Population structure and genetic variation in Nectria

Population structure and genetic variation in Nectria fuckeliana

Can. J. Bot. Downloaded from www.nrcresearchpress.com by Renmin University of China on 06/04/13 For personal use only.

Rimvydas Vasiliauskas and Jan Stenlid

Abstract: Population structure and genetic variation in Necrria fuckeliar~a Booth isolated from Picea abies (L.) Karst. in Sweden and Lithuania was studied using somatic incompatibility tests and DNA fingerprinting. All incompatibility pairings between different isolates of N. fuckeliana resulted in demarcation zones; thus, no vegetative compatibility groups were detected. Each isolate was distinguishable from all other isolates on the basis of banding patterns produced by amplification of DNA using the M I 3 primer. No country-specific markers were observed. Principal component analysis of amplified banding patterns separated the isolates from Sweden and Lithuania into two clusters, showing genetic differentiation between the geographical populations across the Baltic sea. An analysis of similarity matrix, calculated by the program SIMQUAL from the numerical taxonomy package NTSYS-pc, confirmed the separation of the isolates into the two groups. Low genetic differentiation was revealed within both the Swedish and Lithuanian geographical populations of the fungus. Local distances in the forest stand (100 m) had no influence on the genetic similarity of the N. ji~ckeliar~a isolates (R2 = 0.003).

Key words: Necrria fuckeliana, DNA fingerprinting, genetic variation, somatic incompatibility, population structure

RCsumC : Les auteurs ont CtudiC la structure de population et la variation gCnCtique de Necrria jilckeliarla Booth isolCs du Picea abies (L.) Karst., en Subde et en Lithuanie, en utilisanat des tests d'incompatibilitk somatique et les patrons d'ADN. Tous les pairages d'incompatibilitC entre differents isolats du N. jilckeliana ont montrC des zones de demarcation et consCquemment aucun groupe de compatibilitC vCgCtative n'a pu Ctre dCcelC. Chaque isolat peut Ctre distinguC de tous les autres sur la base des patrons de bandes produits par amplification de I'ADN en utilisant l'amorce M13. On observe aucun marqueur specifique au pays d'origine. L'analyse en composantes principales des patrons d'amplification permet de sCparer les isolats de la Subde et de la Lithuanie en deux groupes, ce qui montre I'existence d'une differenciation gCnCtique entre les populations gkographiques au dela de la mer Baltique. L'analyse matricielle de similarit&, calculCe par le programme SIMQUAL, B partir de l'ensemble de taxonomie numCrique NTSYS-pc, confirme la skparation des isolats en deux groupes. Une faible variation gCnCtique se manifeste B l'indrieur des deux groupes, dans les populations gkographiques sutdoises et lithuaniennes du champignon. Les distances locales entre peuplements forestiers (100 m) n'ont pas d'influence sur la similarit6 gknttique des isolats du N. fuckeliana (R2 = 0.003).

Mots clPs : Necrriafuckeliana, patrons ADN, variation gCnCtique, incompatibilitC somatique, structure des populations. [Traduit par la rCdaction]

Introduction The ascomycete Nectriafuckeliana Booth is known to cause bark necrosis of Picea sitchensis (Bong.) Carr. (Zycha 1955) and Picea abies (L.) Karst. (Phillips and Burdekin 1982; Butin 1989). It is also one of the most frequent invaders of spruce stem wounds, detected in 60 -69 % of examined injuries (Bazzigher 1973; Schonhar 1975; Roll-Hansen and RollHansen 1980). Infection of the stem through branch stubs may also be important, since the fungus was repeatedly isolated from dead branches and sound-looking stems of Picea abies (Roll-Hansen and Roll-Hansen 1979; Huse Received January 7, 1997.

R. Vasiliauskas' and J. Stenlid.' Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, P.O. Box 7026, 750 07 Uppsala, Sweden.

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Present address: Department of Plant Protection, Lithuanian University of Agriculture, 4324 Kaunas, Lithuania. Author to whom all correspondence should be addressed. e-mail: [email protected]

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1981). In a recent study, occurrence of N. fuckeliana in wounded Picea abies correlated positively with attack of the spruce bark beetle, Dendroctonus micans Kug., implying possible association between the insect and the fungus (Vasiliauskas et al. 1996). It is already known that some representatives of pine beetles from the genus Dendroctonus can serve as vectors for ascomycetes from the genus Ceratocystis (Cook and Hain 1988). In addition, N. fuckeliana is able to produce large amounts of conidia in a pure culture (RollHansen 1962; Hallaksela 1977), the role of which, if any, in natural environment is not yet known. Both insects and conidia may play an import& part -in spread of the fungus, and therefore, the possibility cannot be excluded that, in nature, populations of N. fuckeliana to some extent consist of clonal lineages. Somatic incompatibility (SI) is the visible reaction line that usually results when isolates of different genets are paired in culture. It has become widespread in recent years as a method to measure clonality and diversity in populations of many plant pathogenic ascomycetes (Leslie 1993). Among wood-inhabiting species the method has succesfully been applied to reveal heterothallism in Daldinia concentrica

