Research Article |
Corresponding author: Z. Wilhelm de Beer ( wilhelm.debeer@fabi.up.ac.za ) Academic editor: Cecile Gueidan
© 2017 Runlei Chang, Tuan A. Duong, Stephen J. Taerum, Michael J. Wingfield, Xudong Zhou, Z. Wilhelm de Beer.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Chang R, Duong TA, Taerum SJ, Wingfield MJ, Zhou X, de Beer ZW (2017) Ophiostomatoid fungi associated with conifer-infesting beetles and their phoretic mites in Yunnan, China. MycoKeys 28: 19-64. https://doi.org/10.3897/mycokeys.28.21758
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The Ophiostomatales is an Ascomycete order of fungi that accommodates several tree pathogens and many species that degrade wood. These fungi are commonly vectored by Scolytine bark and ambrosia beetles. In recent years it has also been shown that hyperphoretic mites on these beetles can vector some Ophiostomatales. Little is known regarding the Ophiostomatales in China and we have consequently explored the diversity of these fungi associated with conifer-infesting beetles and mites in Yunnan province. Galleries and beetles were collected for 17 beetle species, while 13 mite species were obtained from six of these beetle species. Collectively, 340 fungal isolates were obtained, 45 from beetles, 184 from mites, 56 from galleries and 55 isolates where the specific niche was not clear. DNA sequences for five gene regions (ITS, LSU, BT, EF, and CAL) were determined for fungal isolates representing different morphological groups. Phylogenetic analyses confirmed the presence of 19 fungal taxa, including five novel species described here as Ophiostoma acarorum sp. nov., Ophiostoma brevipilosi sp. nov., Graphilbum kesiyae sp. nov., Graphilbum puerense sp. nov., and Leptographium ningerense sp. nov. Ophiostoma ips was the most frequently isolated species, representing approximately 31% of all isolates. Six of 19 taxa were present on mites, beetles and in the galleries of the beetles, while three species were found on mites and galleries. Two species were found only on mites and one species only on a beetle. Although the numbers of beetles and mites were insufficient to provide statistical inferences, this study confirmed that mites are important vectors of the Ophiostomatales in China. We hypothesize that these mites are most likely responsible for horizontal transfer of fungal species between galleries of different beetle species. The fact that half of the fungal species found were new to science, suggests that the forests of east Asia include many undescribed Ophiostomatales yet to be discovered.
Symbiont, species diversity, fungal vector, Ophiostoma quercus
The ophiostomatoid fungi represent a polyphyletic group of tree- or wood-infecting fungi, most often staining freshly exposed sapwood and thus lowering the value of timber (
Bark beetles (Coleoptera: Scolytinae) are well known vectors of ophiostomatoid fungi (
Along with the fungi, mites are also common symbionts of bark beetles (
As is the case with bark beetles, their mite associates can also vector ophiostomatoid fungi (
Research on the interactions between ophiostomatoid fungi and bark beetles have been ongoing for more than a century in North America (
The first new beetle associated ophiostomatoid species described from China for which the description was supported by DNA sequence data, was Grosmannia yunnanensis (
Yunnan province forms part of in the southwestern forest zone, the second largest forest area in China (http://www.china.org.cn/english/shuzi-en/en-shuzi/gq/htm/zrzy-land-sl.htm). This province has a unique geography where three climatic regions meet: the eastern Asia monsoon region, the Tibetan plateau region, and the tropical monsoon region of southern Asia and Indo-China. As a result , species diversity in Yunnan province is high when compared to other parts of China. For example, more than 18000 plant species and 1836 vertebrate species are found here which represent 51.6% and 54.8% respectively of total species numbers in China (
Three surveys were conducted in Yunnan during the flight period of bark beetles in July 2001, July 2002, and between June and September 2010 (Table
Conifer-infesting bark and ambrosia beetles (Scolytinae), weevils (Molytinae), true weevils (Cossoninae), cylindrical bark beetles (Colydiinae), and mites (Acari) collected from Yunnan in this study. Numbers in the table refer to number of mite individuals collected.
Beetle species | Family | Subfamily | Tree host | Origin | Collection dates | Mite species | ||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
a | b | c | d | e | f | g | h | i | j | k | l | m | Total | |||||||
A | Coccotrypes cyperi | Curculionidae | Scolytinae | Pinus kesiya | Ning’er | Sep. 2010 | 16 | 3 | 9 | 0 | 0 | 0 | 2 | 4 | 0 | 0 | 0 | 0 | 3 | 37 |
B | Cyrtogenius luteus | Curculionidae | Scolytinae | P. kesiya | Ning’er | Sep. 2010 | 0 | 2 | 10 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 12 |
C | Hylurgops major | Curculionidae | Scolytinae | Pinus yunnanensis | Zixishan | Jul. 2002 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
D | Ips acuminatus | Curculionidae | Scolytinae | P. kesiya | Ning’er, Puer | Jun. 2010 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 8 |
E | Lasconotus sp. | Zopheridae | Colydiinae | P. kesiya | Ning’er | Jun. 2010 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
F | Orthotomicus angulatus | Curculionidae | Scolytinae | P. kesiya | Ning’er | Sep. 2010 | 0 | 0 | 4 | 13 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 19 |
G | Pissodes sp. | Curculionidae | Molytinae | Pinus armandii | Lijiang, Midu | Jul. 2001 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
H | Polygraphus aterrimus | Curculionidae | Scolytinae | P. kesiya | Ning’er | Jun. 2010 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
I | Polygraphus sp. | Curculionidae | Scolytinae | P. kesiya | Ning’er | Jun. 2010 | 0 | 0 | 0 | 6 | 0 | 4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 10 |
J | P. szemaoensis | Curculionidae | Scolytinae | P. kesiya | Ning’er, Simao | Jun. 2010 | 0 | 0 | 0 | 14 | 1 | 0 | 0 | 0 | 0 | 2 | 2 | 0 | 1 | 20 |
K | P. verrucifrons | Curculionidae | Scolytinae | P. yunnanensis | Lufeng | Jul. 2002 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
L | Stenoscelis sp. | Curculionidae | Cossoninae | P. kesiya | Ning’er | Jun. 2010 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
M | Tomicus minor | Curculionidae | Scolytinae | P. yunnanensis | Zixishan | Jul. 2002 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
N | T. piniperda | Curculionidae | Scolytinae | P. yunnanensis | Lufeng, Zixishan, Changhu | Jul. 2002 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
O | T. brevipilosus | Curculionidae | Scolytinae | P. kesiya | Ning’er | Jun. 2010 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
P | Unknown sp.1 | Curculionidae | Scolytinae | P. semaonensis | Chuxiong | Jul. 2002 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Q | Unknown sp.2 | Curculionidae | Molytinae | Tsuga sp. | Dali | Jul. 2002 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Total | 16 | 5 | 23 | 39 | 1 | 4 | 2 | 4 | 1 | 2 | 2 | 1 | 6 | 106 |
Fungi were isolated from the bark beetles and their galleries following the methods described by
Isolates were grown on 2% MEA in Petri dishes. DNA was extracted using PrepMan Ultra Sample Preparation reagent (Applied Biosystems, Foster City, CA) following the manufacturer’s instructions. The primer pairs ITS1F (
The sequences obtained using the forward and reverse primers were aligned in Geneious Pro v. 7.1.4 (Biomatters, Auckland, New Zealand). ITS sequences were submitted to BLAST searches in NCBI Genbank for preliminary identifications. Based on these results, the ITS data were separated into different data sets according to genus. ITS2-LSU data were used for analyses of taxa residing in Leptographiums.l., while ITS was used for analyses of taxa belonging to the other genera. The BT, EF and CAL data were separated into smaller data sets based on the species complexes as defined by
Isolates of ophiostomatoid fungi obtained from different beetles, their galleries and mites in Yunnan.
