SEARCH

SEARCH BY CITATION

Keywords:

  • biological control;
  • Castanea sativa;
  • chestnut blight disease;
  • dsRNA/RT-PCR/RFLP;
  • epidemiology

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cryphonectria parasitica, the causal agent of chestnut blight, has been present in Slovenia since at least 1950. To improve understanding of its diversity, 254 isolates of the fungus from 11 Slovenian populations were sampled. Fifteen vegetative compatibility (vc) types were identified. The dominant vc type was EU-13, comprising 40·1% of all isolates tested, followed by EU-1 (19·7%), EU-2 (12·2%) and EU-12 (9%). The vc type diversity in the most diverse population sampled in Slovenia was higher than in the populations found previously in northern Italy and Croatia. Both mating types and perithecia were observed in surveyed populations. Natural hypovirulence was found in six out of seven populations tested, with frequencies ranging from 72·2% in the population sampled near the Croatian border to 11·1% in the population sampled near the Austrian border. All identified hypoviral isolates (21) belonged to the Italian subtype of Cryphonectria hypovirus 1 and were closely related to the hypoviruses found in other European countries. Despite the high vc type diversity, incidence of hypovirulence was also high, indicating widespread natural biological control of the disease.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cryphonectria parasitica is the causal agent of chestnut blight, a severe disease responsible for the devastation of chestnut stands in North America and Europe. The ascomycete fungus causes bark cankers that progressively enlarge, girdle and kill branches and trunks. It was accidentally introduced into North America from Asia, and later into Europe, where it was first noticed on European chestnut (Castanea sativa) in Italy (Biraghi, 1946). In North America, the blight epidemic almost completely killed all American chestnut trees (Castanea dentata), while in Europe many chestnut stands spontaneously recovered from the disease as a result of the natural appearance of hypovirulent C. parasitica isolates (Heiniger & Rigling, 1994). Hypovirulent isolates are infected by fungal viruses from the family Hypoviridae that cause a reduction in fungal virulence (hypovirulence) and sporulation. Cryphonectria hypovirus 1 (CHV-1) is the best-characterized hypovirus and the only hypovirus detected in Europe to date (Allemann et al., 1999; Sotirovski et al., 2006; Krstin et al., 2008; Robin et al., 2010). Several subtypes within CHV-1 have been identified in Europe, with the Italian subtype being the most widespread. CHV-1 has been used as a biological control agent for chestnut blight, especially in French orchards (Robin et al., 2010). Biological control with CHV-1, either naturally or applied, is probably successful in Europe because of the relatively low diversity of vegetative compatibility (vc) types in the C. parasitica populations, which enables natural spread of hypovirulence (Heiniger & Rigling, 1994; Robin et al., 2000). The hypovirus can be transmitted from an infected isolate to a virus-free isolate through hyphal anastomosis. Virus transmission occurs between isolates of the same vc type, i.e. between isolates sharing identical alleles at all vegetative incompatibility (vic) loci (Cortesi et al., 2001). Transmission of hypovirus can also occur between isolates of different vc types, but heteroallelism at one or more vic loci strongly decreases the transmission rate (Cortesi et al., 2001; Papazova-Anakieva et al., 2008). In North America, the high vc type diversity in C. parasitica populations is thought to be a major reason for the failure of biological control of chestnut blight (Anagnostakis et al., 1986). The hypovirus is vertically transmitted into asexual conidia but not into sexual ascospores.

In Europe, six vic loci, each with two alleles, have been identified that account for 64 genetically distinct vc types (Cortesi & Milgroom, 1998). Knowledge of the vc type diversity in Europe has improved in recent years as a result of national surveys using common vc type tester isolates (Bissegger et al., 1997; Cortesi & Milgroom, 1998; Robin et al., 2000, 2009; Robin & Heiniger, 2001; Trestićet al., 2001; Sotirovski et al., 2004; Perlerou & Diamandis, 2006; Krstin et al., 2008). Many of the 64 defined vc types have been found in the field in these studies. In addition, several new vc types were recovered that could not be assigned to one of the known vc types (Robin et al., 2000; Bragança et al., 2007; Montenegro et al., 2008). Although more than 70 vc types have been identified across Europe (Robin et al., 2009), diversity of vc types on a local or regional scale is generally much lower and typically comprises of one or few dominant vc types (Robin & Heiniger, 2001).

The dominant vc types in Italy, eastern France, Switzerland, Austria, north-eastern Spain and Germany are EU-1, EU-2 and EU-5 (Cortesi et al., 1998; Robin et al., 2000, 2010; Robin & Heiniger, 2001), whereas EU-1, EU-11, EU-33, EU-66 and EU-72 are dominant in western France, western Spain and Portugal (Robin et al., 2000, 2009; Bragança et al., 2007); vc type EU-12 is dominant in southern and eastern Europe (Sotirovski et al., 2004; Perlerou & Diamandis, 2006).

