Corresponding author: Quan Lu (
Academic editor: M. Stadler
The activity of the pine wood nematode
Wang HM, Lun YY, Lu Q, Liu HX, Decock C, Zhang XY (2018) Ophiostomatoid fungi associated with pines infected by
The pathogenic pine wood nematode (
The ophiostomatoid fungi are one of the most common fungal groups inhabiting wood infected by
The association of
Thus far, the association with
The aims of the current study were to investigate and identify the ophiostomatoid mycobiota associated with the nematode and sawyer in the epidemic forests of Shandong and Zhejiang Provinces in eastern China to facilitate the understanding of pine wilt disease infection and prevalence mechanisms. The two coastal provinces, Shandong and Zhejiang, represent new and historic epidemic areas, with
Fungi were isolated from 98 samples of
Strains of ophiostomatoid fungi isolated from pines infested by
Group | Species | Strain No. | Host | Origin (Latitude, Longitude) | Genbank No. | Collector | |
---|---|---|---|---|---|---|---|
|
β-tubulin | ||||||
A |
|
Yuyao, Zhejiang ( |
|
|
Q. Lu, YY Lun | ||
|
Yuyao, Zhejiang ( |
|
|
||||
|
Yuyao, Zhejiang ( |
|
|
||||
|
Yuyao, Zhejiang ( |
|
|
||||
B |
|
Yuyao, Zhejiang ( |
|
|
|||
|
Yuyao, Zhejiang ( |
|
|
||||
CFCC52170 (CXY1643) |
|
Yuyao, Zhejiang ( |
|
|
|||
C |
|
CXY1628 |
|
Changdao, Shandong ( |
|
|
|
CXY1631 |
|
Zhoushan, Zhejiang ( |
|
|
|||
CXY1635 |
|
Yuyao, Zhejiang ( |
|
|
|||
CXY1638 |
|
Fuyang, Zhejiang ( |
|
|
|||
CXY1639 |
|
Weihai, Shandong ( |
|
|
|||
D |
|
Fuyang, Zhejiang ( |
|
|
|||
|
Yuyao, Zhejiang ( |
|
|
||||
E |
|
CXY1623 |
|
Yuyao, Zhejiang ( |
|
– | |
F |
|
|
Weihai, Shandong ( |
|
– |
Sequences missing data are indicated by [–].
The ophiostomatoid fungal strains were incubated on 2%
A 5-mm mycelium disc was cut from an actively growing fungal colony using a sterile cork borer and placed at the centre of
DNA was extracted from freshly collected mycelia grown in liquid malt medium (20g malt extract in 1000 ml of deionised water) at 25 °C in the dark for 7 d using an Invisorb Spin Plant mini kit (Invitek, Berlin, Germany), following the manufacturer’s instructions. The internal transcribed spacer (
PCR reactions were performed in 25 ml volumes (2.5 mM MgCl2, 1X PCR buffer, 0.2 mM dNTP, 0.2 mM of each primer and 2.5 U of Taq polymerase). The conditions for
Sequencing reactions were performed using CEQ DTCS Quick Start KitH (Beckman Coulter, American), following the manufacturer’s instructions, with the same PCR primers as above. Nucleotide sequences were determined using a CEQ 2000 XL capillary automated sequencer (Beckman Coulter).
Contigs were subjected to BLAST searches of the NCBI GenBank database (
The most parsimonious trees generated by
The general-time-reversible (
For
In total, 240 strains belonging to
The information of references sequences used for phylogenetic analyses in this study.