Taxon | Species | Isolate number1,2 | Host | Location | Beetle | G/B/M3 | GenBank number4 | ||||
---|---|---|---|---|---|---|---|---|---|---|---|
CMW | CBS | ITS/ITS2-LSU5 | BT | EF | CAL | ||||||
Ophiostomatales | |||||||||||
1 | Sporothrix sp. A | 41787 | Pinus kesiya | Ning’er | Coccotrypes cyperi | M | MG205645 | MG205681 | – | – | |
2 | S. nebularis | 11791 | Pinus yunnanensis | Zixishan | Tomicus piniperda | * | MG205646 | MG205682 | – | – | |
41762 | P. kesiya | Ning’er | Co. cyperi | M | MG205647 | MG205683 | – | – | |||
41776 | P. kesiya | Ning’er | Co. cyperi | M | MG205648 | MG205684 | – | – | |||
41779 | P. kesiya | Ning’er | Co. cyperi | M | MG205649 | =MG205684 | – | – | |||
41782 | P. kesiya | Ning’er | Co. cyperi | M | MG205650 | =MG205684 | – | – | |||
41816 | P. kesiya | Ning’er | Cyrtogenius luteus | M | MG205651 | MG205685 | – | – | |||
41819 | P. kesiya | Ning’er | Orthotomicus angulatus | M | MG205652 | =MG205685 | – | – | |||
41835 | P. kesiya | Ning’er | Polygraphus sp. | M | MG205653 | =MG205684 | – | – | |||
3 | Ophiostoma acarorum sp. nov. | 41630 | P. kesiya | Ning’er | Ips acuminatus | G | =MG205656 | MG205686 | – | – | |
41641 | P. kesiya | Ning’er | Polygraphus szemaoensis | G | =MG205656 | =MG205686 | – | – | |||
41642 | P. kesiya | Ning’er | P. szemaoensis | G | =MG205656 | =MG205686 | – | – | |||
41647 | P. kesiya | Pu’er | I. acuminatus | M | =MG205656 | =MG205686 | – | – | |||
41789 | P. kesiya | Ning’er | Cy. luteus | M | =MG205657 | =MG205686 | – | – | |||
41791 | P. kesiya | Ning’er | Cy. luteus | M | =MG205657 | =MG205686 | – | – | |||
41795 | P. kesiya | Ning’er | Cy. luteus | M | =MG205657 | =MG205686 | – | – | |||
41798 | 139643 | P. kesiya | Ning’er | Cy. luteus | M | =MG205657 | =MG205686 | – | – | ||
41812 | 139658 | P. kesiya | Ning’er | Cy. luteus | M | MG205656 | =MG205686 | – | – | ||
41850H | 139748 | P. kesiya | Ning’er | O. angulatus | M | MG205657 | =MG205686 | – | – | ||
41852 | P. kesiya | Ning’er | Co. cyperi | M | =MG205657 | =MG205686 | – | – | |||
41987 | P. kesiya | Ning’er | I. acuminatus | G | =MG205656 | =MG205686 | – | – | |||
4 | O. ips | 41620 | P. kesiya | Ning’er | I. acuminatus | G | MG205658 | MG205687 | – | – | |
41644 | P. kesiya | Pu’er | I. acuminatus | B | =MG205658 | =MG205687 | – | – | |||
4 | O. ips | 41653 | P. kesiya | Ning’er | P. szemaoensis | G | =MG205658 | MG205688 | – | – | |
41695 | P. kesiya | Simao | P. szemaoensis | M | =MG205658 | =MG205688 | – | – | |||
41697 | P. kesiya | Simao | P. szemaoensis | B | =MG205658 | =MG205688 | – | – | |||
41709 | P. kesiya | Simao | P. szemaoensis | B | =MG205658 | =MG205688 | – | – | |||
41745 | P. kesiya | Ning’er | Polygraphus sp. | M | =MG205658 | =MG205688 | – | – | |||
41916 | P. kesiya | Ning’er | P. szemaoensis | G | =MG205658 | =MG205688 | – | – | |||
41993 | P. kesiya | Ning’er | I. acuminatus | M | =MG205658 | =MG205687 | – | – | |||
5 | Ophiostoma sp. B | 12032 | Pinus semaonensis | Chuxiong | Unknown sp.1 | * | MG205659 | MG205689 | MG205731 | – | |
6 | O. brevipilosi sp. nov. | 41624 | 139661 | P. kesiya | Ning’er | Tomicus brevipilosus | B | =MG205660 | MG205690 | MG205732 | – |
41662H | 139659 | P. kesiya | Ning’er | T. brevipilosus | B | MG205660 | =MG205690 | =MG205732 | – | ||
41760 | P. kesiya | Ning’er | T. brevipilosus | B | =MG205660 | =MG205690 | =MG205732 | – | |||
41873 | 139660 | P. kesiya | Ning’er | T. brevipilosus | B | =MG205660 | =MG205690 | =MG205732 | – | ||
41932 | P. kesiya | Ning’er | T. brevipilosus | B | =MG205660 | =MG205690 | =MG205732 | – | |||
41995 | P. kesiya | Ning’er | T. brevipilosus | B | =MG205660 | =MG205690 | =MG205732 | – | |||
7 | O. setosum | 12152 | Tsuga sp. | Dali | Unknown sp.2 | * | MG205661 | MG205691 | MG205733 | – | |
12192 | Tsuga sp. | Dali | Unknown sp.2 | * | =MG205661 | =MG205691 | MG205734 | – | |||
12337 | Tsuga sp. | Dali | Unknown sp.2 | * | =MG205661 | =MG205691 | MG205735 | – | |||
8 | O. quercus | 11747 | P. yunnanensis | Lufeng | Polygraphus verrucifrons | * | =AF198238 | =FJ455565 | MG205736 | – | |
11748 | P. yunnanensis | Lufeng | P. verrucifrons | * | =AF198238 | =FJ455565 | =MG205736 | – | |||
11756 | P. yunnanensis | Lufeng | P. verrucifrons | * | MG205662 | MG205692 | MG205737 | – | |||
11806 | P. yunnanensis | Changhu | T. piniperda | * | =FJ434947 | =FJ455565 | MG205738 | – | |||
11807 | P. yunnanensis | Changhu | T. piniperda | * | =FJ434947 | =AY466647 | =MG205738 | – | |||
11981 | Abies sp. | Chuxiong | Unknown sp.1 | * | =FJ434947 | =FJ455570 | MG205739 | – | |||
12015 | P. semaonensis | Chuxiong | Unknown sp.1 | * | =AY466624 | MG205693 | MG205740 | – | |||
12037 | Unknown | Chuxiong | Unknown sp.1 | * | =AY466624 | MG205694 | MG205741 | – | |||
12039 | Unknown | Chuxiong | Unknown sp.1 | * | =FJ434947 | MG205695 | MG205742 | – | |||
8 | O. quercus | 12122 | Tsuga sp. | Dali | Unknown sp.2 | * | =FJ434947 | MG205696 | MG205743 | – | |
12146 | Tsuga sp. | Dali | Unknown sp.2 | * | =FJ434947 | =MG205696 | =MG205743 | – | |||
12185 | Tsuga sp. | Dali | Unknown sp.2 | * | =FJ434947 | MG205697 | =MG205740 | – | |||