After Italy, Spain and Switzerland, the former Yugoslavia was the fourth country in Europe to be struck by chestnut blight. In Slovenia, one of the six republics of the former Yugoslavia, the disease appeared in 1950 near the town of Nova Gorica, about 4 km from the Italian border (Jurc, 2002). Since the disease was already widespread in the forest area near the Italian border, it was assumed that it had been introduced years before its discovery. An intensive disease survey conducted in 1951 revealed 15 disease foci in the submediterranean region of Slovenia, covering 212 ha. In 1956, new disease foci were detected in the central continental part of Slovenia around Ljubljana and in 1961 near Celje (70 km from Ljubljana). Despite strict quarantine measures all chestnut-growing regions in Slovenia were quickly infested in the following years (Jurc, 2002). In the early 1980s, widespread healing and healed cankers were observed in the submediterranean region of Slovenia. White dsRNA-containing C. parasitica isolates were obtained from those cankers, confirming the presence of natural hypovirulence (Turchetti et al., 2002).

Although the chestnut blight outbreak in Slovenia has been intensively surveyed, the dominant vc types and incidence of the hypovirus are still not known. Studies on vc type diversity were carried out in three countries of former Yugoslavia: Bosnia-Herzegovina, Macedonia and Croatia (Trestićet al., 2001; Sotirovski et al., 2004; Krstin et al., 2008). Research on vc type and mating type diversity, as well as the spread of the hypovirus, are required to assess the state of chestnut blight and the potential of natural hypovirulence for biological control of the disease. In addition, these results will make an important contribution to the current knowledge on epidemiology of chestnut blight in this part of Europe.

The aims of this study were to (i) determine vc and mating types of C. parasitica isolates sampled from 11 Slovenian populations, (ii) estimate vc type diversity and assess the potential for an increase in that diversity, and (iii) investigate the incidence of hypovirus-infected isolates and identify which CHV-1 subtypes are present.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cryphonectria parasitica sampling and isolation

Cryphonectria parasitica was sampled in 2004, 2005 and 2007 from 11 populations in seven regions throughout the growing area of sweet chestnut (C. sativa) in Slovenia (Fig. 1). The Ostrovica population in the Sežana region is located in the submediterranean area in the west of the country, while all the other populations are located in the continental region (central and eastern part of the country). These two areas are divided by a zone of forest approximately 35 km wide zone (at its narrowest point) with calcareous soils that do not sustain the growth of sweet chestnut. The submediterranean area contains approximately 2500 ha of sweet chestnut forests and the continental part approximately 22 500 ha (Jurc, 2002). Each population sample consisted of 18–29 C. parasitica isolates that were collected in an area of 1–3 ha. Populations were at least 2 km apart from each other and all of them were sampled in privately owned coppice stands. All types of canker were sampled, including necrotic, superficial, healed (callused) and active cankers. Only one randomly selected canker per sprout cluster was sampled. Cryphonectria parasitica was isolated from bark samples (5 × 7 cm) taken from the margin of each canker as described by Cortesi et al. (1996). To assess the presence of perithecia, bark samples from all populations were examined under a dissecting microscope. All C. parasitica isolates were assessed for culture morphology on potato dextrose agar (PDA) as described by Krstin et al. (2008). Isolates with a white phenotype were termed white, and were presumed to be infected with CHV-1, whereas isolates with an orange phenotype were presumed to be virus-free. Isolates that could not be clearly assigned to a white or orange cultural type were classified as intermediate.

image

Figure 1.  Geographical origins of Slovenian Cryphonectria parasitica isolates.

Download figure to PowerPoint

Vegetative compatibility assays

A total of 254 C. parasitica isolates were assayed for vegetative compatibility as described by Krstin et al. (2008) using the European vc type testers EU-1 to EU-31 (Cortesi & Milgroom, 1998; Cortesi et al., 1998). Each isolate was first paired with six testers representing the dominant vc types in the neighbouring countries (EU-1, EU-2, EU-5, EU-12, EU-13, EU-17). Isolates incompatible with these dominant vc types were then paired with all the other European testers. Each vc test was done at least in triplicate, and only when replications showed identical results was the isolate assigned to the particular vc type.

Analysis of C. parasitica population diversity

The diversity of vc types in each population was determined by the number of vc types (N), the sample size ratio (S/N), Shannon diversity index (H′) (Anagnostakis et al., 1986) and by genotypic diversity index (G). H′ was calculated as − ∑pi ln pi, in which pi is the frequency of the ith vc type, and G was calculated as inline image, where pi is the frequency of the ith genotype. The distribution of genotypes within the sample, i.e. the evenness index (E5) was estimated according to Grünwald et al. (2003), where E5 = (G − 1)/(eH − 1). Based on the known vic loci for the vc types EU-1 to EU-32 (Cortesi & Milgroom, 1998), allelic diversity at six vic loci was calculated for the Slovenian C. parasitica populations as described by Milgroom & Cortesi (1999).

dsRNA isolation

Isolation of dsRNA was conducted for a subsample of 74 C. parasitica isolates from seven Slovenian populations (Valterski Vrh, Olševec, Zgornji Boč, Hočko Pohorje, Brežice, Gornji Suhor and Kal). Mycelia grown on PDA with a cellophane overlay were lyophilized and ground to a fine powder with a steel ball using a tissue lyser (Qiagen). Isolation of dsRNA from 40 mg mycelial powder by cellulose CF-11 chromatography followed the procedure of Allemann et al. (1999). After DNase treatment, hypoviral genomic dsRNA was analysed by electrophoresis in a 0·8% agarose gel in 1 × TBE (89 mm Tris borate pH 8·0, 89 mm boric acid, 2 mm EDTA) buffer. After staining with ethidium bromide, dsRNA bands were visualized under UV light and photographed by the Kodak EDAS 290 system. Lambda DNA digested with HindIII (Promega) was used as a molecular weight marker.

