Discovery of a new gene pool and a high genetic diversity of the chestnut blight fungus Cryphonectria parasitica in Caucasian Georgia.

In this study, we investigated the population genetic structure and possible origins of the plant pathogen Cryphonectria parasitica in Caucasian Georgia, a region within the centre of origin of the host species Castanea sativa. A total of 427 C. parasitica isolates from nine populations were genotyped at 10 microsatellite loci. A high genetic diversity was detected, but the overall Georgian population was dominated by three haplotypes which were present in most individual populations. Two of them have not been previously found in Europe. Bayesian clustering analysis and principal component analysis could not identify their source population, neither in Asia nor in North America. On the other hand, one haplotype is frequent in Central Europe and probably naturally invaded Caucasian Georgia from neighbouring Turkey. Seventy-three haplotypes were unique to specific populations, and 66 of them were represented by a single isolate. Allele patterns suggest that most of these haplotypes emerged locally through sexual recombination between haplotypes of the Georgian and the central European gene pool. Due to the high incidence of haplotypes not otherwise present in Europe, Caucasian Georgia represents an additional source of diversity for the European C. parasitica population.

Invasion genetics of the chestnut blight fungus Cryphonectria parasitica in Switzerland.

Cryphonectria parasitica is the best-known example of an invasive forest pathogen in Europe. In southern Switzerland, chestnut blight was first reported in 1948 whereas, north of the Alps, it did not appear until the 1980s. Between 1995 and 2008, we sampled 640 C. parasitica isolates from nine populations south of the Alps and nine north of the Alps. Twelve historical isolates, collected between 1950 and 1972 in the south, were obtained from our collection. All 652 isolates were screened at 10 microsatellite loci to test for the existence of divergent genetic pools and to infer possible origins of haplotypes. In total, 52 haplotypes were identified. Structure software analysis indicated that 43 haplotypes (including all historical haplotypes) belonged to a main cluster, 6 haplotypes belonged to a different cluster, and 3 haplotypes had an intermediate allele pattern. All newly founded populations in northern Switzerland were initiated by one or just a few haplotypes from the main cluster, which probably came directly from the populations south of the Alps. Subsequently, genetic diversity increased through mutations, sexual reproduction, or new migrations. The highest increase in diversity was observed in populations where haplotypes from different genetic pools were encountered.

Effect of Watering Regime on Disease Development in Pinus sylvestris Seedlings Inoculated with Bursaphelenchus vallesianus and B. mucronatus.

Several Bursaphelenchus spp. have been detected in declining pine trees in Europe during intensive monitoring for the pine wood nematode B. xylophilus. We investigated the pathogenicity of B. vallesianus and B. mucronatus, isolated from declining Scots pine (Pinus sylvestris) forests in Valais (Switzerland), in relation to drought stress. Four isolates of B. vallesianus and two isolates of B. mucronatus were inoculated into 3-year-old P. sylvestris trees, which were subjected to different watering treatments (50, 100, 150, and 250 ml of water per pot, biweekly). Disease symptoms, plant mortality, nematode population density, and nematode distribution in dead plants were assessed. Both Bursaphelenchus spp. proved highly pathogenic to the seedlings and watering treatment affected disease development in the inoculated pine trees. With decreasing water supply, we observed faster disease progress and higher pine mortality for both Bursaphelenchus spp. The overall mortality 70 days after inoculation was 60, 92, 95, and 100% for B. vallesianus and 40, 95, 100, and 100% for B. mucronatus in the 250-, 150-, 100-, and 50-ml watering treatments, respectively. Both nematode species multiplied in the inoculated plants; however, B. mucronatus had higher population densities than B. vallesianus in all watering treatments (on average, 33,159 versus 14,702 nematodes/dead plant compared with the initial inoculum density of 6,000 nematodes/plant). The highest nematode density was found in the lower part of the stem. About 7 to 16% of the nematodes were extracted from the roots. This study demonstrated that B. vallesianus has a pathogenicity potential comparable with that of B. mucronatus and provided experimental evidence that drought stress can result in increased symptoms caused by either Bursaphelenchus sp.

