DNA sequence comparisons of Ophiostoma spp., including Ophiostoma aurorae sp. nov., associated with pine bark beetles in South Africa.

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.

INTRODUCTION

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 , Jacobs & Wingfield 2001), while many others can cause sapstain on logs and freshly cut wood (Wingfield ). 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 ), 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 ).

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 ). These fungi have been isolated from the insects or their galleries and identified based on their morphological characteristics (Zhou ). Eight of these species belong to the genus Ophiostoma (sensu Zipfel ) or its anamorphs. However especially those of which only the anamorphs were observed remained to be identified to species level (Zhou ). The aim of this study was to use DNA sequence comparisons to confirm the identities of the Ophiostoma spp. (Zipfel ) from South African pine bark beetles, previously identified based only on morphology (Zhou ).

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

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 ). 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.

Table 2.

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

Species	Isolate Number	GenBank no.		Host	Insect vector	Area
		ITS	β-tubulin
Pesotum fragrans	aCMW 19357	DQ396790		Pinus patula	Hylastes angustatus	Mpumalanga
Ophiostoma abietinum	CMW 397	DQ396788		—	Orthotomicus erosus	Western Cape
O. floccosum	CMW 19358	DQ396791		P. elliottii	Hylurgus ligniperda	Kwazulu-Natal
	CMW 19359	DQ396792		P. patula	O. erosus	Mpumalanga
O. pluriannulatum	CMW 19360	DQ396793		P. elliottii	H. ligniperda	Kwazulu-Natal
O. piliferum	CMW 554	DQ396789		—	O. erosus	Western Cape
O. quercus	CMW 19361	DQ396794		P. patula	H. angustatus	Mpumalanga
	CMW 19365	DQ396795		P. elliottii	H. ligniperda	Kwazulu-Natal
O. stenoceras	CMW 544	DQ396799		—	H. angustatus	Western Cape
O. aurorae	CMW 19362	DQ396796	DQ396800	P. elliottii	H. angustatus	Mpumalanga
	CMW 19363	DQ396797	DQ396801	P. elliottii	H. angustatus	Mpumalanga
	CMW 19364	DQ396798	DQ396802	P. elliottii	H. angustatus	Mpumalanga

Culture Collection of the Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, South Africa.

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 ). 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.

Table 3.

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

Species	Strain No.	GenBank no.		Collector / supplier	Origin	Host / insect
		ITS	β-tubulin
Leptographium guttulatum	aCMW 1310	AY649782		J.N. Gibbs	England	Pinus / Tomicus piniperda
	bCMW 742	AY649783		M. Morelet	France	P. sylvestris / Tomicus sp.
Pesotum fragrans	b,cCBS 279.54	AF198248		A. Mathiesen-Käärik	Sweden	P. sylvestris / Ips sexdentatus
Ophiostoma abietinum	bCBS 125.89	AF484453		J.G. Marmolejo	Mexico	Abies vejari / Pseudohylesinus sp.
O. dentifundum	bCBS 115790	AY495434	AY495445	C. Delatour	Hungary	Quercus wood
	CBS 115865	AY495435	AY495446	T. Kowalski	Poland	Quercus robur
O. floccosum	bCBS 799.73	AF198231		A. Käärik	Sweden	Picea or Pinus
O. fusiforme	bCBS 112912	AY280481	AY280461	D.N. Aghayeva	Azerbaijan	Populus nigra
	CBS 112909	AY280482	AY280462	D.N. Aghayeva	Azerbaijan	Castanea sativa
O. ips	bCBS 137.36	AY546704		C.T. Rumbold	U.S.A.	Ips integer
	CMW 6418	AY546702		X.D. Zhou	South Africa	Pinus elliottii / Orthotomicus erosus
O. lunatum	bCBS 112927	AY280485	AY280466	T. Kirisits	Austria	Carpinus betulus
	CBS 112928	AY280486	AY280467	T. Kirisits	Austria	Larix decidua
O. multiannulatum	CBS 357.77			R.W. Davidson	U.S.A.	Pinus sp.
O. narcissi	dC 1648	AF484451		—	U.K.	Narcissus sp.
	bCBS 138.50	AY194510		D.P. Limber	Netherlands	Narcissus sp.
O. nigrocarpum	bCBS 637.66	AY280489	AY280479	R.W. Davidson	U.S.A.	Abies sp.
	CBS 638.66	AY280490	AY280480	R.W. Davidson	U.S.A.	Pseudotsuga menziesii
O. piceae	bCBS 108.21	AF198226		E. Münch	Germany	Abies or Picea
	CMW 7648	AF493249		D.B. Redfern, J.F. Webber	U.K.	Picea sitchensis
O. piliferum	CBS 129.32	AF221070		H. Diddens	—	Pinus sylvestris
O. pluriannulatum	CMW 75			R.W. Davidson	U.S.A.	—
O. pulvinisporum	bCMW 9020	AY546713		X.D. Zhou	Mexico	P. pseudostrobus / Dendroctonus mexicanus
	CMW 9026	AY546715		X.D. Zhou	Mexico	Pinus maximinoi / Ips calligraphus
O. quercus	bCMW 2467	AY466626		M. Morelet	France	Quercus sp.
	CMW 7645	AF493246		T. Kirisits, E. Halmschlager	Austria	Q. robur
O. stenoceras	bCBS 237.32	AY484462	AY280471	H. Robak	Norway	Pinus pulp
	CMW 11193	AY280493	AY280475	R. Farrell	New Zealand	wood
Sporothrix inflata	bCBS 239.68	AY495426	AY495437	W. Gams	Germany	wheatfield soil
	CBS 841.73	AY495431	AY495442	J. Grinbergs	Chile	soil
S. schenckii	CMW 7612	AY280494	AY280476	H.F. Vismer	South Africa	human sporotrichosis
	CMW 7614	AY280495	AY280477	H.F. Vismer	South Africa	human sporotrichosis
	CMW 7615	AY280496	AY280478	H.F. Vismer	South Africa	human sporotrichosis

CMW = Culture Collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa.

Ex-type culture or authentic strain.

CBS = Culture collection of the Centraalbureau voor Schimmelcultures, Utrecht, Netherlands.

C = Culture collection of T.C. Harrington, Department of Plant Pathology, Iowa State University, U.S.A.

All sequences were aligned using MAFFT v. 5.667 (Katoh ). 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 and AY649783). Bootstrap analyses (1000 replicates) were run to determine confidence levels of the branching points (Felsenstein 1985).

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

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 ).

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.

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 ). 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 ). 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.

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.

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

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:

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

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

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 ). 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 ). 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 , Jacobs ).

Ophiostoma aurorae described in this study is morphologically similar to species in the O. stenoceras-complex (De Beer , 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 ). Clearly, the bark beetle/fungal association represents a complex and dynamic environment that deserves further study.