Complementary DNA (cDNA) synthesis, PCR amplification and RFLP analysis

The cDNA was synthesized from 100 ng dsRNA with random primers using AMV reverse transcriptase (Promega). Two regions of the hypoviral genome, one in ORF-A and one in ORF-B, were amplified from the cDNA as previously described (Allemann et al., 1999; Krstin et al., 2008). The two PCR products of each of 21 randomly chosen isolates sampled from six Slovenian populations (Valterski Vrh: V13b, V14, V33, V63; Zgornji Boč: ZB8, ZB18, ZB35, ZB71; Hočko Pohorje: HP22, HP33; Brežice: B11, BI8, BII69; Gornji Suhor: GS7, GS14, GS62; Kal: K1, K5, K32, K47, K50) were subjected to RFLP analysis. Each PCR product was digested with the restriction enzymes BsuRI and HinfI (MBI Fermentas). Restriction fragments were analysed by electrophoresis in 2% agarose gels with a 100-bp DNA Step Ladder (Promega) as the molecular weight marker. Binary data of RFLP analysis (Allemann et al., 1999) were used to construct phylogenetic trees with upgma cluster analysis (unweighted pair-group method with arithmetic averages) in the software program paup 4.0b10 (Swofford, 2003) using the mean character difference among isolates as the distance measure. To assess the reliability of the tree obtained, bootstrap analysis consisting of 1000 replicates was performed. Previously characterized CHV-1 isolates belonging to known subtypes from other European countries were included as references: subtype I (represented by isolates from Macedonia: Sk32MK, J12MK, Os65MK, Vr42MK, Vr25MK, Vr4MK, Os44MK, Vt28MK, J4MK, Po48MK, Po54MK; Italy: 33I, 32I, 38I, 40I, Euro7, 34I, 35I, 39I; Switzerland: 16CH, 5CH, 26CH; Bosnia-Herzegovina: 46BIH, 47BIH; Greece: 53GR; Croatia: 42HR, 43HR, 44HR, PzII34HR, M35HR, HK33HR, M7HR, MM22BHR, S53HR, M37 1HR, S36HR, SA18HR, IS9HR, OS8HR, HK76HR), subtype F1 (France: EP713, 62F, 55F, 56F), subtype F2 (France: 57F), CHV-1 subtype D (Germany: 72D) and subtype E (Spain: 71E) (Allemann et al., 1999; Sotirovski et al., 2006; Krstin et al., 2008).

Mating type assays

A subsample of 63 randomly chosen isolates from 11 populations was assayed for mating type using PCR. Primers M1-GS1 and M1-GS2-rev were used to amplify the MAT1-1 idiomorph and primers M2-GS2 and InvA5n to amplify the MAT1-2 idiomorph (Marra & Milgroom, 1999; McGuire et al., 2001). Amplification was carried out in 50 μL containing 50–100 ng DNA, 2 mm MgCl2, 0·2 mm dNTPs, primers at 0·2 μm each, PCR buffer and 1 U Taq DNA polymerase. Cycling conditions were: 95°C for 3 min, followed by 30 cycles of 0·5 min at 95°C, 1 min at 64°C and 4 min at 72°C, and a final extension at 72°C for 10 min. Amplified DNA fragments were separated by electrophoresis in 1% agarose gels at 90 V for 2 h using the 100-bp DNA Step Ladder (Promega) as a molecular size marker.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Vc type diversity

A total of 15 vc types were identified among the 254 C. parasitica isolates sampled from 11 Slovenian populations (Table 1). The number of vc types identified in each population ranged from four to eight. The dominant vc types were EU-13 and EU-1, which comprised 40·1% and 19·7% of all isolates, respectively. EU-13 was found in eight populations, while EU-1 was found in all populations except in the Olševec one, where EU-13 was the only observed vc type. In spite of the widespread occurrence of EU-1, this vc type was dominant in only three populations (Brežice, Gornji Suhor and Kal) in southeastern Slovenia. More than 12% of the isolates belonged to the vc type EU-2 found in eight populations. EU-12 comprised 9% of the isolates and was present in nine populations. Six vc types (EU-3, EU-4, EU-5, EU-15, EU-17 and EU-21) occurred at frequencies between 0·8% and 5·1% of all isolates. Five vc types (EU-7, EU-10, EU-19, EU-28 and EU-31) were represented by a single isolate.

Table 1.   Vegetative compatibility types and mating types of Cryphonectria parasitica isolates from 11 populations in Slovenia
Region/PopulationYear of samplingN aEU1EU2EU3EU4EU5EU7EU10EU12EU13EU15EU17EU19EU21EU28EU31Cankers with perithecia (%)bMAT c1-1MAT c1-2Both MAT types
  1. aNumber of isolates in each population.

