. Keller A, Schleicher T, Schultz J, Müller T, Dandekar T, Wolf M (2009) 5.8S-28S rRNA interaction and HMM-based ITS2 annotation. Gene 430:50–57View ArticlePubMedGoogle Scholar
  15. Ko WH (2003) Long-term storage and survival structure of three species of Phytophthora in water. J Gen Plant Pathol 69:186–188Google Scholar
  16. Ko WH, Yang CH, Lin MJ, Chen CY, Tsou YJ (2011) Humicola phialophorides sp. nov. from soil with potential for biological control of plant diseases. Bot Stud 52:197–202Google Scholar
  17. Naqvi SKB, Ahmed S, Rauf CA, Naqvi SMS (2013) Application and sequencing of interval transcribed regions 1 & 2, and 5.8S rDNA from local isolates of Fusarium species. Pak J Bot 45:301–307Google Scholar
  18. Pegler DN, Waterston JM (1968) Phellinus noxius No. 195. In: Descriptions of pathogenic fungi and bacteria. Commonwealth Mycological Institute, KewGoogle Scholar
  19. Sawada K (1928) Camphor tree decline. Descr Catal Formos Fungi 4:86–91Google Scholar
  20. Shin KS (2001) Identification of some Phellinus spp. Mycobiology 29:190–195Google Scholar
  21. Tai FL (1979) Sylloge fungorum sinicorum. Science Press, PekingGoogle Scholar
  22. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876-4882. doi:https://doi.org/10.1093/nar/25.24.4876 View ArticlePubMedPubMed CentralGoogle Scholar
  23. Wagner T, Fischer M (2002) Proceeding towards a natural classification of the worldwide taxa Phellinus s. l. and Inonotus s. l., and phylogenetic relationships of allied genera. Mycologia 94:998–1016View ArticlePubMedGoogle Scholar
  24. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of ribosomal RNA genes for applications. In: Innis MA, Gelfand DH, Snirsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego, pp 315–322Google Scholar

Copyright

© The Author(s) 2017