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Chestnut blight in Europe: Diversity of Cryphonectria parasitica, hypovirulence and biocontrol

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Review article] A review of the chestnut blight situation in Europe is given. Since its introduction in 1938 into Italy, the blight has spread throughout Europe with the exception of the UK, the Netherlands and northern France. Recently the blight has begun to have much more impact on chestnut in north-west Spain and Portugal. Hypovirulence is widespread. In most countries healing cankers have been observed and the presence of dsRNA has been confirmed. However, hypovirulence is still absent from newly infected areas in northern France, northern Switzerland, Portugal and north-western Spain. The year of the first observation of healing cankers is weakly correlated with the year of the first occurrence of the blight. Although vc type diversity may be low at a local level, over 40 vc types have been identified in Europe. Vc type EU-2 is dominant in Western Europe whereas EU-12 is dominant in the south. In order not to jeopardise natural biocontrol with hypovirulence, strict quarantine regulations should prevent the introduction of new vc types. 1 Introduction Chestnut blight, caused by Cryphonectria parasitica (Murr.) Barr (Syn. Endothia parasitica [Murr.] And.), is one of the major diseases of chestnut (Castanea spp.) and has caused serious damage in orchards and in forests since its introduction in Europe (ANAGNOSTAKIS 1987). The hypovirulence, which is an attenuation of fungal pathogenis, has reduced the impact of this disease and has been used as a biological control method in Europe (NUSS 1992; HEINIGER and RIGLING 1994). During the COST G4 Action "Multidisciplinary Chestnut Research", the common objective of the 14 participating countries was to update infor -mation on the distribution of the blight and the hypovirulence, to standardize protocols and methods used in different countries to study the biology and vegetative compatibility of the fungus and to improve biological control in Europe. This paper is a short review of the recent findings obtained in Europe during this project.
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361
For. Snow Landsc. Res. 76, 3: 361–367 (2001)
Chestnut blight in Europe: Diversity of Cryphonectria
parasitica, hypovirulence and biocontrol
Cécile Robin1and Ursula Heiniger2
1UMR Santé Végétale, INRA Bordeaux, BP81, F-33 883 Villenave d’Ornon, France
robin@bordeaux.inra.fr
2WSL, Swiss Federal Research Institute, CH-8903 Birmensdorf, Switzerland. heiniger@wsl.ch
Abstract [Review article]
A review of the chestnut blight situation in Europe is given. Since its introduction in 1938 into
Italy, the blight has spread throughout Europe with the exception of the UK, the Netherlands and
northern France. Recently the blight has begun to have much more impact on chestnut in north-
west Spain and Portugal. Hypovirulence is widespread. In most countries healing cankers have
been observed and the presence of dsRNA has been confirmed. However, hypovirulence is still
absent from newly infected areas in northern France, northern Switzerland, Portugal and north-
western Spain. The year of the first observation of healing cankers is weakly correlated with the
year of the first occurrence of the blight. Although vc type diversity may be low at a local level,
over 40 vc types have been identified in Europe. Vc type EU-2 is dominant in Western Europe
whereas EU-12 is dominant in the south. In order not to jeopardise natural biocontrol with
hypovirulence, strict quarantine regulations should prevent the introduction of new vc types.
Keywords: hypovirus, vegetative compatibility, epidemiology, Castanea sativa
1 Introduction
Chestnut blight, caused by Cryphonectria parasitica (Murr.) Barr (Syn. Endothia parasitica
[Murr.] And.), is one of the major diseases of chestnut (Castanea spp.) and has caused serious
damage in orchards and in forests since its introduction in Europe (ANAGNOSTAKIS 1987).
The hypovirulence, which is an attenuation of fungal pathogenis, has reduced the impact of
this disease and has been used as a biological control method in Europe (NUSS 1992;
HEINIGER and RIGLING 1994). During the COST G4 Action “Multidisciplinary Chestnut
Research”, the common objective of the 14 participating countries was to update infor -
mation on the distribution of the blight and the hypovirulence, to standardize protocols and
methods used in different countries to study the biology and vegetative compatibility of the
fungus and to improve biological control in Europe. This paper is a short review of the
recent findings obtained in Europe during this project.