  2. bPercentage of cankers with sexual structures (perithecia) of C. parasitica.

  3. cA random subsample of isolates in each population was analysed for mating type.

  4. dNumber of populations in which each vc type was found.

Kranj
 Valterski Vrh (V)20042312 1   112 5  1 6·225 
 Olševec (OL)200418        18      9·4311
Ljubljana
 Janče (JČ)200723151  1 114      5·713 
 Zgornja Jevnica (ZJ)2007296 1    4161 1   10·311 
Celje
 M. R. Mrzlica (CE)20072234 61 124 1    11·922 
Maribor
 Zgornji Boč (ZB)20042422     51212    5·913 
 Hočko Pohorje (HP)20042211 2   313 1 1  7·363 
Brežice
 Brežice (B)200422125     3  2    6·573 
Novo Mesto
 Gornji Suhor (GS)200425119 12     1 1  12·514 
 Kal (K)20041893 1   2  1 1 115·355 
Sežana
 Ostrovica (OC)2005284      2138  1  31·21 1
Total25450312113112310210131411 30312
Percentage 19·712·20·84·31·20·40·4940·13·95·10·41·60·40·4    
Populationsd 1082521198271411    
No. of white isolates 2412191001635180101    

The diversity of vc types, calculated with the Shannon diversity index (H′), varied from 1·16 in Brežice to 1·89 in M. R. Mrzlica (Table 2). The evenness index (E5) of Slovenian populations ranged from 0·55 (Hočko Pohorje population) to 0·85 (M. R. Mrzlica population), with a mean value of 0·69.

Table 2.   Richness, diversity and evenness of Cryphonectria parasitica vegetative compatibility (vc) types in Slovenia
PopulationNRichness, diversity and evenness
SaHbGcinline image
  1. aNumber of vc types found in each population.

  2. bShannon diversity index H ′ = − Σpi ln pi, where pi is the frequency of the i th vc type.

  3. cGenotypic diversity was calculated as inline image, where pi is the frequency of the i th genotype.

  4. dEvenness was estimated as described in Grünwald et al. (2003), E5 = (G − 1)/(eH ′ − 1).

Valterski Vrh2371·422·980·62
Olševec1810  
Janče2361·192·350·59
Zgornja Jevnica2961·272·700·66
M. R. Mrzlica2281·895·760·85
Zgornji Boč2461·423·160·68
Hočko Pohorje2271·362·600·55
Brežice2241·162·660·76
Gornji Suhor2561·312·990·72
Kal1871·533·300·63
Ostrovica2851·293·080·78

Allelic diversity at six vic loci was calculated for all 11 populations using known vic genotypes (Table 3). Five polymorphic vic loci were found in eight populations. One population (Brežice) had four and one population (M. R. Mrzlica) six polymorphic vic loci. With only one vc type (EU-13) present, the Olševec population lacked allelic diversity at all vic loci. The vic3 locus was polymorphic in the M. R. Mrzlica population, but not in the other ten populations. Allelic diversity for the other five vic loci ranged from 0·08 (Gornji Suhor, vic1 and vic7) to 0·52 (Valterski Vrh, vic4).

Table 3.   Allelic diversity at six vic loci in Slovenian populations of Cryphonectria parasitica
PopulationNvic1vic2vic3vic4vic6vic7MeanNo. of polymorphic vic lociMax. no. of vc typesa
  1. aMaximum number of vc types assuming sexual recombination at all polymorphic vic loci.

Valterski Vrh230·490·4900·520·300·30·35532
Olševec18000000001
Janče230·470·4000·490·440·440·37532
Zgornja Jevnica290·410·2400·480·370·370·31532
M. R. Mrzlica220·510·480·090·360·480·480·40664
Zgornji Boč240·430·4800·510·280·290·33532
Hočko Pohorje220·310·4500·480·310·360·32532
Brežice220·240·51000·360·360·25416
Gornji Suhor250·080·5100·220·150·080·17532
Kal180·360·5000·200·360·290·28532
Ostrovica280·510·1300·340·250·300·26532

Mating types and presence of perithecia

Both MAT idiomorphs were detected in 11 Slovenian C. parasitica populations (Table 1). Among the 63 isolates tested for mating type, 30 belonged to MAT1-1 while 31 belonged to MAT1-2. Two isolates, one from population Janče and one from Zgornja Jevnica, showed amplification of both mating types. The presence of perithecia was quantified for all 11 populations (Table 1). The incidence of cankers with perithecia in these populations varied from 5·7% (Janče) to 31·2% (Ostrovica), with a weighted mean of 14·8%.