Saprophytic Activity and Sporulation of Cryphonectria parasitica on Dead Chestnut Wood in Forests with Naturally Established Hypovirulence.

ABSTRACT Sustainable biological control of the chestnut blight fungus Crypho-nectria parasitica with hypovirulence depends on the production and dissemination of hypovirus-infected propagules of the pathogen. We investigated the ability of C. parasitica to sporulate and produce hypo-virus-infected spores on recently dead chestnut wood in coppice stands in southern Switzerland where hypovirulence has been naturally established. The number and type (active, inactive, or none) of cankers was assessed on experimentally cut and stacked stems, firewood stacks, and natural dead wood. Hypovirus-free and hypovirus-infected strains readily survived for more than 1 year in the chestnut blight cankers of the stacked stems. Sporulation of C. parasitica was observed on the surface of preexisting inactive and active cankers, as well as on newly colonized bark areas and was significantly more abundant than on comparable cankers on living stems. On all types of dead wood, we observed more stromata with perithecia than with pycnidia; however, a large proportion of the stromata was not differentiated. All perithecia examined yielded only hypovirus-free ascospores. The incidence of pycnidia that produced hypovirus-infected conidia ranged from 5% on natural dead wood to 41% on the experimental stacks. The mean virus transmission rate into conidia was 69%. Our study demonstrates a considerable saprophytic activity of C. parasitica on recently dead chestnut wood and supports the hypothesis of a role of this saprophytic phase in the epidemiology of hypovirulence.

Sequence variation and evolution of Cryphonectria hypovirus 1 (CHV-1) in Europe.

Cryphonectria hypovirus 1 (CHV-1) acts as a naturally occurring biological control agent for chestnut blight, a destructive fungal disease of chestnut trees, which has been introduced into Europe in the 1930s. We have determined partial nucleotide and deduced amino acid sequences of the ORF A of 47 CHV-1 isolates collected in Europe over a period of 28 years. Phylogenetic analysis revealed the presence of four groups or single viruses, which showed sequence divergences ranging from 11 to 19%. These results confirm the previous subtype classification based on RFLP markers, with the exception of the two CHV-1 subtypes E and D, which appear to be related closer than anticipated previously. Dates of divergences between CHV-1 subtypes, calculated from nucleotide substitution rates, indicate that the CHV-1 subtypes diverged several hundreds years ago. Our results suggest that the genetic variation among CHV-1 subtypes did not evolve in Europe and support the hypothesis of multiple introductions of CHV-1 into Europe.

Genetic variation of Cryphonectria hypoviruses (CHV1) in Europe, assessed using restriction fragment length polymorphism (RFLP) markers.

A total of 72 hypovirus-infected isolates of the chestnut blight fungus Cryphonectria parasitica were sampled from nine European countries between 1975 and 1997. The double-stranded RNA of the Cryphonectria hypoviruses (CHV1) was isolated and reverse transcription (RT)-PCR products were obtained for two different regions of the viral genome (ORF A and ORF B) using primer sequences of the type species CHV1-EP713. Both PCR products of each viral isolate were digested with four restriction endonucleases recognizing sequences of four nucleotides. The restriction fragment length polymorphism (RFLP) analysis revealed 41 genetically distinct RFLP types of CHV1 with 10 types occurring more than once. Identical RFLP types were detected nine times among viruses collected in the same location. Cluster analysis based on the RFLP banding patterns separated the viral isolates into five CHV1 clusters or subtypes. Most viral isolates (64 out of 72) grouped into one large cluster which comprised all viruses from Italy (including CHV1-EP747), Switzerland, Crotia, Bosnia, Hungary, Greece, and the French island Corsica, as well as five out of 11 isolates from continental France. Two additional subtypes of CHV1 were found in France (one related to CHV1-EP713) and one each in Spain and Germany. The Swiss samples collected over a period of 20 years showed that very little RFLP variation has evolved during this time. The results of this study are consistent with the hypothesis of multiple introductions of CHV1 into Europe.