Species | Strain No. | Host/insect | Country | Genbank No. | Reference | |
---|---|---|---|---|---|---|
|
β-tubulin | |||||
|
CBS125.89 |
|
Mexico |
|
|
de Beer et al. 2003 |
|
CMW19362 |
|
South Africa |
|
|
|
|
Soil | Brazil |
|
|
|
|
Soil | Brazil |
|
|
|
||
|
Ss383 |
|
Brazil |
|
|
|
|
Soil | Spain |
|
|
|
|
Soil | Spain |
|
|
|
||
|
CMW13016 | Hungary |
|
|
|
|
CMW13017 | Poland |
|
|
|
||
|
|
Argentina |
|
|
|
|
|
CPC 24638 |
|
Western Australia |
|
N/A |
|
|
CMW23057 |
|
South Africa |
|
|
|
|
CMW12529 | Soil | Canada |
|
|
|
CMW12527 | wheat-field soil | Germany |
|
|
|
|
|
CMW27319 |
|
Spain |
|
N/A |
|
CMW27900 |
|
Spain |
|
N/A |
|
|
|
CBS131.56 |
|
Japan |
|
|
|
CBS150.87 |
|
Japan |
|
|
|
|
|
CMW23049 |
|
South Africa |
|
|
|
|
CMW20676 |
|
South Africa |
|
|
|
|
CMW1103 |
|
South Africa |
|
|
|
|
MITS2474 | N/A | Mexico |
|
N/A |
|
Human | Franch |
|
N/A |
|
||
|
CMW9968 |
|
Azerbaijan |
|
|
|
|
CMW10563 |
|
Austria |
|
|
|
|
CBS138.50 | N/A | Canada |
|
|
|
|
CMW872 |
|
South Africa |
|
|
|
|
CMW2524 |
|
South Africa |
|
|
de Beer et al. 2003 |
CBS237.32 | pine pulp | Norway |
|
N/A | de Beer et al. 2003 | |
|
CMW38930 |
|
South Africa |
|
|
|
CMW38929 |
|
South Africa |
|
|
|
|
|
CMW14543 | Pine utility poles | Australia |
|
|
|
|
CMW135 |
|
USA |
|
N/A |
|
|
CBS185.86 |
|
Europe |
|
N/A |
|
|
Zoq16 | N/A | N/A |
|
N/A |
|
CBS186.86 |
|
USA |
|
|
|
|
|
CBS492.77 | USA |
|
|
|
|
|
CMW26484 |
|
South Africa |
|
|
|
CMW26483 |
|
South Africa |
|
|
|
|
|
C1084 |
|
Italy |
|
N/A |
|
|
|
Iberian Peninsula |
|
|
|
|
|
C1194 |
|
USA |
|
N/A |
|
|
WIN(M)41 | N/A | N/A |
|
N/A |
|
|
UM56 |
|
CanadaCanada |
|
|
|
|
C01-021 | Girdled |
Canada |
|
N/A |
|
C1086 | Soil | Sweden |
|
N/A |
|
|
|
CMW26813 |
|
South Africa |
|
|
|
CMW26818 |
|
South Africa |
|
|
|
|
|
CMW23196 |
|
Finland |
|
|
|
|
C1183 |
|
India |
|
N/A |
|
C1306 |
|
India |
|
N/A |
|
|
|
CMW7075 | N/A | USA |
|
N/A |
|
CMW22843 |
|
N/A |
|
N/A |
|
|
|
YCC099 | N/A | N/A |
|
N/A |
|
|
DAOM 229701 | Austria |
|
|
Jacobs and Kirisits 2013 | |
DAOM 229702 | Austria |
|
|
Jacobs and Kirisits 2013 | ||
K6/3/2 | Austria |
|
|
Jacobs and Kirisits 2013 | ||
|
PIR 18S | N/A | N/A |
|
N/A |
|
CMW22802 |
|
N/A |
|
N/A | Romón et al. 2005 | |
RJ-T144 | Poland |
|
N/A |
|
||
CMW28117 | Russia |
|
|
|
||
AU58.4 |
|
Canada |
|
N/A |
|
|
DAOM 212686 | N/A | Canada |
|
|
Jacobs and Kirisits 2013 | |
|
|
China |
|
|
|
|
|
CMW13221 | USA |
|
N/A |
|
|
CMW13222 | Canada |
|
N/A |
|
||
|
USA |
|
|
|
||
CMW651 |
|
USA |
|
|
|
|
|
|
China |
|
|
|
|
|
C1185 |
|
Russia |
|
N/A |
|
C510 |
|
USA |
|
N/A |
|
|
|
CXY1404 | China |
|
|
|
|
CXY1405 | China |
|
|
|
||
CXY1410 | China |
|
|
|
||
|
CMW23279 | Finland |
|
N/A |
|
|
CMW23278 | Finland |
|
|
|
||
|
C1087 | N/A | Germany |
|
N/A |
|
C1246 |
|
USA |
|
N/A |
|
|
|
92-634/302/6 | Canada |
|
|
|
|
D48/3 | N/A | Canada |