Culture morphology and hypovirulence

The sample of 254 Slovenian C. parasitica isolates yielded 110 (43·3%) white, 34 (13·3%) intermediate and 110 (43·3%) orange isolates on PDA (Table 4). The highest incidence of white isolates (72·2%) was found in Kal. Five other populations had more than 50% white isolates: Valterski Vrh 52·5%, Zgornja Jevnica 55·2%, Janče 56·5%, M. R. Mrzlica 59·1%, and Brežice 59·1%. The lowest incidences of white isolates were found in Olševec (11·1%) and Ostrovica (17·4%). White isolates were present in the dominant vc types (EU-13, EU-1, EU-12, EU-2) as well as in less frequent vc types (EU-4, EU17), but not in EU-7 EU-10, EU-19 and EU-28. The highest percentage of white EU-13 isolates was recorded in Janče (78·5%), while the highest percentage of white EU-1 isolates was detected in Kal (77·7%).

Table 4.   Culture morphology of Cryphonectria parasitica isolates, dsRNA and RT-PCR/RFLP analysis of hypovirus-(un)infected isolates
PopulationNMorphology of collected isolatesaWhite isolates (%)No. of isolates tested for dsRNADetected dsRNA and morphology of tested isolatesbNo. of dsRNAs analysed by RT-PCR/RFLP
  1. aNo. of isolates showing white (w), intermediate (i) or orange (o) morphology in culture on potato dextrose agar.

  2. bNo. of isolates in which dsRNA was detected, out of the total tested, by colour in culture (w, white; i, intermediate; o, orange).

Valterski Vrh2312 w 4 i 7 o52·277/7 w0/0 i0/0 o4
Olševec182 w 1 i 15 o11·180/0 w 0/1 i 0/7 o0
Janče2313 w 7 i 3 o56·5000
Zgornja Jevnica2916 w 1 i 12 o55·2000
M. R. Mrzlica2213 w 2 i 7 o59·1000
Zgornji Boč246 w 6 i 12 o25134/4 w 2/3 i 0/6 o4
Hočko Pohorje227 w 3 i 12 o31·8115/5 w 2/2 i 0/4 o2
Brežice2213 w 4 i 5 o59·11310/10 w 2/2 i 0/1 o3
Gornji Suhor2511 w 4 i 10 o44107/7 w 1/1 i 0/2 o3
Kal1813 w 1 i 4 o72·2128/8 w 1/1 i 0/3 o5
Ostrovica284 w 1 i 23 o17·4000
Total254110 w 34 i 110 o43·37441/41 w 8/10 i 0/23 o21

Out of 74 isolates (41 white, ten intermediate and 23 orange) that were tested for the presence of dsRNA, 49 isolates (41 white and eight intermediate) contained a large dsRNA molecule. Estimation using lambda DNA/HindIII marker indicated that the dsRNA was approx. 12–13 kbp in length, which corresponds well with the known length of 12·7 kbp of the hypoviral genome. No dsRNA was detected in isolates showing the orange culture morphology. Hypoviral dsRNA was found in all Slovenian populations where white isolates were included in the analysis (Valterski Vrh, Zgornji Boč, Hočko Pohorje, Brežice, Gornji Suhor and Kal). RT-PCR of regions in ORF-A and ORF-B of the hypoviral dsRNA amplified fragments of approx. 1·4 and 1·7 kbp, respectively. RFLP banding patterns, obtained after digestion of the PCR products with two restriction enzymes, grouped all Slovenian hypoviruses to the Italian subtype of CHV-1 (Fig. 2). Within this subtype two groups of Slovenian CHV-1 isolates having identical restriction patterns in both ORFs were formed. The larger group contained 11 isolates and all of these isolates shared identical patterns with reference isolates from Croatia (CR23HR, HK27HR, 44HR), Macedonia (Sk32MK), Greece (53GR), Bosnia-Herzegovina (46BIH) and Italy (33I). The smaller group comprised four viral isolates, which had identical patterns with reference isolates from Croatia (S53HR), Switzerland (16CH) and Italy (32I). Five isolates (B11SL, K32SL, V33SL, HP33SL and K1SL) had unique restriction patterns.