Population Structure and Disease Development of Cryphonectria parasitica in European Chestnut Forests in the Presence of Natural Hypovirulence.

ABSTRACT The Cryphonectria parasitica populations in two 6-year-old European chestnut (Castanea sativa) coppices were investigated in southern Switzerland over a period of 4 years. Occurrence of white isolates indicating an infection with Cryphonectria hypovirus, vegetative compatibility groups (VCGs), hypovirulence conversion capacity, and mating types were used to characterize the populations. Sampling of randomly chosen cankers in the first year yielded 59% white isolates in one and 40% in the other population. The distribution of the VCGs and mating types was similar among white and orange isolates, indicating a homogeneous infection of the two populations by the hypovirus. Fourteen VCGs were found in the first population, 16 VCGs in the second. Altogether, 21 VCGs were determined. The same three VCGs dominated in both populations, comprising more than 60% of all isolates. Several VCGs were represented only by white isolates. Five of the six most common VCGs were clustered in two hypovirulence conversion groups, with almost 100% hypovirus transmission within each cluster. Repeated sampling of the same cankers in 1990, 1992, and 1994 did not reveal an increase of white isolates. The portion of blighted stems rose from 37% to about 60% in both plots within 4 years. In this time, chestnut blight killed 15% and competition an additional 21% of the sprouts. Predominantly, sprouts with low diameters at breast height were killed. The growth rate of new cankers was high in their first year and decreased gradually in the following years. A role of hypovirulence in the decline of disease severity was evident since (i) cankers yielding white isolates grew slower and killed considerably fewer sprouts than cankers with orange isolates; and (ii) the majority of the cankers yielded white isolates at least once during the 4-year observation period.

Extra- and Intracellular Laccases of the Chestnut Blight Fungus, Cryphonectria parasitica.

A double-stranded RNA virus of the chestnut blight pathogen, Cryphonectria parasitica, has been shown previously to reduce accumulation of mRNAs of extracellular laccase (laccase A) produced by this fungus. Both extra- and intracellular laccases have been detected after growth of the fungus in liquid culture. In addition to cellular localization, the two laccases are distinguishable by time of appearance during growth and electrophoretic mobility. Laccase A was purified from the culture filtrate by standard protein purification procedures. The enzyme was characterized as a glycoprotein with a molecular mass of approximately 77 kDa. Both laccase A and laccase B activities were significantly reduced in the hypovirulent (double-stranded RNA-infected) strain UEP1 compared with the isogenic virulent (double-stranded RNA-free) strain EP155/2.

Regulation of laccase biosynthesis in the plant-pathogenic fungus Cryphonectria parasitica by double-stranded RNA.

Transmissible hypovirulence of the chestnut blight fungus, Cryphonectria parasitica, is associated with cytoplasmic double-stranded-RNA (dsRNA) viruses. The fungal laccase has attracted interest because its activity is reduced in hypovirulent dsRNA-containing strains. A laccase cDNA clone was isolated by screening a cDNA expression library with antibodies against the purified extracellular laccase. The amino acid sequence deduced from part of the cDNA clone revealed high homology to other fungal laccases, especially to the Neurospora crassa laccase. A major laccase transcript 2.3 kb in length was detected in Northern (RNA) blots. In liquid culture, extracellular laccase activity was reduced by about 75% in the hypovirulent (dsRNA-free) strain EP155/2. In contrast, production of biomass was not affected by the dsRNA. Northern blot analysis indicated that dsRNA down regulates laccase biosynthesis by reducing laccase mRNA accumulation. The laccase gene is one of several developmentally regulated genes affected by the presence of dsRNA.