Specifi c Primers for the Differentiation of Heterobasidion annosum ( s . str . ) and H . parviporum Infected Stumps in Northern Europe

Heterobasidion annosum (Fr.) Bref. sensu lato is a pathogenic basidiomycete, which causes root and butt rot in conifers of northern temperate and boreal forests. The species has been divided to intersterility groups (IS groups), two of which (S and P) occur in northern Europe (Korhonen 1978). Although few natural intergroup hybrids have been reported between IS groups S and P in North America (Garbelotto et al. 1996), and compatible matings have been observed in pairing experiments (Korhonen 1978, Olson and Stenlid 2001, Stenlid and Karlsson 1991), the two IS groups do not seem to hybridize naturally in Europe. Therefore the fungi occurring in Europe were recently named as H. parviporum (IS group S) and H. annosum (Fr.) Bref. sensu stricto (Niemelä and Korhonen 1998). H. parviporum (Niemelä and Korhonen) attacks mainly Picea abies (L.) Karst., and H. annosum s.str. prefers Pinus sylvestris (L.), but occurs also on several other tree species including P. abies. Heterobasidion sp. spreads to new sites by basidiospore infections to fresh stumps, from which the mycelium grows vegetatively to living Specifi c Primers for the Differentiation of Heterobasidion annosum (s.str.) and H. parviporum Infected Stumps in Northern Europe


Introduction
Heterobasidion annosum (Fr.)Bref.sensu lato is a pathogenic basidiomycete, which causes root and butt rot in conifers of northern temperate and boreal forests.The species has been divided to intersterility groups (IS groups), two of which (S and P) occur in northern Europe (Korhonen 1978).Although few natural intergroup hybrids have been reported between IS groups S and P in North America (Garbelotto et al. 1996), and compatible matings have been observed in pairing experiments (Korhonen 1978, Olson and Stenlid 2001, Stenlid and Karlsson 1991), the two IS groups do not seem to hybridize naturally in Europe.Therefore the fungi occurring in Europe were recently named as H. parviporum (IS group S) and H. annosum (Fr.)Bref.sensu stricto (Niemelä and Korhonen 1998).H. parviporum (Niemelä and Korhonen) attacks mainly Picea abies (L.) Karst., and H. annosum s.str.prefers Pinus sylvestris (L.), but occurs also on several other tree species including P. abies.
Heterobasidion sp.spreads to new sites by basidiospore infections to fresh stumps, from which the mycelium grows vegetatively to living Specifi c Primers for the Differentiation of Heterobasidion annosum (s.str.) and

H. parviporum Infected Stumps in Northern Europe
trees via root contacts.This can be controlled by conducting fellings only during the wintertime when no basidiospores are formed, or by treating the fresh stumps by biological or chemical control agents (Risbeth 1952).In sites where infection has been established, the fungus may grow vegetatively from old infected stumps to the next tree generation using root contacts (Stenlid 1987, Piri 1996, Piri and Korhonen 2001).This kind of dispersal can be avoided by using a non-host tree species for forest regeneration.
In Finland, regeneration of heavily contaminated spruce stand with spruce is not recommended if the site supports growth of any other tree species.If the pathogen on the site belongs to H. parviporum, then pine, birch or aspen can all be planted.However, if H. annosum has contaminated the site, then both pine and spruce should be avoided.Therefore, it should be confi rmed that H. annosum is not present on the site when Scots pine is planned to be used for the regeneration.
The two Heterobasidion species can be differentiated by pectic isozyme profi les (Karlsson and Stenlid 1991), fatty acid and sterol (FAST) profi les (Müller et al. 1995), as well as using several DNA-based techniques, including randomly amplifi ed DNA (RAPD) analysis (Fabritius and Karjalainen 1993, Garbelotto et al. 1993, Karjalainen 1996), M13 minisatellite fi ngerprinting (Karlsson 1994, Stenlid et al. 1994), ribosomal DNA sequencing (Kasuga et al. 1993) and random amplifi ed microsatellite (RAMS or ISSR) fi ngerprints (Vainio and Hantula 1999).All these methods as well as traditional pairing tests (Korhonen 1978) are based on mycelial cultures.However, in northern Europe a method allowing identifi cation of H. annosum directly from infected wood would be ideal for practical purposes.It would also be useful if the occurrence of H. parviporum could be recognized in the same test.
PCR-based identifi cation methods have been described for various fungi.The methods may be based on Internal Transcribed Spacer (Lindqvist et al. 1998), ribosomal RNA genes (Edel et al. 2000) or other DNA (Paavolainen et al. 2000).In this report we describe the development and testing of a PCR based tool for the identifi cation of H. annosum and H. parviporum.