12195 | Tsuga sp. | Dali | Unknown sp.2 | * | =FJ434947 | =MG205697 | =MG205740 | – | |||
12286 | Tsuga sp. | Dali | Unknown sp.2 | * | =FJ434947 | =FJ455570 | MG205744 | – | |||
12350 | Tsuga sp. | Dali | Unknown sp.2 | * | =FJ434947 | MG205698 | MG205745 | – | |||
12359 | Tsuga sp. | Dali | Unknown sp.2 | * | =AF198238 | =FJ455570 | MG205746 | – | |||
12364 | Tsuga sp. | Dali | Unknown sp.2 | * | =AF198238 | MG205699 | MG214780 | – | |||
12370 | Tsuga sp. | Dali | Unknown sp.2 | * | =AF198238 | =MG205698 | MG205747 | – | |||
12382 | Tsuga sp. | Dali | Unknown sp.2 | * | =AF198238 | =FJ455570 | MG205748 | – | |||
41659 | P. kesiya | Pu’er | I. acuminatus | G | MG205664 | MG205700 | MG205749 | – | |||
41693 | P. kesiya | Simao | P. szemaoensis | G | =MG205664 | =MG205700 | =MG205749 | – | |||
41715 | P. kesiya | Simao | P. szemaoensis | B | =MG205664 | =MG205700 | =MG205749 | – | |||
41718 | P. kesiya | Simao | P. szemaoensis | B | =MG205664 | =MG205700 | =MG205749 | – | |||
41724 | P. kesiya | Ning’er | P. szemaoensis | B | MG205665 | MG205701 | MG205750 | – | |||
41732 | P. kesiya | Ning’er | Cy. luteus | G | =MG205664 | =MG205700 | =MG205749 | – | |||
9 | O. tsotsi | 41730 | P. kesiya | Ning’er | Co. cyperi | G | =FJ441284 | MG205704 | MG205755 | – | |
41731 | P. kesiya | Ning’er | Co. cyperi | G | =FJ441284 | MG205705 | MG205756 | – | |||
41733 | P. kesiya | Ning’er | Cy. luteus | G | =FJ441284 | =MG205704 | =MG205755 | – | |||
41734 | P. kesiya | Ning’er | Cy. luteus | G | =FJ441284 | =MG205704 | =MG205755 | – | |||
41735 | P. kesiya | Ning’er | Co. cyperi | M | =FJ441284 | =MG205704 | =MG205755 | – | |||
41742 | P. kesiya | Ning’er | Co. cyperi | M | =FJ441284 | =MG205705 | =MG205756 | – | |||
41746 | P. kesiya | Ning’er | Co. cyperi | M | =FJ441284 | =MG205704 | =MG205755 | – | |||
41758 | P. kesiya | Ning’er | Co. cyperi | M | =FJ441284 | =MG205705 | =MG205756 | – | |||
10 | Ophiostoma sp. C | 12150 | Tsuga sp. | Dali | Unknown sp.2 | * | MG205666 | MG205709 | MG205762 | – | |
11 | Graphilbum fragrans | 11778 | P. yunnanensis | Zixishan | Tomicus minor | * | MG205667 | MG205710 | – | – | |
12 | Gra. kesiyae sp. nov. | 41626 | P. kesiya | Ning’er | P. szemaoensis | M | =MG205669 | – | – | – | |
41657 | 139639 | P. kesiya | Ning’er | Polygraphus sp. | M | MG205668 | =MG205714 | – | – | ||
12 | Gra. kesiyae sp. nov. | 41686 | 139641 | P. kesiya | Simao | P. szemaoensis | M | =MG205668 | MG205711 | – | – |
41691 | 139642 | P. kesiya | Simao | P. szemaoensis | M | =MG205669 | =MG205713 | – | – | ||
41703 | P. kesiya | Simao | P. szemaoensis | G | – | MG205712 | – | – | |||
41716 | 139657 | P. kesiya | Simao | P. szemaoensis | M | =MG205669 | =MG205712 | – | – | ||
41729H | 139652 | P. kesiya | Ning’er | P. szemaoensis | G | MG205669 | MG205713 | – | – | ||
41774 | 139653 | P. kesiya | Ning’er | Polygraphus aterrimus | B | MG205668 | MG205714 | – | – | ||
46468 | P. kesiya | Ning’er | P. aterrimus | G | MG205668 | =MG205714 | – | – | |||
46469 | P. kesiya | Ning’er | P. aterrimus | B | MG205668 | =MG205714 | – | – | |||
13 | Gra. puerense sp. nov. | 41619 | P. kesiya | Ning’er | P. szemaoensis | B | =MG205670 | MG205715 | – | – | |
41667 | 139651 | P. kesiya | Pu’er | I. acuminatus | G | MG205670 | MG205716 | – | – | ||
41670 | P. kesiya | Ning’er | I. acuminatus | B | =MG205670 | – | – | – | |||
41671 | P. kesiya | Ning’er | I. acuminatus | B | =MG205670 | MG205717 | – | – | |||
41673 | 139640 | P. kesiya | Ning’er | I. acuminatus | B | =MG205670 | MG205718 | – | – | ||
41942H | 139650 | P. kesiya | Ning’er | P. szemaoensis | G | MG205671 | MG205719 | – | – | ||
41971 | P. kesiya | Ning’er | P. szemaoensis | M | =MG205671 | MG205720 | – | – | |||
41996 | P. kesiya | Ning’er | I. acuminatus | M | =MG205671 | MG205721 | – | – | |||
41998 | P. kesiya | Ning’er | I. acuminatus | G | =MG205670 | =MG205715 | – | – | |||
14 | Leptographium gracile | 12305 | Pinus armandii | Lijiang | Pissodes sp. | * | MG205672 | MG205722 | MG205763 | MG205782 | |
12397 | P. armandii | Midu | Pissodes sp. | * | =MG205672 | =MG205722 | =MG205763 | =MG205782 | |||
12399 | P. armandii | Midu | Pissodes sp. | * | =MG205672 | =MG205722 | =MG205763 | =MG205782 | |||
12404 | P. armandii | Midu | Pissodes sp. | * | =MG205672 | =MG205722 | =MG205763 | =MG205782 | |||
12407 | P. armandii | Midu | Pissodes sp. | * | =MG205672 | =MG205722 | =MG205763 | =MG205782 | |||
12412 | P. armandii | Midu | Pissodes sp. | * | =MG205672 | =MG205722 | =MG205763 | =MG205782 | |||
15 | Grosmannia radiaticola | 12323 | Tsuga sp. | Dali | Unknown sp.2 | * | MG205673 | MG205723 | MG205764 | – | |
16 | L. ningerense sp. nov. | 41773 | P. kesiya | Ning’er | Co. cyperi | M | – | =MG205724 | MG205765 | =MG205783 | |
41786H | 139663 | P. kesiya | Ning’er | Co. cyperi | M | MG205674 | MG205724 | =MG205765 | MG205783 | ||
41831 | 139664 | P. kesiya | Ning’er | O. angulatus | M | MG205675 | =MG205724 | =MG205765 | =MG205783 | ||
17 | G. yunnanensis | 41622 | P. kesiya | Ning’er | P. szemaoensis | B | MG205676 | MG205725 | MG205766 | – | |
