Development of polymorphic microsatellite markers for the tree pathogen and sapstain agent

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Ophiostoma spp. associated with two pine-infesting bark beetles from Chile

Bark beetles (Coleoptera: Scolytidae) are common vectors of Ophiostoma spp., which include primary tree pathogens as well as important sapstain agents. In Chile, Hylurgus ligniperda and Hylastes ater, which are native to Europe, commonly occur on the exotic Pinus radiata . Little research has been done on Ophiostoma spp. associated with bark beetles in Chile and especially those carried by introduced pine-infesting insects. We recently obtained specimens of these bark beetles and their galleries, and the aim of this study was to isolate and identify Ophiostoma spp. associated   with   the   two   beetle   species. Identification was achieved using morphological characteristics and where appropriate, DNA sequencing. A total of five ophiostomatoid species, Ceratocystiopsis minuta, 0. galeiformis, 0. huntii, 0. ips, and 0. quercus, were found associated with the bark beetles, all of which are recorded from Chile for the first time .
Keywords : Leptographium, Ascomycetes, Scolytidae, Hylurgus, Hylastes.

INTRODUCTION

Pinus spp . are native to the Northern Hemisphere, where species diversity is most pronounced in Central America and Southeast Asia (Richardson,  1998). Many Pinus spp., however, have been introduced into Southern Hemisphere countries such as New Zealand, Australia, Chile and South Africa. In these countries, some pine species are grown in very large commercial plantations (Le Maitre, 1998; Richardson, 1998).
Many bark beetle species (Coleoptera: Scolytidae) infest Pinus spp. (Wood & Bright, 1992). Most of these  bark  beetles  are  not  considered  as  pests  in  their  native  environment,  but  when introduced into new areas and particularly where uniform stands of Pinus spp. are planted, they ,can become problematic (Wingfield & Swart,  1994; Wingfield et al., 2001).In Chile, Hylurgus /igniperda (Fabricius) and Hylastes ater (Paykull) are exotic pests of European origin which infest exotic P. radiata ‘(Wood & Bright,  1992; Billings,  1993).Both of these insects can infest fresh stumps and slash shortly after trees are felled (Ciesla, 1988).
Many  bark  beetles  are   also  vectors  of ophiostomatoid fungi,which include a number of primary pathogens and sapstain agents (Whitney, 1982; Harrington,1988; Seifert,1993; Brasier & Mehrotra, 1995; Paine, Raffa & Harrington, 1997). In Chile, at least eight ophiostomatoid species have been reported from different hosts (Table 1).Little research has, however, been conducted on the fungal associates of pine-infesting bark beetles in this country.
In Chile, as is the case in South Africa, exotic pine plantations constitute a significant section of the forestry industry. Considerable research has been conducted on pine bark beetle-associated fungi in exotic pine plantations of South Africa in recent years (Wingfield & Swart, 1989; Zhou et al., 2001, 2002).   A comparison of the fungi associated with introduced bark beetles in Chile, with fungi from the same niche in South Africa, could provide insight into the spread of the bark beetles and their fungi south of the equator.
Recently, we have had the opportunity to examine bark beetles and their galleries from Chile, and to  isolate Ophiostoma spp.  occurring on the beetles and in their galleries. The aim of this study was to identify these fungi based on morphology and comparisons of ITS rDNA sequences.

MATERIALS AND METHODS

Isolation offungi

In  the  Valdivia area of Chile,3 4 specimens  of H. ater  were  collected from  roots  of dying radiata trees, and 80 specimens of H. /igniperda were collected from felled trees of the same species in log stacks. Four fungal  isolates were collected directly from  the galleries of H.  ater.
Isolation of fungi    from  bark beetles and their galleries was conducted  in a similar way to that described by Zhou et al.  (2001). All  cultures used  in  this  study are maintained  in the culture collection  (CMW) of Forestry and Agricultural Biotechnology Institute (F ABI), University of Pretoria, Pretoria, South Africa.