DNA Isolations
DNA from both cultured isolates (Table 1) and wood chips (Table 2) was used.For most mycelial samples previously isolated DNA was applied (Table 1), and the DNA from new isolates was isolated basically as described in Vainio et al. (1998).DNA from Heterobasidion-infected wood chips (Table 2) was isolated with either the method described in Vainio and Hantula (2000a) (early part of this investigation) or using the UltraClean TM Soil DNA Isolation Kit (Mo Bio Laboratories Inc.).

RAMS-Reactions
For the cloning of Heterobasidion-specifi c fragments we amplifi ed RAMS markers from fungal DNA as described in Vainio and Hantula (2000b).The RAMS-primers used were CT-primer: VDV(CT) 7 C and CCA-primer: 5´DDB(CCA) 5 , where D = A/G/T, B = C/G/T and V = A/C/G.Amplifi cation products were separated by electrophoresis and visualised using UV light (Vainio and Hantula 1999).

The Design of Specifi c Primers and PCR-Conditions
Amplifi cation products CT1350 and CCA1210 specifi c to H. annosum and H. parviporum (Vainio and Hantula 1999) were cloned from isolates 36 and 29, respectively, to Escherichia coli as described previously (Vainio and Hantula 2000b).
The selection of fragments was based on standard agarose gel electrophoresis.The cloned inserts were sequenced by A.L.F.DNA Sequencer TM (Pharmacia Biotech, Uppsala, Sweden) using M13 reverse and forward primers and the Thermo Sequenase fl uorescent labelled primer cycle sequencing kit (Amersham Pharmacia Biotech, England).Primers MJ-F and MJ-R (Table 3) were designed based on the sequence of CT1350 (Gen Bank accession number AY211523) in order to amplify an approximately 100 bp fragment from isolates belonging to H. annosum, and primers  b) More detailed information about these isolates has been given previously (Vainio and Hantula 1999).c) More detailed information about these isolates has been given previously (Vainio and Hantula 2000a).KJ-F and KJ-R (Table 3) were designed based on the sequence of CCA1210 (accession number AY211522) in order to amplify an approximately 350 bp fragment from isolates belonging to H. parviporum.The amplifi cation conditions for these primers were optimised using pure cultures so that both primer pairs could be used in the same PCR-reaction.As a result, the amplifi cation was carried out using a 'hot start' protocol where DNA samples and primers (each in a concentration of 0.5 uM) were denaturated at +95 °C for 10 minutes, after which a dNTP-mix (each deoxynucleotide in a concentration of 0.2 mM) and 20 U/ml of Dynazyme thermostable DNA-polymerase (Finnzymes Ltd, Espoo, Finland) were added.Then 40 cycles of amplifi cation (30 s denaturation at 95 °C, 35 s annealing at 67 °C, 1 min extension at 72 °C) and a fi nal extension of 7 minutes were carried out.The buffer conditions were the same as suggested by the manufacturer of the Dynazyme polymerase.

Tests for the Specifi city of the Primers
The specifi city of the primers was tested using DNA from 14 isolates of H. parviporum, 13 isolates of H. annosum and 35 isolates of other fungal species (Table 1).Mixtures of H. annosum and H. parviporum DNA were used as templates in order to test the capability of primers to detect both species simultaneously.In addition, the primers were tested on 33 wood chip samples, fi ve of which were known to be colonised by H. parviporum (fi ve samples), 18 by H. annosum (18 samples), and nine samples by unidentifi ed Heterobasidion sp.(from a forest stand known to be infected by H. parviporum).In addition, one  a) Samples with abbreviations including KK, TP or KL were provided by Kari Korhonen, Tuula Piri and Katriina Lipponen, respectively.Other samples were collected by the authors.All samples originateed from southern Finland.b) Based on the principles described previously (Korhonen 1978).c) Based on the principles described previously (Vainio and Hantula 2000a).
Table 3.The sequences of the primers designed.