2 Distribution of chestnut blight
C. parasitica, the causal agent of chestnut blight was first observed in Europe in 1938 near
Genova, Italy. From Genova this disease spread rapidly within Italy and to neighboring
chestnut (Castanea sativa Mill.) growing areas (for a review see HEINIGER and RIGLING
1994). A recent survey in France has revealed a significant northward progression of the
blight (DEVILLEBONNE 1998). Similarly, areas with only scattered chestnut stands, such as
Swiss chestnut stands north of the Alps (HEINIGER and STADL ER 1990) and in the Rhine
valley in Germany (SEEMANN et al. 2001), are also infected. The disease is also spreading
and gaining high impact in the northwest regions of Spain and in Portugal. Today only the
scattered stands of chestnuts in the Netherlands and the coppice stands in southern UK are
free of the blight (Fig. 1).
362 Cécile Robin, Ursula Heiniger
3 Distribution of hypovirulence
Unlike in the USA where chestnut blight virtually eliminated the American chestnut (C.
dentata Borkh.), the European chestnut recovered from the disease due to the natural
occurrence of hypovirulence, caused by the dsRNA hypovirus CHV1 (HEINIGER and
RIGLING 1994, ALLEMANN et al. 1999). In most countries healing cankers were observed and
the presence of dsRNA was confirmed (Table 1).
There is a weak correlation between the year of the first observation of chestnut blight
and the appearance of healing cankers (Fig. 2). The hypovirus did not reach all the chestnut
blight affected areas and is still absent from newly infected areas in northern France and
northern Switzerland, Portugal, and north-western Spain (Pereira and Heiniger, unpup -
lished results). In Germany only one hypovirulent isolate was found in one of several blight
infested areas (SEEMANN et al. 2001).
ALLEMANN et al. (1999) reported considerable molecular variability within the European
CHV1 population, which led to the definition of five different CHV1 sub-types. Tracking
hypovirus is now possible using molecular markers, which allow the assessment of the
spread of hypovirulent strains used as biocontrol agents (HOEGGER et al. 1998; ROBIN et al.
2000b).
1938
1956
1950
1955
1969
1970
1992
1948
?
1974
1984
1968
1967 1963
1989
1947
1961
?
1992
1976
Fig. 2. Correlation of year of first canker report and year of first hypovirulence (hyv) report in
European countries (data from authors cited in Table 1).
Fig. 1. Map of Europe showing the
presence of chestnut blight and the
year of its first observation.
Spain
Greece
Hungary
Turkey
Austria
Albania
Croatia
Switzerland
Bosnia-Herzegovina
Germany
year of first canker
year of first report of hyv
2010
2000
1990
1980
1970
1960
Italy France
1930 1940 1950 1960 1970 1980 1990 2000
R2 = 0.47
Slovakia
Rep. Macedonia
363
For. Snow Landsc. Res. 76, 3 (2001)
The natural spread of CHV1 is still not fully understood. Although CHV1 infested C.
para sitica strains grow well in vitro, their growth is limited in the chestnut bark and sporu -
lation is rare. However, PROSPERO et al. (1998) found heavy sporulation of virulent as well
as hypovirulent strains on fallen wood and stacked fire wood.
Biological control by applying hypovirulent strains to growing cankers is practised in
France using Grente’s method (GRENTE and BERTHELAY-SAURET 1978). Selected mixtures
of hypovirulent strains were produced by a private company using a slightly different proto-
col and applied by chestnut growers in orchards. In Hungary, Greece, the Slovak Republic
and Switzerland local virulent strains converted with French or local hypovirulent strains
are used as biocontrol agents, produced using Grente’s method and applied by specialized
technicians or by scientists (JUHASOVA et al. 1998; RADÓCZ 1998; S. Diamandis, personal
communication).
Table 1. The chestnut blight epidemic in Europe. Years of first observation of chestnut blight and healing
cankers. 1% of chestnut area affected; 2Year of first observation of chestnut blight; 3Year of first obser-
vation of healing cankers; 4Year of first isolation of white C. parasitica strains; 5dsRNA confirmed.