Morphological studies

Both teleomorph  and  anamorph  fruiting  structures,  when present,  were mounted  in lactophenol cotton blue on glass slides, examined microscopically, and characteristic structures measured To induce the production of perithecia, isolates presenting only anamorphs were grown on 2 % W A (20 g Biolab agar and 1000 ml distilled water), to which sterilised pine twigs had been added.

DNA sequencing and phylogenetic analysis

Isolates used. Some isolates produced only a Pesotum-like anamorph in culture, resembling that of 0. piceae (Munch)  H. &  P.  Sydow, 0.quercus (Georgevitch) Nannfeldt,  and  0. jloccosum Mathiesen. These  isolates were  difficult to  identify based  on morphology,  and  for two of them (CMW9480 and CMW9481), single hyphal tip cultures, were prepared for sequencing (Table 2).

DNA extraction

Each culture was grown in 50 ml of malt extract broth (20 g Biolab malt extract, and  1000 ml distilled water) at 25  ° C in the dark for 10 days. Mycelium was then harvested by filtration (Whatman no. 1 filter paper) and freeze-dried.
DNA was extracted using a modified version of the extraction method developed by Raeder and  Broda  (1985) . Freeze-dried  mycelium  was  grounded  to fine powder  in liquid nitrogen. Approximately 0.5 ml of mycelial powder was suspended in 800 ~l of extraction buffer (200 mM Tris-HCI pH 8.0,150 mM NaCI, 25 mM EDT A pH 8.0, 0 .5 % SDS). Phenol (500 ~l) and 300 ~l of chloroform were added to the suspension, and the mixture was votexed, then centrifuged in a Beckman JA 25.50 rotor (12,000 rpm, 60 min, 4 ° C). The upper aqueous layer was transferred to sterilized  Eppendorf tubes.  200  ~l of phenol  and  an  equal  volume  of chloroform  were  added, vortex ed, and then centrifuged for 5 minutes . The aqueous phase was transferred again, and the chloroform extraction (400 ~l) was repeated once or twice until the interface was clear.            Nucleic acid was then precipitated with  0.1 vol. of 3 M NaAc (pH 5.4) and  1 vol.  of isopropanol. The ,nucleic acid was pelleted using centrifugation (12,000 rpm, 30 min, 4 ° C), and the salt removed by washing with 70 % ethanol The vacuum-dried pellet was resuspended in 50 ~l of sterile water and 2 III of RNAase (10 mg / ml, Roche Molecular Biochemicals) was added to digest any RNA.
The reaction was incubated in a water bath overnight at 37 ° C.  Agarose (Promega, Madison, CT, USA) gel electrophoresis (l %) was used to determine the presence of the DNA. The DNA was visualized using Ethium bromide and UV light.The concentration of the DNA was determined using UV spectroscopy (Beckman Du Series 7500 Spectrophotometer).

Acknowledgements 
Preface
LITERATURE REVIEW
Chapter 1 The occurrence of ophiostomatoid fungi in the Southern Hemisphere, with special reference to species associated with pine bark beetles
TAXONOMY
Chapter 2 Ophiostomatoid fungi associated with three pine-infesting bark beetles in South Africa
Chapter 3 Ophiostoma spp. associated with two pine-infesting bark beetles from Chile
Chapter 4 Characterisation of Ophiostoma spp. associated with pine bark beetles from Mexico, including 0. pulvinisporum sp. nov
Chapter 5 A new Leptographium species associated with Tomicus piniperda in South-western China
Chapter 6 Epitypification of Ophiostoma galeiformis and phylogeny of species in the 0. galeiformis
complex
PATHOLOGY
Chapter 7 Pathogenicity of Ophiostoma ips, Leptographium serpens and L. lundbergii to pines in
South Africa
POPULATION BIOLOGY
Chapter 8 .Development of polymorphic microsatellite markers for the tree pathogen and sapstain
agent, Ophiostoma ips
Chapter 9 .Microsatellite (SSR) markers reveal genetic diversity among isolates of Ophiostoma ips
from South Africa, Chile, Europe, and the USA
Summary 
Opsomming

Philosophiae Doctor Natural and Agricultural Sciences

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