image

Figure 2.  Dendrogram derived by upgma cluster analysis based on RT-PCR/RFLP fragments of Cryphonectria hypovirus 1 (CHV-1) isolates from Slovenia (marked by the extension SL) and other European countries. Reference isolates in CHV-1 subtype I were from Croatia (HR), Bosnia-Herzegovina (BIH), Greece (GR), Italy (I; including Euro7), Macedonia (MK), and Switzerland (CH). CHV-1 subtypes F1, E, D, and F2 were represented by isolates from France (F; including EP713), Spain (E) and Germany (D). Reference isolates from Croatia and Macedonia were from Krstin et al. (2008) and Sotirovski et al. (2006), respectively; all others from Allemann et al. (1999). Bootstrap values >50% (1000 replicates) are shown above the main branches of the upgma dendrogram.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fifteen vc types were recorded within the 11 populations of C. parasitica sampled in Slovenia. With the exception of Olševec, where only one vc type was found, the number of vc types within a local population ranged from four to eight. In all these populations the value of the H′ diversity index was higher than 1·15. Such relatively high vc type diversity has also been reported in some C. parasitica populations in neighbouring Croatia (H′ = 0·63–1·69) and northern Italy (H′ = 0·9–1·7), where 4–8 and 4–16 vc types were found, respectively (Cortesi et al., 1996; Krstin et al., 2008). Nevertheless, the most diverse population in Slovenia (H′ = 1·89) had a higher Shannon index than populations in northern Italy and Croatia. The number of vc types and the diversity index was higher in the Slovenian populations than in the populations in southern Italy (H′ = 0·4–0·8) and Macedonia (H′ = 0·1–0·7) (Cortesi et al., 1996; Sotirovski et al., 2004). In contrast, H′ values of Slovenian populations were lower than in two populations in southern Switzerland that comprised 14 and 16 vc types and H′ values of 1·9 and 2·1 (Bissegger et al., 1997). In Bosnia-Herzegovina, vc type diversity was high in the north-western region, but low in the eastern, central and southwestern regions (Trestićet al., 2001). In European countries, the value of the H′ index for local populations was generally lower than two (Sotirovski et al., 2004). High vc type diversity is typically found in regions with a long history of chestnut blight, such as northern Italy, southwestern France (Dordogne), southern Switzerland, and Croatia, and also where sexual reproduction of C. parasitica is frequent (Cortesi et al., 1996; Bissegger et al., 1997; Robin et al., 2000; Krstin et al., 2008). However, low vc type diversity was detected in Switzerland north of the Alps (Hoegger et al., 2000), Germany (Seemann et al., 2001), Turkey (Gurer et al., 2001), Macedonia (Sotirovski et al., 2004), Slovakia (Juhásováet al., 2005), Greece (Perlerou & Diamandis, 2006), Portugal (Bragança et al., 2007) and northwestern Spain (Montenegro et al., 2008), where chestnut blight has been present for a relatively short time. The present study shows that Slovenia groups with regions showing a relative high vc type diversity in Europe, which is in accordance with the early appearance of the disease in these countries in the 1950s.

All vc types found in Slovenia could be assigned to one of the 64 genetically defined EU vc types (Cortesi & Milgroom, 1998). The dominant vc type in Slovenia was EU-13 (40·1%), followed by EU-1 (19·7%), EU-2 (12%) and EU-12 (9%). EU-13 was the most frequent vc type in eight populations but was absent from three populations, all located in the southeast of the country. EU-13 is relatively rare in other European countries. It was found in northwestern Bosnia-Herzegovina (Trestićet al., 2001), Hungary (Radócz, 2001), Slovakia (Adamčíkováet al., 2006) and in some populations in northern Italy, Switzerland, Czech Republic and Croatia (Cortesi et al., 1998; Haltofová, 2006; Krstin et al., 2008). The other three main vc types in Slovenia were also frequent in north-western Bosnia-Herzegovina and Croatia (Trestićet al., 2001; Krstin et al., 2008). EU-1 and EU-2 are dominant in northern Italy, southern France, Switzerland, eastern Spain and Croatia (Robin & Heiniger, 2001; Krstin et al., 2008). The fourth most frequent vc type in Slovenia, EU-12, is dominant in southeastern Europe, including Greece, Macedonia, Bulgaria, Romania and southern Italy (Sotirovski et al., 2004; Perlerou & Diamandis, 2006). EU-12 is also frequent in Bosnia-Herzegovina, Croatia and other eastern European countries (Radócz, 2001; Robin & Heiniger, 2001; Trestićet al., 2001; Krstin et al., 2008). The other vc types found in Slovenia occur with much lower frequencies and are generally also rare in other European countries.

The dominance of EU-13 in Slovenia might be the result of a founder effect by which a relatively rare vc type was introduced by chance and spread across the country. It is very likely that EU-13 was responsible for the first disease foci observed in the western and central parts of the country in the 1950s. From Slovenia, EU-13 probably spread further into neighbouring Hungary, where the disease was first recorded in 1969 and where EU-13 is also a frequent vc type (Robin & Heiniger, 2001). EU-12 might have migrated from southeastern Europe via Bosnia-Herzegovina and Croatia into Slovenia, while EU-1 and EU-2 have probably spread from northern Italy into Slovenia. All rare vc types in Slovenia, except EU-10 could have been generated by recombination of the dominant vc types (EU-13 with EU-1 or EU-2). The presence of perithecia and both mating types indicate that sexual reproduction of C. parasitica occurs in Slovenia. It is interesting to note that EU-12 is also among the recombinant vc types that will be produced in sexual crosses between EU-13 and EU-2 (Cortesi & Milgroom, 1998). Thus, the possibility cannot be excluded that EU-12 originated locally from such crossing events. On the other hand, EU-10 is most likely a true immigrant, because it has the allele vic3-2, whereas all other Slovenian vc types have vic3-1. The result of the present study suggests that the vc type diversity currently observed in Slovenia is the result of the immigration of at least three vc types and subsequent sexual recombination among these vc types.

Since the vic genotypes are known for all vc types found in Slovenia, it is possible to determine the potential number of vc types according to the current situation. The most common vc types differed at five vic loci. Thus, through sexual recombination at all these polymorphic vic loci, the number of vc types could reach 32 in most of the Slovenian C. parasitica populations. In one population (M. R. Mrzlica), where six vic loci are polymorphic, a maximum number of 64 vc types could be expected in the future.

