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Stud Mycol 55(1): 269-277 2006
Copyright © 2006 CBS Fungal Biodiversity Centre
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DNA sequence comparisons of Ophiostoma spp., including Ophiostoma aurorae sp. nov., associated with pine bark beetles in South Africa

Xudong Zhou1,*, Z. Wilhelm de Beer2 and Michael J. Wingfield1

1 Tree Protection Co-operative Programme (TPCP), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, Republic of South Africa
2 Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, 0002, Republic of South Africa

* Correspondence: Xudong Zhou, Xu.Zhou{at}fabi.up.ac.za


   Abstract
TOP
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 TAXONOMY
 DISCUSSION
 References
 
Bark beetles (Coleoptera: Scolytinae) are well-recognized vectors of Ophiostoma species. Three non-native bark beetle species infest various Pinus species in South Africa, and they are known to carry at least 12 different species of ophiostomatoid fungi. Some of these fungi have not been identified to species level. The aim of this study was to determine or confirm the identities of Ophiostoma species associated with bark beetles in South Africa using comparisons of DNA sequence data. Identities of Ophiostoma ips, O. floccosum, O. pluriannulatum, O. quercus and O. stenoceras were confirmed. Ophiostoma abietinum, O. piliferum and Pesotum fragrans are recognised for the first time and the new species, O. aurorae sp. nov., is described from pine-infesting bark beetles in South Africa.

Taxonomic novelty: Ophiostoma aurorae X.D. Zhou & M.J. Wingf. sp. nov.

Keywords Bark beetle / Ophiostoma / phylogeny / taxonomy


   INTRODUCTION
TOP
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 TAXONOMY
 DISCUSSION
 References
 
Conifer-infesting bark beetles (Coleoptera: Scolytinae) are economically important forest insects. They include many primary pest species, which can attack healthy living trees and have caused significant economic losses to the global forestry industry (Wood & Bright 1992). In South Africa, three non-native bark beetle species, Hylastes angustatus, Hylurgus ligniperda, and Orthotomicus erosus infest various Pinus spp. (Tribe 1992). They are generally considered as secondary pests, although H. angustatus may undergo maturation feeding on healthy living seedlings causing significant losses during plantation establishment (Tribe 1992).

Bark beetles are well-known vectors of fungi, especially Ophiostoma species (Six 2003, Kirisits 2004, Harrington 2005). The ophiostomatoid fungi are a polyphyletic group of morphologically similar fungi, adapted for insect dispersal. Several ophiostomatoid fungi are important pathogens of conifers (Harrington & Cobb 1988, Wingfield et al. 1993b, Jacobs & Wingfield 2001), while many others can cause sapstain on logs and freshly cut wood (Wingfield et al. 1993b). The group includes the genera Ceratocystis Ellis & Halst., Gondwanamyces G.J. Marais & M.J. Wingf., Sphaeronaemella P. Karst. and Cornuvesica C.D. Viljoen, M.J. Wingf. & K. Jacobs and their anamorphs in the Microascales (Spatafora & Blackwell 1994, Hausner et al. 2000), and Ophiostoma Syd. & P. Syd., Grosmannia Goid. and Ceratocystiopsis H.P. Upadhyay & W.B. Kendr., with their Pesotum J.L. Crane & Schokn., Leptographium Lagerb. & Melin, Sporothrix Hektoen & C.F. Perkins and Hyalorhinocladiella H.P. Upadhyay & W.B. Kendr. anamorphs in the Ophiostomatales (Zipfel et al. 2006).

More than 30 ophiostomatoid fungi have been reported from South Africa (Table 1), of which at least 12 are associated with the three exotic pine-infesting bark beetle species in the country (Zhou et al. 2001). These fungi have been isolated from the insects or their galleries and identified based on their morphological characteristics (Zhou et al. 2001). Eight of these species belong to the genus Ophiostoma (sensu Zipfel et al. 2006) or its anamorphs. However especially those of which only the anamorphs were observed remained to be identified to species level (Zhou et al. 2001). The aim of this study was to use DNA sequence comparisons to confirm the identities of the Ophiostoma spp. (Zipfel et al. 2006) from South African pine bark beetles, previously identified based only on morphology (Zhou et al. 2001).


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  		Table 1. Ophiostomatoid fungi, including species with affinities to both the Microascales and Ophiostomatales, reported from South Africa. Currently accepted species names are listed first, with the name used in the original report in square brackets. Species reported as associates of bark or ambrosia beetles are printed in blue.