|
|
|
|
|
|
|
Zambia |
|
|
|
|
CMW9022 | Mexico |
|
|
|
|
|
|
China |
|
|
|
|
|
C970 |
|
United Kingdom |
|
N/A |
|
C969 |
|
United Kingdom |
|
N/A |
|
|
C1085 |
|
Germany |
|
N/A |
|
|
|
CBS434.77 | Woodpulp | USA |
|
|
|
|
CMW29497 | Finland |
|
|
|
|
CMW28135 |
|
Russia |
|
N/A |
|
|
|
Ophi 1B | N/A | N/A |
|
N/A |
|
Ophi 1A | N/A | N/A |
|
N/A |
|
|
|
AU160-38 |
|
North America |
|
N/A |
|
CMW12378 | China |
|
|
|
||
|
CBS189.86 |
|
USA |
|
|
|
|
C00-027a |
|
Canada |
|
NA |
|
C00-003 |
|
Canada |
|
|
|
|
|
C1182 |
|
Netherlands |
|
N/A |
|
|
|
Spain |
|
N/A |
|
|
|
C1224 |
|
Sweden |
|
N/A |
|
|
YCC612 | Japanese larch logs | Japan |
|
N/A |
|
|
N/A | Japan |
|
N/A |
|
|
|
N/A | N/A |
|
N/A |
|
|
|
DAOM 64746 | N/A | N/A |
|
|
|
|
WIN(M)1435 |
|
South Africa |
|
N/A |
|
N/A= represents information that are not available.
For each phylogenetic tree,
Phylograms of fungal associates of pine infected by
According to the
Phylogenetic inferences based on
Considering morphological differences, strains in groups A, B and D represent three undescribed species of
Based on the phylogenetic signals of the
The epithet reflects Zhejiang Province in China where the species was first collected.
Sexual morph perithecial: Perithecia occasional on 2%
Asexual morph: pesotum-like and sporothrix-like.
Pesotum-like: Conidiophores macronematous, synnematous, abundant in 2%
Light micrographs of
Colonies on 2%
Galleries of
CHINA, Zhejiang, Yuyao City, from
The epithet reflects the white colour of the colonies.
Sexual form: Unknown. Asexual form:
Light micrographs of
Colonies on 2%
Galleries of
CHINA, Zhejiang, Yuyao City, from
The epithet reflects the host tree,
Sexual form: Unknown. Asexual form:
Light micrographs of
Colonies on 2%
Galleries of
CHINA, Zhejiang Province, Yuyao City, from
In the current study, six ophiostomatoid species were found associated with pines infected by
In China,
Members of
Although
In the current study,
According to
The
According to both
In the current study, a relatively large number of ophiostomatoid fungal species associated with
This work was supported by the National Key R&D Programme of China (2017YFD0600103) and the National Natural Science Foundation of China (Project No.: 31770682). Cony Decock gratefully acknowledges the financial support received from the Belgian State (Belgian Federal Science Policy through the BCCMTM research programme). We are grateful to Professor Yuichi Yamaoka and Hugo Madrid for their invaluable suggestions to improve the manuscript.
Figure S1. Phylogram of fungal associates of pine infected by
Data type: phylogenetic data
Explanation note: The phylogram was generated after
Figure S2. Phylograms of fungal associates of pine infected by
Data type: phylogenetic data
Explanation note: The phylograms were generated after
Figure S3. Three
Data type: phylogenetic data