41627 | P. kesiya | Ning’er | P. szemaoensis or I. acuminatus | M | – | =MG205725 | – | – | |||
41633 | P. kesiya | Ning’er | P. szemaoensis or I. acuminatus | M | – | MG205726 | – | – | |||
41635 | P. kesiya | Ning’er | Lasconotus sp. | B | – | =MG205726 | MG205767 | – | |||
41636 | P. kesiya | Ning’er | Lasconotus sp. | B | – | =MG205726 | MG205768 | – | |||
41666 | P. kesiya | Ning’er | Polygraphus sp. | M | – | =MG205725 | – | – | |||
41687 | P. kesiya | Simao | P. szemaoensis | M | – | =MG205725 | MG205769 | – | |||
41694 | P. kesiya | Simao | P. szemaoensis | M | – | =MG205725 | – | – | |||
41707 | P. kesiya | Simao | P. szemaoensis | B | – | =MG205725 | MG205770 | – | |||
41720 | P. kesiya | Simao | P. szemaoensis | G | – | =MG205725 | MG205771 | – | |||
41721 | P. kesiya | Ning’er | P. szemaoensis | M | – | =MG205725 | MG205772 | – | |||
41726 | P. kesiya | Ning’er | P. szemaoensis | M | – | =MG205725 | – | – | |||
41728 | P. kesiya | Ning’er | P. szemaoensis | M | – | =MG205726 | MG205773 | – | |||
41777 | P. kesiya | Ning’er | Co. cyperi | M | – | MG205727 | MG205774 | – | |||
41778 | P. kesiya | Ning’er | Co. cyperi | M | – | MG205728 | =MG205774 | – | |||
41781 | P. kesiya | Ning’er | Co. cyperi | M | – | =MG205727 | =MG205774 | – | |||
41783 | P. kesiya | Ning’er | Co. cyperi | M | – | =MG205725 | MG205775 | – | |||
41805 | P. kesiya | Ning’er | Polygraphus sp. | M | – | =MG205725 | – | – | |||
41814 | P. kesiya | Ning’er | Polygraphus sp. | M | – | =MG205725 | – | – | |||
41858 | P. kesiya | Ning’er | P. szemaoensis | G | – | =MG205725 | MG205776 | – | |||
41863 | P. kesiya | Ning’er | P. szemaoensis | G | MG205677 | =MG205725 | MG205777 | – | |||
41945 | P. kesiya | Ning’er | P. szemaoensis | M | – | =MG205725 | – | – | |||
41963 | P. kesiya | Ning’er | P. szemaoensis | M | – | =MG205725 | – | – | |||
41990 | P. kesiya | Ning’er | I. acuminatus | G | – | =MG205725 | =MG205776 | – | |||
17 | G. yunnanensis | 41992 | P. kesiya | Ning’er | I. acuminatus | G | – | =MG205725 | MG205778 | – | |
41999 | P. kesiya | Ning’er | P. szemaoensis | B | – | =MG205725 | – | – | |||
42000 | P. kesiya | Ning’er | P. szemaoensis | B | – | – | =MG205778 | – | |||
18 | L. conjunctum | 11782 | P. yunnanensis | Zixishan | Hylurgops major | * | MG205678 | MG205729 | MG205779 | – | |
41761 | P. kesiya | Ning’er | Polygraphus sp. | M | MG205679 | MG205730 | MG205780 | – | |||
Microascales | |||||||||||
19 | Graphium pseudormiticum | 41665 | P. kesiya | Pu’er | I. acuminatus | M | MG205680 | – | MG205781 | – |
Growth studies were conducted on three isolates of each novel taxon. Mycelium-covered agar plugs were transferred from the actively growing margins of one-week-old cultures and placed at the centers of 90 mm Petri dishes containing 2% MEA. Cultures were incubated in the dark at temperatures ranging from 5–35 °C at 5 °C intervals. Three replicates were used for each isolate at each temperature. Colony diameters were measured every day until hyphae reached the edges of the Petri dishes. Optimum and maximum growth temperatures were calculated for each species.
To facilitate morphological descriptions of new taxa, asexual and sexual structures (where present) were mounted in lactophenol on glass slides, covered with a coverslip and examined with a Zeiss Axioskop2 Plus compound microscope or a Zeiss Discovery V12 dissection microscope with an Axiocam digital camera (Axiovision 3.1) (München-Hallbergmoos, Germany). Measurements were made for each taxonomically characteristic structure. The measurements are presented in the format (minimum–) mean minus standard deviation–mean plus standard deviation (–maximum). For reference to asexual states that resemble morphological features of well-known asexual genera, we followed the reference style (e.g. hyalorhinocladiella-like) suggested by
Frequencies of isolation of the ophiostomatoid species in all samples were calculated as follows: F = (NF/NT) × 100, where F represents the frequency of isolation (%), NT represents the total number of isolates collected, and NF represents the number of isolates of a particular fungal species.
Collectively, 17 beetle species belonging to four sub-families were collected from conifer hosts at 10 sites (Table
A total of 340 fungal isolates were obtained, 54 from beetles in 2001 and 2002, and 286 isolated from beetles, galleries and mites in 2010 (Tables
From the total of 340 isolates obtained in the study, DNA sequences were generated for 134 isolates (Table
The 340 isolates were separated in 19 taxa (Taxa 1 to 19) based on DNA sequences (Table
Two species of Sporothrix were collected (Suppl. material 2: Figure S3). Taxon 1 was represented by a single isolate and both ITS and BT data suggested that this was of an undescribed species. Taxon 2 included 45 isolates, eight for which sequence data were included in our analyses (Suppl. material 2: Figure S3). In the ITS tree, these isolates grouped in a monophyletic clade that included the ex-type isolates of S. nebularis and S. nigrograna. In the BT tree these isolates again grouped with S. nebularis.