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Ces. & de Not. (Sharland and Rayner 1986), clonality in


Rosellinia desmazieresii (Berk. & Br.) Sacc. (Sharland et al. 1988), and outcrossing in Hypoxylon spp. (Sharland and Rayner 1989) and Phomopsis spp. (Brayford 1990). In a pine canker pathogen, Fusarium subglutinarzs (Wollenw. & Reinking) P.E. Nelson, Toussoun, & Marasas f.sp. pini Correll et al., SI tests revealed limited diversity and absence of sexual reproduction in California (Gordon et al. 1996). In contrast, evidence for partial clonality in natural populations was obtained for the chesnut blight fungus, Cqphonectria parasitica (Murill) Barr (Milgroom et al. 199 1). Furthermore, in Ophiostorna ultni (Buisman) Nannf., vegetative compatibility groups (VCGs) were detected both within a piece of infected bark (Webber et al. 1987) and on an intercontinental scale, revealing the presence of mainly outcrossing populations in Europe as compared with prevailing clonality of the fungus in North America (Mitchell and Brasier 1994). Owing to a limited number of alleles present at the SI loci, VCGs do not necessarily represent clonal lines. Higher resolution in genetic markers may be provided by DNA fingerprinting to confirm that VCGs correspond to clonal lineages (Leslie 1993; Anderson and Kohn 1995). SI in combination with randomly amplified polymorphic DNA (RAPD) fingerprinting were previously used in population studies of the plant pathogenic ascornycetes Phomopsis subordinaria (Desm.) Trav. (Meijer et al. 1994), Sclerotinia sclerotiorum (Lib.) deBary (Kohli et al. 1995), and Fusarium oxysporum f.sp. cubense (E.F. Sm.) W.C. Snyder & H.N. Hansen (Bentley et al. 1995). In the two latter studies, each clone was distinguishable from other clones on the basis of DNA fingerprints, and SI tests were consistent with clonal assignments based on DNA fingerprints. However, in the case of Phomopsis subordinaria, SI tests revealed a generally higher diversity level than RAPD analysis (Meijer et al. 1994). The capability of SI data to delineate genets having identical nuclear DNA markers was also demonstrated in a study of the basidiomycete Artnillaria ostoyae (Romagn.) Herink (Rizzo et al. 1995). The results of these studies suggest that SI and DNA fingerprinting measure genetic variation independently, complementing each other. During earlier studies, RAPD analysis alone has also been used to determine genetic diversity and pathotypes within several ascomycetes causing diseases in agricultural crops (Goodwin and Annis 1991; Meyer et al. 1992; Van Der Vlugt-Bergmans et al. 1993; Nicholson and Rezanoor 1994). In tree-inhabiting species, arbitrary priming of DNA revealed considerable genetic differences between isolates of Gremmeniella abietina (Lagerb.) Morelet, distinguishing them into two ecotypically distinct groups (Hellgren and Hogberg 1995). Moreover, RAPD data supported the separation both of Ophiostoma piceae (Miinch) H. & P. Sydow and 0 . ulmi into two different species, respectively, and showed North American and Eurasian races of Ophiostorna novo-ulmi Brasier to be distinct biological entities (Pipe et al. 1995a, 1995b). Various degrees of geographical differentiation within ascomycete species were detected by several other studies based on DNA fingerprinting (Goodwin and Annis 1991 ; Hellgren and Hogberg 1995; Anderson and Kohn 1995). Earlier work on N. fuckeliana has already shown high within-species variation of fatty acid and sterol profiles

(Miiller and Hallaksela 1994). The purpose of this paper was to check possible clonality by means of SI and DNA fingerprinting and to determine the extent of genetic variation in two populations of N. fi~ckeliana separated by the Baltic sea.