Primer
Sequence (starting from 5´end) sample from a healthy tree (both spruce and pine) was used as a negative control.The sensitivity of the detection was tested using known amounts of H. annosum and H. parviporum DNA in PCR-reactions.We also conducted PCR-reactions, where different amounts (ratios) of both H. annosum and H. parviporum DNA was added to reactions with primer pairs detecting only one species (either MJ-F and MJ-R or KJ-F and KJ-R).

Results and Discussion
Initial tests with the two primer pairs were carried out separately.PCR reactions with primers MJ-F and MJ-R resulted in successful amplifi cation of an approximately 100 bp fragment only when DNA from H. annosum was used as a template (not shown).In contrast, the amplifi cation of 350 bp fragment with primers KJ-F and KJ-R was successful only using template DNA from H. parviporum (not shown).No unspecifi c fragments with unexpected sizes were observed in these reactions.
After the initial trials, both primer pairs were combined in a single PCR reaction.Amplifi cation products were observed only if Heterobasidion DNA was present, and their sizes were in accordance with the origin of the template DNA (i.e.H. annosum or H. parviporum; Fig. 1).If DNA from both species was present, both bands appeared (Fig. 1).No amplifi cation products were observed in amplifi cations with DNA from the other species tested (not shown).
Finally we made tests with wood chip samples known to be infected by H. annosum or H. parviporum.In all cases a positive identifi cation was made; and in all cases the identifi cation agreed with the pre-existing information about the fungal species (Fig. 1; Table 1).No amplifi cation products were obtained from the negative control.
In sensitivity tests primer pair MJ-F and MJ-R was able to detect even 15 × 10 -3 pg of H. annosum DNA.Primer pair KJ-F and KJ-R was less sensitive, and detected only amounts higher than 15 ng of H. parviporum DNA.In competition experiments both species were detected even if the ratio of the two templates was as skewed as 1:10.
The primers pairs developed were based on RAMS-primers.In Blast searches no homologies exceeding 20 bp were observed for CCA1210.Therefore it was not possible to conclude on what type of DNA primers KJ-F and KJ-R are based.In contrast, Blast search conducted with CT1350 showed high levels of similarity with the ribosomal gene cluster, and therefore we could conclude that both primers MJ-F and MJ-R are based on Intergenic Spacer Region.This may explain why the detection of H. annosum was more sensitive, as there are several copies of ribosomal gene cluster in the genome.
There is plenty of molecular data, which would probably also enable the differentiation of the two Heterobasidion species directly from a wood chip (Karjalainen 1996, Kasuga and Mitchelson 2000, Kasuga et al. 1993).In this work a new set of primers was designed to allow the detection of the two species directly from decaying wood material in one PCR-reaction without any additional analyses (except electrophoresis).This makes the As a conclusion, we developed a primer system which can be used for the detection and identifi cation of H. annosum and H. parviporum directly from the decaying wood in northern Europe.However, our identifi cation test was developed for research and practical usage in Finland (and northern Europe) and therefore it was not tested with H. abietinum, which occurs commonly in southern Europe, or with Heterobasidion species (IS-groups) occurring in Asia or North America.Therefore the molecular identifi cation tool described here should only be used in northern Europe.

Fig. 1 .
Fig. 1.Examples of amplifi cation products obtainedwith the developed primers.The samples from left to the right are as follows: wood chip KL12, wood chip KK2, wood chip 3.4.9,wood chip 3.5.11,a mixture of isolates 29 and 52, isolate 52, isolate 32, isolate 29 and isolate 9. On the left is a DNA ladder, where the band sizes from the bottom to the fi rst double intensity band are 100, 200, 300, 400 500 and 600 bp.

Table 2 .
Information about the wood chips, and amplifi cation products obtained.