Taxon 3 included 29 isolates (Table
Numbers of fungal isolates per species obtained from beetles (B), their galleries (G) or mite (M) associates
Year of survey → | 2010 | 2001 | 2002 | |||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Host tree species1 → | I | II | III | IV | V | Total | ||||||||||||||||||||||||||||||||
Beetle species2 → | A | B | D | E | F | H | I | J | L | O | G | C | K | M | N | P | Q | |||||||||||||||||||||
B | G | M | B | G | M | B | G | M | B | G | M | B | G | M | B | G | M | B | G | M | B | G | M | B | G | M | B | G | M | * | * | * | * | * | * | * | ||
Taxon. Fungus species↓ | ||||||||||||||||||||||||||||||||||||||
1. Sporothrix sp. A | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
2. S. nebularis | 0 | 0 | 4 | 0 | 0 | 7 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 18 | 0 | 0 | 0 | 0 | 0 | 5 | 1 | 3 | 2 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 45 |
3. Ophiostoma acarorum sp. nov. | 0 | 0 | 21 | 0 | 0 | 2 | 0 | 2 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 29 |
4. O. ips | 0 | 0 | 1 | 0 | 1 | 17 | 6 | 11 | 14 | 0 | 0 | 0 | 0 | 0 | 13 | 0 | 1 | 0 | 0 | 0 | 2 | 9 | 15 | 5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 3 | 0 | 4 | 4 | 0 | 106 |
5. Ophiostoma sp. B | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 |
6. O. brevipilosi sp. nov. | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 8 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 8 |
7. O. setosum | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 5 | 5 |
8. O. quercus | 0 | 1 | 0 | 0 | 1 | 7 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 6 | 1 | 6 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 3 | 0 | 2 | 4 | 11 | 43 |
9. O. tsotsi | 0 | 2 | 0 | 0 | 2 | 8 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 12 |
10. Ophiostoma sp. C | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 |
11. Graphilbum fragrans | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 1 |
12. Gra. kesiyae sp. nov. | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 1 | 0 | 0 | 0 | 2 | 0 | 2 | 5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 11 |
13. Gra. puerense sp. nov. | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 2 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 11 |
14. Leptographium gracile | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 10 | 0 | 0 | 0 | 0 | 0 | 0 | 10 |
15. G. radiaticola | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 |
16. L. ningerense sp. nov. | 0 | 0 | 2 | 0 | 0 | 8 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 13 |
17. G. yunnanensis | 0 | 0 | 0 | 0 | 0 | 6 | 0 | 2 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 3 | 4 | 3 | 17 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 39 |
18. L. conjunctum | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 2 |
19. Graphium pseudormiticum | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
Total | 0 | 3 | 28 | 0 | 4 | 56 | 10 | 19 | 18 | 2 | 0 | 0 | 0 | 0 | 34 | 2 | 2 | 0 | 0 | 0 | 12 | 21 | 28 | 36 | 1 | 0 | 0 | 9 | 0 | 0 | 10 | 1 | 6 | 1 | 8 | 12 | 17 | 340 |
Taxon 4 formed part of the O. ips complex (Fig.
Taxon 5, including only one isolate, together with Taxon 6 that included eight isolates (Table
Five isolates comprised Taxon 7 (Tables
There were 55 isolates (Table
For Graphilbum, results from the analyses of the ITS and BT sequences (Fig.
In Leptographiums.l. (Fig.
A single isolate (Taxon 15) grouped in the Grosmannia galeiformis complex (Fig.
Taxon 16 (Fig.
Forty one isolates (Tables
A single isolate represented Taxon 19 (Table
The origin and sources of the 340 isolates representing 19 taxa are presented in Tables
Grosmannia yunnanensis, O. ips, O. quercus, S. nebularis and Taxon 3 were the most frequently isolated species, representing 12.5%, 31.2%, 12.6%, 13.2% and 8.5% of the isolated fungi respectively. The remaining species were isolated only occasionally. O. ips was isolated from the niches (beetles, galleries, mites) of ten different bark beetles species. However, most of the O. ips were found associated with niches of I. acuminatus, P. szemaoensis and C. cyperi representing approximately 9.7%, 8.5% and 5.3%, respectively. S. nebularis was also isolated from the niches of ten beetle species and the highest frequency of isolation was 5.5%, associated with the niche of O. angulatus.
The number of fungal species isolated from different beetle or weevil niches varied between different species. There were ten fungal species, representing about 27.9% of total fungal isolates associated with the niche of P. szemaoensis. Among them, O. ips and L. yunnanense were the most frequently isolated, representing about 8.5% and 7.1%, respectively. There were eight fungal species, representing about 15.3%, associated with Co. cyperi and of these, the most frequently isolated fungus was O. ips with the frequency of 5.3%.
Only 13.2% of the total number of isolates were obtained directly from beetles (Table
Approximately 54% of all isolates were collected from mites (Table
Eight of the 19 taxa obtained in the present study represented undescribed species. For three of these, only a single isolate was obtained and we have chosen not to formally describe these. The remaining five taxa including two Ophiostoma, two Graphilbum, and one Leptographium species, are described as follows:
The epithet acarorum refers to the subclass Acari in the Arachnida to which all mite species belong from which 25 of the 29 isolates of this species were isolated.
Sexual state not observed. Hyalorhinocladiella-like asexual state: conidiophores (7–) 18–76.5 (–140) μm long; cells arising directly from the hyphae, (10.5–) 13.5–24.5 (–31) × (1–) 1.5–2 (–2) μm; conidia hyaline, smooth, oblong, (3–) 3.5–5 (–6.5) × (0.7–) 1–1.5 (–2.5) μm.
Colonies hyaline at the beginning, becoming white to dark brown with age. Mycelium superficial on the 3% OA. Colony margin smooth. Colonies on 2% MEA flat, reaching 69 mm diam in 13 d at 30 °C. No growth observed at 5 °C. Optimal temperature for growth 25 °C.
CHINA, Yunnan Province, Puer City, from Insectolaelaps sp. 1 in Orthotomicus angulatus gallery on Pinus kesiya bark, 17 Sep. 2010, S.J.Taerum, herbarium specimen of dried culture, PREM 61539 (holotype), CMW41850 = CBS139748 (ex-holotype culture).
CHINA, Yunnan Province, Puer City, from Histiostoma cf. sapromyzarum in Cyrtogenius luteus gallery on Pinus kesiya bark, 16 Sep. 2010, S.J.Taerum, PREM 61540, CMW41812 = CBS139658; from Histiostoma cf. sapromyzarum in Cyrtogenius luteus gallery on Pinus kesiya bark, 16 Sep. 2010, S.J.Taerum, CMW41798 = CBS139643.
Pinus kesiya.
Ips acuminatus, Polygraphus szemaoensis.
Histiostoma cf. sapromyzarum (phoretic on Cyrtogenius luteus), Insectolaelaps sp. 1 (phoretic on Ips acuminatus and Orthotomicus angulatus).
At present known only from Yunnan, China.
The hyalorhinocladiella-like asexual state of O. acarorum resembles that of O. pallidulum (
The epithet brevipilosi refers to the bark beetle vector Tomicus brevipilosus from which all eight isolates of this taxon were obtained.