This study revealed that the hypovirus CHV-1 is widespread in the Slovenian C. parasitica populations. White isolates were found in all main vc types and in all populations tested, with frequencies ranging from 72·2% in the Kal population near the Croatian border to 11·1% in the Olševec population near the Austrian border. The actual incidence of the hypovirus is even higher since hypoviral dsRNA was also found in several fungal isolates with intermediate culture morphology. In Europe, a high prevalence of hypovirulence was generally observed in regions with a long history of chestnut blight, where hypovirulence was naturally established (Heiniger & Rigling, 1994; Bissegger et al., 1997; Robin & Heiniger, 2001), and in areas with low vc type diversity (Sotirovski et al., 2006). In contrast, low incidences of hypovirulence are attributed to recent introduction of the hypovirus in a given area. In Slovenia, the hypovirus is well established in spite of the relatively high vc type diversity. This is in concordance with the findings of Krstin et al. (2008) for Croatia and Robin et al. (2010) for France and indicates that the level of vc type diversity observed in many regions in Europe does not pose a major barrier for hypovirus spread. Furthermore, Carbone et al. (2004) demonstrated that the hypovirus is frequently transmitted in nature, even between isolates of different vc types.

All identified Slovenian hypovirus isolates were assigned to the Italian subtype of CHV-1. This subtype is widespread in Europe and is the only hypoviral subtype present in Croatia, Bosnia and Herzegovina, Macedonia, Greece, Hungary, Italy and Switzerland (Allemann et al., 1999; Sotirovski et al., 2006; Krstin et al., 2008). The fact that restriction patterns of many Slovenian CHV-1 isolates were identical to those of isolates from Croatia, Macedonia, Greece, Italy and Switzerland supports the hypothesis of Sotirovski et al. (2006) that this virus hyplotype has migrated throughout southern Europe. The presence of natural hypovirulence in Slovenia could have a positive impact on biological control of chestnut blight and attenuation of disease severity. On the other hand, sexual reproduction and increase of vc type diversity by recombination could obstruct natural biological control. Since sustainable biological control of chestnut blight requires a comprehensive knowledge of the dynamics of the Cryphonectria–hypovirus interaction, additional population studies of the fungus and the hypovirus will be necessary.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We would like to thank Dr Paolo Cortesi and Dr Michael G. Milgroom for providing EU reference isolates for vc types EU-1 to EU-64 and the company Tanin for collecting bark samples. This research was supported by the company Tanin Sisak, the Ministry of Science, Education and Sports of the Republic of Croatia (project no. 119-1191192-1215) and the Swiss National Science Foundation (SCOPES projects IB73A0-111089 and IZ73Z0-12792/1).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Adamčíková K, Juhásová G, Kobza M, 2006. Genetic diversity of Cryphonectria parasitica population in the Štiavnicko-Krupinská subpopulation in Slovakia. Plant Protection Science 42, 11924.
  • Allemann C, Hoegger P, Heiniger U, Rigling D, 1999. Genetic variation of Cryphonectria hypoviruses (CHV1) in Europe, assessed using restriction fragment length polymorphism (RFLP) markers. Molecular Ecology 8, 84354.
  • Anagnostakis SL, Hau B, Kranz J, 1986. Diversity of vegetative compatibility groups of Cryphonectria parasitica in Connecticut and Europe. Plant Disease 70, 5368.
  • Biraghi A, 1946. Il cancro del castagno causato da Endothia parasitica. L′Italia Agricola 7, 406.
  • Bissegger M, Rigling D, Heiniger U, 1997. Population structure and disease development of Cryphonectria parasitica in European chestnut forests in the presence of natural hypovirulence. Phytopathology 87, 509.
  • Bragança H, Simões S, Onofre N, Tenreiro R, Rigling D, 2007. Cryphonectria parasitica in Portugal – diversity of vegetative compatibility types, mating types, and occurrence of hypovirulence. Forest Pathology 37, 391402.
  • Carbone I, Liu YC, Hillman BI, Milgroom MG, 2004. Recombination and migration of Cryphonectria hypovirus 1 as inferred from gene genealogies and the coalescent. Genetics 166, 161129.
  • Cortesi P, Milgroom MG, 1998. Genetics of vegetative incompatibility in Cryphonectria parasitica. Applied and Environmental Microbiology 64, 298894.
  • Cortesi P, Milgroom MG, Bisiach M, 1996. Distribution and diversity of vegetative compatibility types in subpopulations of Cryphonectria parasitica in Italy. Mycological Research 100, 108793.
  • Cortesi P, Rigling D, Heiniger U, 1998. Comparison of vegetative compatibility types in Italian and Swiss subpopulations of Cryphonectria parasitica. European Journal of Forest Pathology 28, 16776.