 


   MATERIALS AND METHODS
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 TAXONOMY
 DISCUSSION
 References
 
Fungal isolates
Twelve isolates (Table 2) used in this study originated from a previous investigation of ophiostomatoid fungi associated with the three pine-infesting bark beetle species in South Africa (Zhou et al. 2001). All cultures are maintained in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa. A relevant sub-set of cultures has been deposited with the Centraalbureau voor Schimmelcultures (CBS), Utrecht, Netherlands.


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  		Table 2. Fungal isolates from pine bark beetles in South Africa used in this study.

 

DNA sequencing and phylogenetic analyses
Single hyphal-tip cultures from the 12 isolates were grown on 2 % MEA (20 g Biolab malt extract, 20 g Biolab agar, and 1000 mL deionised water). DNA was extracted using PrepMan Ultra Sample reagent (Applied Biosystems) as described by Aghayeva et al. (2004). The ITS (internal transcribed spacer) region of the ribosomal RNA operon was amplified using primers ITS1-F (Gardes & Bruns 1993) and ITS4 (White et al. 1990). PCR products were sequenced with the same primers. Conditions for PCR amplification and sequencing reactions were as described by Zhou et al. (2004b). For comparisons, ITS sequences of closely related taxa (Table 3) were obtained from GenBank.


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  		Table 3. Isolates of selected species of Ophiostoma used for comparative purpose in this study.

 

All sequences were aligned using MAFFT v. 5.667 (Katoh et al. 2002). Phylogenetic relationships among the isolates were determined using distance analyses in MEGA3 (http://www.megasoftware.net/). Trees were constructed using the Neighbour-joining tree-building algorithm (Saitou & Nei 1987) and rooted using GenBank sequences of Leptographium guttulatum M. J. Wingf. & K. Jacobs (AY649782 [GenBank] and AY649783 [GenBank] ). Bootstrap analyses (1000 replicates) were run to determine confidence levels of the branching points (Felsenstein 1985).


Figure 1
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  		Fig. 1. Neighbour-joining tree of Ophiostoma species associated with bark beetles in South Africa based on ITS sequences (ITS1 and ITS2 regions, as well as 5.8S rRNA gene). Isolates sequenced in this study are printed in bold. Bar = total nucleotide differences between taxa. Bootstrap values (1000 replicates) are indicated above the branches.

 
Three of the 12 isolates (CMW 19362, CMW 19363, and CMW 19364) grouped in a clade separate from the other isolates, all of which grouped with known taxa. For these three isolates, part of the β-tubulin gene was amplified using primers Bt2a and Bt2b (Glass & Donaldson 1995). For each of the two regions, phylogenetic analyses were done separately, followed by a distance analysis of the combined data set. A partition homogeneity test was performed in PAUP v. 4.0b8 (Phylogenetic Analyses Using Parsimony) (Swofford 2002) to determine the congruence of the two data sets.

Morphology
Isolates (CMW 19362, CMW 19363, and CMW 19364) that resided in a defined phylogenetic clade of unknown identity were grown on 2 % WA (20 g Biolab agar and 1000 mL deionised water) with sterilised pine twigs, and on 1.5 % oatmeal agar (15 g oats powder, 20 g Biolab agar and 1000 mL deionised water) to induce production of perithecia. Perithecia with ascospores were formed in two isolates (CMW 19362 and CMW 19363) on oatmeal agar. Thirty measurements were made for each structure, and the ranges and averages were computed. Anamorph structures were observed on 7-d-old slide cultures (Riddell 1950), mounted in lactophenol.


   RESULTS
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
TAXONOMY
 DISCUSSION
 References
 
DNA Sequence analyses
PCR of the ITS regions delivered products ranging from about 530 to 610 bp in size. Comparison of the ITS sequences with GenBank sequences confirmed the identities of seven Ophiostoma spp. (Fig. 1). These included O. stenoceras (Robak) Nannf., O. abietinum Marm. & Butin, O. piliferum (Fr.) Syd. & P. Syd., O. pluriannulatum (Hedgc.) Syd. & P. Syd., O. quercus (Georgev.) Nannf., O. floccosum Math.-Käärik, and Pesotum fragrans (Math.-Käärik) G. Okada & Seifert. The identity of O. ips (Rumbold) Nannf. (also included in the study) had previously been confirmed based on DNA sequence comparisons (Zhou et al. 2004a).