Sexual state not observed. Pesotum-like macronematal asexual state predominant. Synnemata simple, dark brown at the base, (179.5–) 227–468 (–667) μm long including conidiogenousapparatus, (22–) 32.5–58 (–69) μm wide at base; cells (13–) 16–26 (–32.5) μm long, conidia hyaline, 1-celled, smooth, oblong, (3–) 3–4.5 (–5.5) × (1.5–) 1.5–2.5 (–3) μm. Hyalorhinocladiella-like asexual state: conidiophores (14.5–) 33–115 (–145) μm long; cells arising directly from the hyphae, (12–) 15–38 (–47) × (1.1–) 1.5–2 (–2.5) μm; conidia hyaline, smooth, obovoid, (2.5–) 3–5.5 (–8) × (1.5–) 2–2.5 (–3) μm.
Morphological characters of asexual structures of Ophiostoma brevipilosi sp. nov. a fourteen days old culture on OA b–d Hyalorhinocladiella-like asexual state and condia ePesotum-like macronematal asexual state f cells of Pesotum-like macronematal asexual state g conidia. Scale bars: a–g = 10 μm.
Colonies hyaline at the beginning, then becoming white to dark. Mycelium superficial on the 3% OA. Colony margin smooth. Colonies on 2% MEA flat, reaching 67 mm diam in 11 d at 25 °C. No growth observed at 5 and above 30 °C. Optimal temperature for growth 20 and 25 °C.
CHINA, Yunnan Province, Puer City, from Tomicus brevipilosus on Pinus kesiya bark, 27 Jun. 2010, S.J.Taerum, herbarium specimen of dried culture, PREM 61537 (holotype), CMW41873 = CBS139660 (ex-holotype culture).
CHINA, Yunnan Province, Puer City, from Tomicus brevipilosus on Pinus kesiya bark, 27 Jun. 2010, S.J.Taerum, PREM 61538, CMW41624 = CBS139661; CHINA, Yunnan Province, Puer City, from Tomicus brevipilosus on Pinus kesiya bark, 27 Jun. 2010, S.J.Taerum, CMW41662 = CBS139659.
Pinus kesiya.
Tomicus brevipilosus.
At present known only from Yunnan, China.
The synnematous asexual state of O. brevipilosi corresponds with similar structures of O. brunneo-ciliatum as described by
The epithet kesiyae refers to the tree host of all beetles and mites from which the 12 isolates of this species were collected.
Sexual state not observed. Pesotum-like macronematal asexual states predominant. Synnemata simple, dark brown at the base, (85.5–) 112.5–173 (–203) μm long including conidiogenous apparatus, (9–) 14–45.5 (–65.5) μm wide at base; cells (8.5–) 10–18.5 (–25.5) μm long; conidia hyaline, 1-celled, smooth, oblong, (3.5–) 4–5 (–5.5) × (1.5–) 1.5–2 (–2.5) μm. Hyalorhinocladiella-like asexual state: conidiophores (22–) 38–101.5 (–166) μm long; cells arising directly from the hyphae, (10–) 12–27(–40) × (1.2–) 1.5–2 (–2.5) μm; conidia hyaline, smooth, obovoid, (3.5–) 4–5.5 (–8.5) × (1–) 1.5–2 (–3) μm.
Morphological characters of asexual structures of Graphilbum kesiyae sp. nov. a fourteen days old culture on OA b–e Hyalorhinocladiella-like asexual state and conidia fPesotum-like macronematal asexual state g cells of Pesotum-like macronematal asexual state h conidia. Scale bars: a–h = 10 μm.
Colonies hyaline. Mycelium superficial on the 3% OA. Colony margin smooth. Colonies on 2% MEA flat, reaching 85 mm diam in 10 d at 25 °C. No growth observed at 5 and 35 °C. Optimal temperature for growth 25 °C.
CHINA, Yunnan Province, Puer City, from Polygraphus szemaoensis gallery on Pinus kesiya bark, 12 Aug. 2010, S.J.Taerum, herbarium specimen of dried culture, PREM 61541 (holotype), CMW41729 = CBS139652 (ex-holotype culture).
CHINA, Yunnan Province, Puer City, from Insectolaelaps sp. 1 in Polygraphus szemaoensis gallery on Pinus kesiya bark, 10 Aug. 2010, S.J.Taerum, CMW41691 = CBS139642; CHINA, Yunnan Province, Puer City, from Proctolaelaps nr. hystrix in Polygraphus szemaoensis gallery on Pinus kesiya bark, 11 Aug. 2010, S.J.Taerum, PREM 61542, CMW41716 = CBS139657.
Pinus kesiya.
Polygraphus aterrimus, Polygraphus szemaoensis.
Mite vectors. Proctolaelaps nr. hystrix (phoretic on Polygraphus szemaoensis), Insectolaelaps sp. 1 (phoretic on Polygraphus szemaoensis).
At present known only from Yunnan, China.
Graphilbum kesiyae and Gra. puerense can be distinguished from Gra. crescericum by the presence of both synnematous and hyalorhinocladiella-like asexual states in culture. Gra. crescericum produces only the hyalorhinocladiella-like asexual state. The optimal temperature for growth of Gra. puerense is 30 °C while that for Gra. kesiyae is 25 °C, and synnemata of Gra. puerense reach double the length of those of Gra. kesiyae.
The epithet puerense refers to the city from which this species was collected.
Sexual state not observed. Pesotum-like macronematal asexual states predominant. Synnemata simple, dark brown at the base, (187.5–) 206– 357(–437.5) μm long including conidiogenous apparatus, (12–) 15.5–45 (–61) μm wide at base; conidiogenous cells (15.5–) 18.5–30.5 (–34) μm long, conidia hyaline, 1-celled, smooth, oblong, (4–) 4–5 (–5.5) × (1–) 1.5–2 (–2.5) μm. Hyalorhinocladiella-like asexual state: conidiophores (17–) 3–140 (–232.5) μm long; cells arising directly from the hyphae, (6.5–) 10–25.5 (–42.5) × (1–) 1–2 (–3) μm; conidia hyaline, smooth, obovoid to oblong, (3.5–) 4–8 (–12) × (1–) 1.5–2.5 (–3) μm.
Morphological characters of asexual structures of Graphilbum puerense sp. nov. a fourteen days old culture on OA b–d Hyalorhinocladiella-like asexual state e conidia fPesotum-like macronematal asexual state g–h cells of Pesotum-like macronematal asexual state and conidia. Scale bars: a–h = 10 μm.
Colonies hyaline. Mycelium superficial on the 3% OA. Colony margin smooth. Colonies on 2% MEA flat, reaching 76 mm diam in 5 d at 30 °C. No growth observed at 5 °C. Optimal temperature for growth 30 °C.
CHINA, Yunnan Province, Puer City, from Polygraphus szemaoensis gallery on Pinus kesiya bark, 29 Jun. 2010, S.J.Taerum, herbarium specimen of dried culture, PREM 61543 (holotype), CMW41673 = CBS139640 (ex-holotype culture).
CHINA, Yunnan Province, Puer City, from Ips acuminatus gallery on Pinus kesiya bark, 4 Jul. 2010, S.J.Taerum, PREM 61544, CMW41667 = CBS139651; CHINA, Yunnan Province, Puer City, from Ips acuminatus gallery on Pinus kesiya bark, Jul 2010, S.J.Taerum, CMW41942 = CBS139650.
Pinus kesiya.
Ips acuminatus, Polygraphus szemaoensis.