  • Cortesi P, McCulloch CE, Song H, Lin H, Milgroom MG, 2001. Genetic control of horizontal virus transmission in the chestnut blight fungus Cryphonectria parasitica. Genetics 159, 10718.
  • Grünwald NJ, Stephen BG, Milgroom MG, Fry WE, 2003. Analysis of genotypic diversity data for populations of microorganisms. Phytopathology 93, 73846.
  • Gurer M, Ottaviani M, Cortesi P, 2001. Genetic diversity of subpopulations of Cryphonectria parasitica in two chestnut-growing regions in Turkey. Forest Snow and Landscape Research 76, 3836.
  • Haltofová P, 2006. Vegetative compatibility groups of Cryphonectria parasitica (Murrill) M.E. Barr in the Czech Republic. Advances in Horticultural Science 20, 558.
  • Heiniger U, Rigling D, 1994. Biological control of chestnut blight in Europe. Annual Review of Phytopathology 32, 58199.
  • Hoegger PJ, Rigling D, Holdenrieder O, Heiniger U, 2000. Genetic structure of newly established populations of Cryphonectria parasitica. Mycological Research 104, 110816.
  • Juhásová G, Kobza M, Adamčíková K, 2005. Diversity of Cryphonectria parasitica (Murr.) Barr vegetative compatibility (vc) in Slovakia. Acta Horticulturae 693, 63540.
  • Jurc D, 2002. An overview of the history of chestnut blight epidemic in Slovenia. Zbornik Gozdarstva in Lesarstva 68, 3359.
  • Krstin L, Novak-Agbaba S, Rigling D, Krajačić M, Ćurković Perica M, 2008. Chestnut blight fungus in Croatia: diversity of vegetative compatibility types, mating types and genetic variability of associated Cryphonectria hypovirus 1. Plant Pathology 57, 108696.
  • Marra RE, Milgroom MG, 1999. PCR amplification of the mating type idiomorphs in Cryphonectria parasitica. Molecular Ecology 8, 194750.
  • McGuire IC, Marra RE, Turgeon BG, Milgroom MG, 2001. Analysis of mating-type genes in the chestnut blight fungus, Cryphonectria parasitica. Fungal Genetics and Biology 34, 13144.
  • Milgroom MG, Cortesi P, 1999. Analysis of population structure of the chestnut blight fungus based on vegetative incompatibility genotypes. Proceedings of the National Academy of Sciences, USA 96, 1051823.
  • Montenegro D, Aguín O, Sainz MJ, Hermida M, Mansilla JP, 2008. Diversity of vegetative compatibility types, distribution of mating types and occurrence of hypovirulence of Cryphonectria parasitica in chestnut stands in NW Spain. Forest Ecology and Management 256, 97380.
  • Papazova-Anakieva I, Sotirovski K, Cortesi P, Milgroom MG, 2008. Horizontal transmission of hypoviruses between vegetative compatibility types of Cryphonectria parasitica in Macedonia. European Journal of Plant Pathology 120, 3542.
  • Perlerou C, Diamandis S, 2006. Identification and geographic distribution of vegetative compatibility types of Cryphonectria parasitica and occurrence of hypovirulence in Greece. Forest Pathology 36, 41321.
  • Radócz L, 2001. Study of subpopulations of the chestnut blight (Cryphonectria parasitica) fungus in the Carpathian basin. Forest Snow and Landscape Research 76, 36872.
  • Robin C, Heiniger U, 2001. Chestnut blight in Europe: diversity of Cryphonectria parasitica, hypovirulence and biocontrol. Forest Snow and Landscape Research 76, 3617.
  • Robin C, Anziani C, Cortesi P, 2000. Relationship between biological control, incidence of hypovirulence, and diversity of vegetative compatibility types of Cryphonectria parasitica in France. Phytopathology 90, 7307.
  • Robin C, Capdevielle X, Martin M, Traver C, Colinas C, 2009. Cryphonectria parasitica vegetative compatibility type analysis of populations in south-western France and northern Spain. Plant Pathology 58, 52735.
  • Robin C, Lanz S, Soutrenon A, Rigling D, 2010. Dominance of natural over released biological control agents of the chestnut blight fungus Cryphonectria parasitica in south-eastern France is associated with fitness-related traits. Biological Control 53, 5561.
  • Seemann D, Bouffier V, Kehr R, Schröder T, Unger J, 2001. Die Esskastanie (Castanea sativa Mill.) in Deutschland und ihre Gefährdung durch den Kastanienrindenkrebs (Cryphonectria parasitica (Murr.) Barr). Nachrichtenblatt des Deutschen Pflanzenschutzdienstes 53, 4960.
  • Sotirovski K, Papazova-Anakieva I, Grünwald NJ, Milgroom MG, 2004. Low diversity of vegetative compatibility types and mating type of Cryphonectria parasitica in the southern Balkans. Plant Pathology 53, 32533.
  • Sotirovski K, Milgroom MG, Rigling D, Heiniger U, 2006. Occurrence of Cryphonectria hypovirus 1 in the chestnut blight fungus in Macedonia. Forest Pathology 36, 13643.
  • Swofford DL, 2003. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sunderland, MA, USA: Sinauer Associates.
  • Trestić T, Ušćuplić M, Colinas C, Rolland G, Giraud A, Robin C, 2001. Vegetative compatibility type diversity of Cryphonectria parasitica in Bosnia-Herzegovina, Spain and France. Forest Snow and Landscape Research 76, 3916.
  • Turchetti T, Maresi G, Biagioni P, 2002. Blight and ink disease as constraint factors in chestnut stands of Mediterranean and central Europe. Zbornik Gozdarstva in Lesarstva 68, 12947.