Fragments 541 bp in size from the ITS region, and 345 from the partial β-tubulin gene were amplified for the three unidentified isolates (CMW 19362, CMW 19363, and CMW 19364). The β-tubulin region included intron 5, but no intron 4 was present. This corresponds with species in the O. stenoceras -complex (Zipfel et al. 2006). Sequences of isolates representing the majority of species in this complex were thus selected for further phylogenetic analyses, with O. nigrocarpum as outgroup. Ophiostoma spp. from outside the O. stenoceras -complex were not included in these analyses because of the presence of intron 4, but no intron 5 (Zipfel et al. 2006). The partition homogeneity test (P = 0.003) confirmed that the ITS and β-tubulin data sets were congruent. Distance analyses for the combined data set showed that the three unidentified isolates grouped together with a bootstrap support of 100 % (Fig. 2), and that they either represented an undescribed species or a species for which no sequence data are available.


Figure 2
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  		Fig. 2. Neighbour-joining tree of the Ophiostoma stenoceras - Sporothrix schenckii complex of species, including O. aurorae based on the combined ITS and β-tubulin sequences. Isolates sequenced in this study are printed in bold. Bar = total nucleotide differences between taxa. Bootstrap values (1000 replicates) are indicated above the branches.

 

Morphology
The three isolates (CMW 19362, CMW 19363, and CMW 19364) are morphologically similar to each other and different from any other described Ophiostoma species. They produced a typical Sporothrix anamorph in culture with swollen clavate conidia. Two of the isolates produced ascomata with allantoid rounded ascospores.


   TAXONOMY
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 TAXONOMY
DISCUSSION
 References
 
Based on combined sequence comparisons of the ITS regions and partial β-tubulin gene, as well as morphology, we conclude the three isolates from H. angustatus infesting pines in South Africa represent an undescribed taxon. This is described as follows:

Ophiostoma aurorae X.D. Zhou & M.J. Wingf., sp. nov. MycoBank MB500888. Figs 3A–3F.


Figure 3
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  		Fig. 3. Ophiostoma aurorae (CMW 19362) on 1.5 % oatmeal agar. A. Perithecium with long neck. (Scale bar = 190 µm). B. Apex of the neck with ostiolar hyphae. (Scale bar = 15 µm). C. Allantoid round ascospores. (Scale bar = 1.5 µm). D. Conidiophore. (Scale bar = 3.5 µm). E. Conidiogenous cell. (Scale bar = 1.5 µm). F. Clavate conidia. (Scale bar = 1.5 µm).

 
Anamorph: Sporothrix (Fig. 3D–F).

Etymology: The type locality of this species is in Mpumalanga Province, South Africa. In siSwati, the name of the province means "the place where the sun rises". Aurora was the Roman (Latin) goddess of dawn, so the specific epithet is an oblique reference to the type locality.

Coloniae in agaro 1.5 % avenae in medio 45 mm diam aetate duarum hebdomadum in 25 °C, laete hyalinae vel albae. Mycelium aerium adest. Ascomata superficialia vel subimmersa in agaro 1.5 % avenae. Bases perithiciorum globosae, obscurae, 130–220(–350) µm diam, hyphis laete griseis 65–150(–280) µm longis, 1.5–2.0 (–2.5) µm latis ornatae. Colla peritheciorum brunnea vel nigra, laevia, 340–800 (1415) µm longa, ad basim 35–42(–58) µm, ad apicem 12–15(–27) µm lata. Hyphae ostiolares adsunt. Ascosporae hyalinae, non septatae, allantoideae, in sectione transversali rotundae, 2–3(–3.5) x 1–1.5(–2) µm.

Cellulae conidiogenae micronematae, mononematae, hyalinae, 12–60(–85) x 1.5–2(–2.5) µm, ad apicem incrassatum denticulos acres perferentes; conidia hyalina, unicellularia, clavata vel guttuliformia, 3–4.5(–8) x 1–1.5(–2.5) µm.

Ascomata with globose bases, dark, 130–220(–350) µm diam (Fig. 3A), ornamented with light grey hyphae, 65–150(–280) µm long, 1.5–2(–2.5) µm wide. Perithecial necks brown to black, smooth, 340–800 (1415) µm long, 35–42(–58) µm wide at the base, 12–15(–27) µm at the apex (Fig. 3A, B). Ostiolar hyphae present (Fig. 3B). Ascospores hyaline, aseptate, allantoid, round in side view, 2–3(–3.5) x 1–1.5(–2) µm (Fig. 3C).