Proctolaelaps nr. hystrix (phoretic on Ips acuminatus), Insectolaelaps sp. 1 (phoretic on Ips acuminatus), and Uropodoidea sp. 2 (phoretic on Polygraphus szemaoensis).
At present known only from Yunnan, China.
See comparison between Gra. kesiyae and Gra. puerense above under notes of Gra. kesiyae.
The epithet ningerense refers to the Ning’er county where all isolates of this taxon were collected.
Sexual state not observed. Asexual state, conidiophores occurring singly or in groups of up to 3, macronematous, mononematous, erect, arising directly from the mycelium, (93.5–) 141.5–195.5 (–210.5) μm long. Rhizoids present. Stipes dark olivaceous, 4–6 septa, not constricted at septa, (66–) 119.5–142 (–159) μm long. Apical cells not swollen at apex, (3–) 5–6.5 (–7) μm wide. Basal cells occasionally swollen at apex, (5.5–) 7–10 (–11.5) μm wide. Conidiogenous apparatus (28–) 35–58 (–70) μm long, excluding the conidial mass, with multiple series of cylindrical branches. Primary branches olivaceous, smooth, cylindrical, not swollen at apex, aseptate, arrangement of primary branches was Type B—two or more branches, (12.5–) 14.5–18 (–19.5) × (3.5–) 4–5.5(–6.5) μm. Secondary branches light olivaceous, frequently swollen at apex, aseptate, (6.5–) 9–13(–15) × (3.5–) 4–5 (–5.5) μm. Tertiary branches light olivaceous, aseptate, (7–) 8–10 (–12) × (3–) 3.5–4.5 (–5) μm. Conidiogenous cells discrete, hyaline, 2–3 per branch, aseptate, cylindrical, tapering slightly at the apex, (10.5–) 12–17.5 (–20.5) × (2–) 2–2.5 (–3) μm. Conidia hyaline, aseptate, elliptical, (3–) 3.5–5.5 (–6.5) × (1.5–) 2–3 (–4) μm.
Colonies on 3% OA flat, hyaline at the beginning, then becoming light olivaceous to dark olivaceous. Colonies hyaline at the beginning, then becoming dark olivaceous. Mycelium superficial on the 3% OA with olivaceous aerial mycelium. Colony margin smooth. Conidiophores forms abundantly in clusters on OA. Colonies on 2% MEA flat, reaching 76 mm diam in 10 d at 25 °C. No growth observed at 5 and 35 °C. Optimal temperature for growth 25 °C, reaching 30.5 mm in diam. in 7 days.
CHINA, Yunnan Province, Puer City, from Schwiebea (Jacotietta) taiwanensis hyperphoretic on Coccotrypes cyperi on Pinus kesiya bark, 16 Sep. 2010, S.J.Taerum, herbarium specimen of dried culture, PREM 61545 (holotype), CMW41786 = CBS139663 (ex-holotype culture).
CHINA, Yunnan province, Puer City, from Insectolaelaps sp. 1 in Orthotomicus angulatus on Pinus kesiya bark, 17 Sep. 2010, S.J.Taerum, PREM 61546, CMW41831 = CBS139664.
Pinus kesiya.
Polygraphus szemaoensis.
Dendrolaelaps sp. 1 (phoretic on Coccotrypes cyperi), Dendrolaelaps sp. 2 (phoretic on Coccotrypes cyperi and Cyrtogenius luteus), Schwiebea (Jacotietta) taiwanensis (phoretic on Coccotrypes cyperi), Insectolaelaps sp. 1 (Orthotomicus angulatus).
At present known only from Yunnan, China.
L. ningerense is morphologically similar to L. pineti, but grows much more rapidly, reaching 30 mm in 7 d on 2% MEA at 25 °C while L. pineti reaches a diameter of only 15 mm in 6 d. However, the two species are best distinguished with BT, EF and CAL sequences.
This study resulted in a total of 340 fungal isolates of ophiostomatoid fungi obtained from the beetles and weevils, their galleries and phoretic mites in a province of China where these fungi are poorly known. The fungi resided in the two phylogenetically unrelated Microascales and Ophiostomatales and included a species of Graphium (Microascales) as well as species of Sporothrix, Graphilbum, Leptographium and Ophiostoma (Ophiostomatales). Analysis showed that these isolates belonged to 19 distinct taxa, eight of which represented undescribed species of which five were provided with names. Of the remaining 11 species, 10 had previously been reported from China, with only S. nebularis representing a new report for this country. This study also includes the first records of fungi associated with the Scolytine beetle species Coccotrypes cyperi, Cyrtogenius luteus, and Tomicus brevilopus and it is the first time that fungal associates are reported from beetle-associated mites in China.
Surveys in this study aimed to explore fungal diversity in conifer-infesting beetle ecosystems in Yunnan. This is in contrast to determining the specificity of these interactions where a much more focused and systematic sampling would have been required. The exact nature of the interactions between the beetles and mites, mites and fungi, beetles and fungi, and all of these with their host trees, thus remains largely unknown. The discussion below therefore focuses on the fungi collected in these surveys, with limited notes on the hosts, beetles and mites and some general observations.
The two Sporothrix species collected in this study were primarily from mites. Sporothrix nebularis was the second most abundant species and 36 of the 45 isolates came from five different mite species phoretic on five different beetle species. The fungus was first described from Hylastes attenuatus infesting Pinus radiata in Spain (
Ophiostoma acarorum grouped peripheral to the well-known species complexes in Ophiostomas.str., and closest to, but distinct from O. pallidulum and O. saponiodorum. O. pallidulum is known from ten different bark beetle and one weevil species infesting Pinus sylvestris in Finland (
Ophiostoma ips was the species most frequently isolated in the study, constituting 31% of the 340 isolates. It was also the fungus found in association with the largest number of beetle species i.e. 10 of 17, and with three of the five host trees in central and southern Yunnan. This is consistent with current knowledge of the fungus that is known to have a global distribution (
Two taxa from Yunnan formed part of the O. clavatum complex as recently defined by
Only a single species of the O. piceae complex, O. setosum, was obtained during the surveys. Five isolates of this species were from Pissodes galleries on Tsuga dumosa, confirming a previous report from the same host in China (
The third most abundant species collected in this study was O. quercus that forms part of the O. ulmi species complex (
The 43 O. quercus isolates obtained in this study came from four of the host trees distributed in eight of the ten study sites in both central and southern Yunnan. The O. quercus isolates were found in association with eight beetle species, with 13 of the isolates coming from three mite species. To the best of our knowledge, this represents the first records of this fungus from mites. However, spores of the closely related and morphologically similar O. novo-ulmi have been observed in the guts and sporothecae of mites phoretic on Scolytus beetle vectors of the Dutch Elm Disease fungi (
Ophiostoma tsotsi was the second species in the O. ulmi complex obtained in this study. This species, which is morphologically indistinguishable from O. quercus, is known from wounds on planted Eucalyptus and Acacia mearnsii trees in Africa (
A single isolate referred to as Ophiostoma sp. C (Taxon 10) came from an unknown beetle gallery on Tsuga. This isolate was clearly distinct from all other species in the O. ulmi complex. Although we have chosen not to describe it as new, together with O. quercus and O. tsotsi, this is only the third of the 18 species currently recognized in the complex to be reported from conifer hosts.