Materials and methods Isolates of N. fuckelianci were collected from three sample plots located in Sweden (A, B, C) and six sample plots located in Lithuania (G, H, I, K, L, M) (Fig. I). Woody pieces were taken from living Picea abies trees that had been damaged by moose. An increment borer was inserted 6-8 cm deep into stems at the vicinity ( 1 -3 cm) of open bark peeling wounds. Core samples were brought to the laboratory in sterilized glass tubes. Sampled trees in Swedish plots A, B, and C were numbered and mapped, and distances (2- 100 m) between every isolation within these plots were estimated. In the laboratory, all samples were surface sterilized by flaming and placed on Petri dishes containing vegetable juice agar (VA) medium: 5 g glucose, 200 mL Graninimvegetable juice, 25 g agar, 800 mL H,O (pH 5.5). Colonies of N. fuckeliana were identified and subcultured after 10- 15 days of growth (Vasiliauskas et al. 1996). Seventy-four N. fuckeliana isolates were obtained, 40 from Sweden and 34 from Lithuania: 18 from the sample plot A, 13 From B, 9 from C, 6 from G, 7 from H, 5 from I, 5 from K, 3 from L, and 8 from M. SI tests were performed by cutting 4-mm discs of mycelium + VA from the margin of actively growing colonies and placing them pairwise 1.5-2.0 cm apart in the centre of 9-cm Petri dishes containing approximately 20 mL VA. These were incubated up to 60 days at room temperature (18-23°C) and cxamined periodically. Isolates were paired in all combinations within each sample plot. Additionally, every isolate was self-paired as two pieces from the same mycelium. Prior to DNA extraction, the isolates were cultured in liquid VA media for 2 weeks, harvested by vacuum filtration, and lyophilized. DNA was extracted and amplified using the core sequence of MI3 minisatelite DNA (5' GAGGGTGGCGGTTCT 3') as a primer in accordance with the methods published by Stenlid et al. (1994) and Hogberg et al. (1995). The cycling parameters were 45 cycles of denaturing at 93°C for 20 s, annealing at 48OC for 1 min, extension at 72°C for 20 s, and final extension at 72OC for 6 min. The amplified products were subjected to electrophoresis on 1.4% agarose gels, stained with ethidium bromide, and visualized under UV light (Stenlid et al. 1994). Owing to concern of some authors about reproducibility and reliability of RAPD fingerprinting (Ellsworth et al. 1993; Tommerup et al. 1995), we verified our results as follows: six of the tested strains were run in two separate amplifications under the same conditions and then in duplicate during the same amplification. Duplicate samples did not differ in amplified products in any of these controls. To test the sensitivity of the method for DNA concentration differences, 5 ng of DNA sample from the same six strains was diluted 10- and 100-fold. The banding patterns using the diluted DNA samples did not differ following amplification showing a low sensitivity to dilution of N. fuckeliatza DNA in this concentration range. All interpretations were made from photoof amplified fragments were graphic prints. Presence a-bsence scored in relation to molecular weight markers. Only clear and distinct bands were included in the analysis. For every pair of N. fuckeliana isolates, a similarity index (S) was calculated as the number of shared bands (S,,) divided by the number of bands in strain X (S,) and strain Y (S,) from the formula (Lynch 1990):

Corresponding similarity matrix was constructed using the program @ 1997 NRC Canada

Vasiliauskas and Stenlid

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Fig. 1. Partial map of the Baltic sea region showing sampling localities of Nectria fuckeliana. The bottom panel shows boundary of


forest area with sample plots and represents enlargement of the boxed area in the top panel.

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Fig. 2. Somatic incompatible reaction in pairing between different isolates (A) and somatic compatible reaction in self-pairing (B) of Nectria fickeliana.

SIMQUAL from the numerical taxonomy package NTSYS-pc, version 1.70 (Rohlf 1992). Similarity between the isolates was then expressed in percentage as the band sharing index (BSI), calculated as 100 times the value from the similarity matrix. Average band sharing index (ABSI) was calculated for all strain comparisons within and between any two populations (Gilbert et al. 1990). ABSI within a population is the mean of all BSIs for individual strain comparisons from that population, whereas ABSI between two populations is the mean of all BSI comparisons between individual strains from these two populations (Stenlid et al. 1994). Principal component analysis (PCA; Sirius for Windows 1993) was carried out to determine how the various isolates and populations of the fungus relate to each other.

Results All SI pairings between different isolates of N. fuckeliana resulted in a demarcation zone consisting of white aerial

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Fig. 3. Gel of amplified DNA from different isolates of Nectria fickeliana. Letters above the lanes indicate populations from which each isolate was obtained, as given in Fig. 1. I, molecular weight marker.