Conidiogenous cells (Fig. 3D–E), micronematous, mononematous, hyaline, 12–60(–85) x 1.5–2(–2.5) µm, sharp denticles present in the swollen apical part. Conidia (Fig. 3F) hyaline, single 1–celled, clavate to guttuliform, 3–4.5(–8) x 1–1.5(–2.5) µm.

Cultural characteristics: Colonies on 1.5 % oatmeal agar reaching on average 45 mm diam in two weeks at 25 °C. Colonies light hyaline to cotton–white. Aerial mycelium present. Perithecia produced superficially on or partially immersed in 1.5 % oatmeal agar.

Substrates: Hylastes angustatus and infested bark of Pinus patula.

Distribution: Mpumalanga Province, South Africa.

Specimens examined: South Africa, Mpumalanga Province, Hylastes angustatus, Sep. 1999, X.D. Zhou, holotype PREM 58886, culture ex-type CBS 118837 = CMW 19362; paratype PREM 58887, culture ex-paratype = CMW 19363; paratype PREM 58888, culture ex-paratype CBS 118827 = CMW 19364.


   DISCUSSION
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 TAXONOMY
 DISCUSSION
References
 
Results of this study have confirmed the identities of five Ophiostoma spp. associated with the non-native pine-infesting bark beetles H. angustatus, H. ligniperda, and O. erosus in South Africa. These fungi are O. ips, O. floccosum, O. pluriannulatum, O. quercus and O. stenoceras. In addition, O. abietinum, O. piliferum and P. fragrans are recognised for the first time from South Africa. One of the fungi associated with these bark beetles represents an undescribed taxon, for which the name O. aurorae has been provided.

The three fungal species O. abietinum, O. piliferum and P. fragrans reported from South Africa for the first time, are well-known associates of conifer timber. Ophiostoma abietinum was first described from Abies vejari attacked by a Pseudohylesinus sp. in Mexico (Marmolejo & Butin 1990), and was considered as an intermediate between O. stenoceras and O. nigrocarpum (R. Davidson) De Hoog (De Beer et al. 2003a). Ophiostoma piliferum is considered economically important to the forestry industry, and a colourless mutant of this species has been marketed as biocontrol agent against sapstaining fungi (Farrell et al. 1993). Pesotum fragrans was described from galleries of Ips sexdentatus infesting Pinus sylvestris in Sweden (Mathiesen-Käärik 1953), and the species has been reported from Australia, California, Canada, and New Zealand (Harrington et al. 2001, Jacobs et al. 2003).

Ophiostoma aurorae described in this study is morphologically similar to species in the O. stenoceras-complex (De Beer et al. 2003a, Aghayeva et al. 2004, 2005). Species in the complex have typical orange-section-shaped ascospores and Sporothrix anamorphs. Ophiostoma aurorae can be distinguished from other species in the complex by its very obviously rounded ascospores and swollen clavate conidia. Its association with the root feeding scolytid bark beetle H. angustatus also appears to be a useful characteristic that might be applied in identification. In addition to its morphologically unique nature, analyses of ITS and partial β-tubulin gene sequences confirmed that O. aurorae resides in a phylogenetic clade, distinct from all morphologically similar Ophiostoma spp. for which sequence data are available.

Results of this study emphasise that a surprisingly large number of Ophiostoma spp. are associated with the three non-native conifer-infesting bark beetles accidentally introduced into South Africa. They also highlight the fact that the introduction of what might initially appear to be a single organism (plant, insect, fungus) is often considerably more complex. It seems likely that most of the fungi treated in this study are specifically associated with the insects in their areas of origin and like their insect vectors, they are also introduced exotics.

Species such as O. quercus that have a wide distribution on many woody substrates in South Africa could have invaded the bark beetle niche. It would be interesting to understand the long-term changes in such vector/fungus relationships, as has recently been found with Tomicus piniperda (Linnaeus) and Leptographium wingfieldii M. Morelet in the United States (Jacobs et al. 2004). Clearly, the bark beetle/fungal association represents a complex and dynamic environment that deserves further study.


   Acknowledgments
 
We thank the National Research Foundation (NRF), members of Tree Protection Co-operative Programme (TPCP) and the THRIP initiative of the Department of Trade and Industry, South Africa for financial support. We also acknowledge Sappi for a fellowship awarded to the first author and Dr Hugh F. Glen for providing the Latin diagnosis.


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 INTRODUCTION
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 TAXONOMY
 DISCUSSION
 References
 

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