Three of the taxa collected in the surveys resided in the genus Graphilbum that includes 10 known species and several undescribed taxa (
Graphilbum fragrans found in this study had previously been reported from T. yunnanensis on Pinus yunnanensis, and Pissodes spp. on Tsuga dumosa and P. armandii in China (
Five species of fungi residing in Leptographiumsensu lato were collected in this study, including one novel species. Ten isolates representing Taxon 14 were from Pissodes sp. on P. armandii and were shown to represent L. gracile. This species was described from the same insects and tree host as the isolates in the present study, as well as from Ips typographus on Picea koraiensis (
A single isolate from the gallery of an unknown bark beetle on Tsuga grouped with Grosmannia radiaticola. This species was originally described from stained Pinus radiata wood imported from New Zealand to Korea (
Of the 13 isolates of L. ningerense collected in this study one isolate was from the gallery of Polygraphus szemaoensis on Pinus kesiya, while the remaining 12 isolates were from four mite species associated with Coccotrypes cyperi, Cyrtogenius luteus and Orthotomicus angulatus. This species grouped closest to L. pineti, which was described from the gallery of an Ips sp. under the bark of Pinus merkusii growing on the island of Sumatra, Indonesia (
The fourth most prevalent species collected in this study was G. yunnanensis including 39 isolates, 27 of which were vectored by seven mite species. G. yunnanensis was originally described from Tomicus yunnanensis on P. yunnanensis in China (
Two isolates of L. conjunctum, including one from a mite, were collected in the present study. L. conjunctum has not previously been reported from any location other than in the case of the original study in which it was described from Hylurgops major on Pinus yunnanensis in China (
The single Graphium isolate arising from this study was from a mite on Ips acuminatus, and had identical sequences to Gr. pseudormiticum. This fungus was first described from South Africa in association with Orthotomicus erosus on pine bait logs (
Three surveys of pine-infesting bark beetles in Yunnan revealed several new fungal species and new beetle-fungus associations. This supports the view that the diversity of fungi associated with bark beetles in China is high and that it deserves further exploration. The results of the 2010 survey that included isolations from mites, revealed that many of the ophiostomatoid species often considered as beetle associates can also be isolated frequently from mites. It has been suggested that mycetophagous mites are often generalists with the ability to feed on and vector several fungal species (
One of the most surprising and intriguing outcomes of the study was that fungal species such as O. quercus and O. tsotsi, which were considered to be primarily hardwood-infecting species, are prevalent on several of the pine-specific beetles and their associated mites. Bark beetles commonly have broad host ranges on either the hardwoods or conifers, but not across these groups. The question thus arises how these hardwood-infecting fungi can move between these host groups. One possibility is that they simply move with mites from one tree in a forest to another, crossing the forest floor and carrying the fungi with them. However, it is also known that most bark beetle-associated mites are not monospecific and may be common on other non-bark beetle hosts as well (e.g. Tenebrionidae, Cleridae, Histeridae, Elateridae) (
None of the known fungal species collected in the present study are considered pathogens based on current published knowledge, and we have not found any evidence that any of the novel taxa are pathogens. However, the study underscores the possibility that many ophiostomatoid fungi that have been considered beetle associates in the past, might actually have a closer association with mites, or that mites at least have the ability to vector them. Generally, bark beetles and their fungal associates are considered serious threats with invasive potential when they are introduced into new environments through the movement of wood and wood products (
We acknowledge members of Tree Protection and Cooperation Programme (TPCP), Center of Excellence in Tree Health Biotechnology (CTHB), the National Research Foundation (NRF), the Department of Science and Technology (DST)/NRF and the University of Pretoria, South Africa. We are deeply grateful to Prof. Zhao of the Yunnan Academy of Forestry for his support during the collection surveys. We also thank Prof. Yihe Zhao, Dr Mingliang Yin and Miss Dongxia Xu for assistance with the fieldwork, Mr Deer Konkarn for assistance with sequencing, and Drs Roger Beaver and Eddie Ueckermann for beetle and mite identifications.
Table S1–S3
Data type: species data
Explanation note:
Table S1. Numbers of ophiostomatoid fungal isolates obtained from different mite species in this study.
Table S2. Haplotypes of Ophiostoma quercus.
Table S3. Haplotypes of Ophiostoma tsotsi.
Figure S1–S11
Data type: occurence
Explanation note:
Figure S1. Map of Yunnan Province in China showing the sites and tree hosts from which samples were collected during the three surveys in 2001, 2002 and 2010.
Figure S2. ML trees of the genus Graphium generated from DNA sequences of the ITS and BT regions. Bold branches indicate posterior probabilities values ≥ 0.95. Bootstrap values ≥ 70 % are recorded at nodes as ML/MP. T = ex-type isolates.
Figure S3. ML trees of the genus Sporothrix generated from DNA sequences of ITS and BT regions. Bold branches indicate posterior probabilities values ≥ 0.95. Bootstrap values ≥ 70 % are recorded at nodes as ML/MP. T = ex-type isolates.
Figure S4. ML trees of the O. ips complex generated from DNA sequences of ITS and BT regions. Bold branches indicate posterior probabilities values ≥ 0.95. Bootstrap values ≥ 70 % are recorded at nodes as ML/MP. T = ex-type isolates.
Figure S5. ML trees of the O. piceae complex generated from DNA sequences of BT and EF regions. Bold branches indicate posterior probabilities values ≥ 0.95. Bootstrap values ≥ 70 % are recorded at nodes as ML/MP. T = ex-type isolates.
Figure S6. ML tree of the O. quercus complex generated from DNA sequences of ITS region. Bold branches indicate posterior probabilities values ≥ 0.95. Bootstrap values ≥ 70 % are recorded at nodes as ML/MP. T = ex-type isolates.
Figure S7. ML tree of the O. quercus complex generated from DNA sequences of BT region. Bold branches indicate posterior probabilities values ≥ 0.95. Bootstrap values ≥ 70 % are recorded at nodes as ML/MP. T = ex-type isolates.
Figure S8. ML tree of the O. quercus complex generated from DNA sequences of EF region. Bold branches indicate posterior probabilities values ≥ 0.95. Bootstrap values ≥ 70 % are recorded at nodes as ML/MP. T = ex-type isolates.
Figure S9. ML trees of the L. procerum complex generated from DNA sequences of BT, EF and CAL regions. Bold branches indicate posterior probabilities values ≥ 0.95. Bootstrap values ≥ 70 % are recorded at nodes as ML/MP. T = ex-type isolates.
Figure S10. ML trees of the G. galeiformis complex generated from DNA sequences of BT and EF regions. Bold branches indicate posterior probabilities values ≥ 0.95. Bootstrap values ≥ 70 % are recorded at nodes as ML/MP. T = ex-type isolates.
Figure S11. ML trees of the L. lundbergii complex generated from DNA sequences of BT and EF regions. Bold branches indicate posterior probabilities values ≥ 0.95. Bootstrap values ≥ 70 % are recorded at nodes as ML/MP. T = ex-type isolates.