mvcelium, and the reaction was interpreted as incompatible (Fig. 2 ~ ) In : contrast, all ~ e l f - ~ a i r controls in~ formed no reaction zone and mycelia intermingled freely (Fig. 2B). Thus, no VCGs of N. fuckeliana were detected in our study. This was further supported by DNA fingerprinting data, where no pair of isolates exhibited identical banding patterns. Amplification of N. fuckeliana DNA using the M13 primer produced a total of 29 different fragments between 517 and 2035 basepairs (bp) in size, and there was a distinct pattern of bands amplified from each individual strain (Fig. 3). Banding patterns were most distinct and reproducible in the size interval of 1018-2035 bp, so fragments from that interval (17 in total) were included in further analyses. Two bands from those were amplified in all 74 isolates from both Sweden and Lithuania. One band was present in all except two strains. Another fragment was present in all Lithuanian strains, but it was also very common among Swedish ones. There were two uncommon markers for Sweden, each noted only once per single isolate, but these fragments also rarely appeared in Lithuanian populations of N. filckeliana. Therefore, no country-specific markers were observed, although there was one band present in all isolates from Swedish sample plot B that was totally absent among isolates from ~ithuancansample plots H, I, and L. The isolates of N. fuckeliana from Sweden and Lithuania were separated into two clusters by the PCA (Fig. 4), showing genetic differentiation between two geographical populations. However, there w s no separation regarding sample plots within each population. Principal components 1, 2 , and 3 contained 19.36, 16.17, and 12.13% of the variation, respectively. Analysis of ABSIs provided further support for the PCA data, as genetic similarity between Swedish and Lithuanian populations (ABSI = 73.9%) appeared to be significantly ( t test; p < 0.001) lower when compared with similarity existing within each country (77.0 and 80.0%, respectively). Within each country differentiation, expressed as comparison of similarity within and between sample plots, was low and statistically nonsignificant in both geographical areas. @ 1997 NRC Canada


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Fig. 4. Principal component analysis of Nectrinfuckelinnn isolates from Sweden (S) and Lithuania (L). Each plus sign indicates a single isolate.


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Spatial distribution of stems from which N. fickeliana was isolated in stands A, B, and C is shown in Fig. 5. Local distances had no influence on the genetic similarity of the occurring N. fickeliana individuals (R2 = 0.003).

Discussion Earlier studies have shown that species of ascomycetes can exhibit various degrees of clonality. Ophiostoma iilmi was introduced intercontinentally along with plant material and later spread clonally over large geographical areas via insects as vectors, also possessing ability for sexual outcrossing (Webber et al. 1987; Mitchell and Brasier 1994; Pipe et al.

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1 9 9 5 ~ ) Fusarium . oxysporittn f.sp. cubetzse and F. subglutinuns f.sp. pini have also- b e e u p r e a d far with contaminated plants or soil, but once established in new areas the local populations maintained clonal structure by dispersal of asexually produced conidia (Bentley et al. 1995; Gordon et al. 1996). Sclerotinia sclerotiorum can be dispersed clonally on a local scale by asexually produced sclerotia, through cultivation or movement of infested seed, irrigation water, or agricultural machinery; however, since the fungus is homothallic, clonal genotypes can also be propagated on macrogeographical scale via airborne ascospores (Anderson and Kohn 1995). Leptosphaeria maculans (Desm.) Ces. & de Not. can quickly spread long distances by movement of infected O 1997 NRC Canada

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Fig. 5. Spatial distribution of Nectriafickeliana in sample plots A, B, and C. Open circles indicate stems from which the fungus was isolated.

spores are the major source of inoculum, but some clonality may arise because of spread of the fungus via conidia that can only disperse relatively short distances compared with ascospores (Millgroom et al. 1991). In the present study, no evidence was obtained to indicate clonal spread of N. fuckeliana, and therefore, conidia are not likely to influence population structure of the fungus in the field. Both SI tests and DNA fingerprinting provided data suggesting that each isolate of N. fuckeliana is a genetically distinct individual and that sexual outcrossing is common in this species. Our sampling method was not designated to study the distribution of N. fuckeliana individuals occupying one spruce stem, but data obtained by Miiller and Hallaksela (1994) indicate multiple infections of the fungus into the same stem. In general, N. fuckeliana exhibited a high degree of genetic diversity. Differences in genetic structure between Lithuanian and Swedish populations of the fungus suggest that two populations are sufficiently separated to diverge on a significant level. The Baltic sea therefore seems to present a considerable geographical barrier for spread of N. fuckeliana. On the other hand, it is known that fruit bodies of ascomycetes can produce large amounts of ascospores ( 3 x lo7 from a single aphothecium of S. sclerotiorum or lo8 per day from one perithecial stroma of D. concentrica) and that various fungal spores can be lifted up above 1 km in the atmosphere and travel more than 700 km over seas (Lacey 1996). Nevertheless, DNA fingerprinting revealed certain geographical differences in populations of airborne ascospore spread ascomycete G. abietina (Hellgren and Hogberg 1995). However, higher genetic similarity of N. fuckeliana within sample plots and within both Swedish and Lithuanian populations is probably due to widespread distribution of the species and the prolific formation of perithecia on colonized plants.

Acknowledgement This study was financed by a scholarship for R.V. from funds of Bilateral Research Cooperation with Eastern Europe at the Swedish University of Agricultural Sciences.

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