Country ha1Chestnut Healing White dsRNA5Communicated by
blight2cankers3isolates4
Albania 1967 1983 1984 yes L. Bashkim
Austria 45 1970 1993 1993 no E. Wilhelm, U. Kudera
Bosnia-Herzegovina 100 1961 1980 1980 yes M. Uscuplic
Croatia 50 1955 1978 1978–1980 yes S. Novak Agbaba
France 90 1956 1964 1964 yes C. Robin
Germany 0.5 1992 1992 1992 yes D. Seemann
Greece 100 1963 1986 1986 yes S. Diamandis
Hungary 100 1969 1951 1964 yes L. Radócz
Italy 95 1938 1951 1964 yes P. Cortesi
Portugal 10 1989 1992 no no C. Abreu
Rep. of Macedonia 1974 1995 1995 yes K. Sotirovski
Romania 55 1984 yes D. Floarea
Slovak Republic 8 1976 1992 1999 yes G. Juhásová
Slovenia 1950 1985 1985 A. Solar
Spain 75 1947 1992 1992 yes C. Colinas
Switzerland 100 1948 1975 1975 yes U. Heiniger
Turkey 35 1968 1999 1999 yes M. Gurer, M. Çeliker
4 Diversity of vegetative compatibility types of C. parasitica
Vegetative compatibility (vc) is a major obstacle to the diffusion of the hypovirus. For a
Europe-wide comparison, 31 European vc types (EU-1 to EU-31) were identified and tester
sets supplied (CORTESI et al. 1998). These testers were used to study the genetic control of
vegetative compatibility in C. parasitica (CORTESI and MILGROOM 1998). 33 additional vc
types were obtained after crosses between the first 31 vc types and were added to the tester
set (EU-32 to EU-64). The vic genotype was determined for these 64 vc types, with 6 vic
genes each having two alleles. In France and Switzerland, 10 vc types incompatible with all
these 64 testers were found, showing that at least one additional vic gene or one additional
allele is needed to entirely describe the genetic determinism of vc types (ROBIN et al. 2000a).
The set of vc tester strains has to be increased accordingly.
364 Cécile Robin, Ursula Heiniger
Studies on vc type diversity were carried out in several European countries with the
available EU testers and results were used to update the map of the dominant vc types in
Europe (Fig. 3), already set up by HEINIGER et al. (1998). C. parasitica populations showed a
clear geographic distribution at the European level. The vc type EU-12 is the dominant vc
type in southern and eastern Europe, apart from in Turkey (M. Gurer, personal communi -
cation), whereas in western and north-western Europe, the vc type EU-2 is dominant.
The number of vc types found per country varies considerably. Over 40 vc types were
found in France, whereas in some countries only 4 vc types were identified, and in Turkey
only one (Table 2). However, sampling sizes greatly varied between countries, and diversity
indices (number of vc types/number of studied isolates or Shannon Index) must be used
with care to compare populations. However, a low vc type diversity is found in two cases:
in areas or countries where C. parasitica had only recently been introduced, e.g. Germany
(SEEMANN et al. 2001), Portugal, northern Switzerland (HOEGGER et al. 2000),
in countries where no perithecia were observed, e.g. Greece and Macedonia (S.
Diamandis and K. Sotirovski, personal communications).
A significant diversity between C. parasitica populations may be found within countries. E.g.
in northern Italy the vc type diversity is much greater than in southern Italy (CORTESI et al.
1996), and in Bosnia-Herzegovina 2 to 29 vc types were found per site (TRESTIC et al. this
volume). Considerable variation is also observed in Spain, where in the Atlantic regions vc
types incompatible with the most common European vc types were found (ROBIN et al.
2000a, COLINAS and USCUPLIC 1998, TRESTIC et al. this volume). Although the number of
European vc types may locally still be low, on a European level it is quite high, indicating the
potential of locally increasing vc type diversity.
1992 ?
1
12
12
12
12
12
12
2,
1,
5
2,
1,
5
2,
1
2
13,
17
2,
1,
5
?
?
?
?
1,
6,
12,
13
12,
1
33
IC
IC
Fig. 3. Map of Europe showing for each country the dominant vc types of C. parasitica populations.
IC = vc types incompatible with main EU testers.
365
For. Snow Landsc. Res. 76, 3 (2001)
Table 2. Dominant vc types of European C. parasitica populations. 1year of first observation of chestnut
blight; 2vc types comprising more than 50% of the isolates; 3+: found, – : not found, ?: no information.
Country Chestnut Number of Major vc types Perithecia Personal communication
blight1vc types present (>50%)2found in or reference
the field3
Austria 1970 15 EU-2 E. Wilhelm, U. Kudera
Bosnia-Herzegovina 1961 27 EU-12, EU-1 + M. Uscuplic
France 1956 40 + EU-2, EU-5, EU-1,
unknown EU-33 + C. Robin
Germany 1992 4 EU-2 D. Seemann
Greece 1963 4 EU-12 S. Diamandis,
C. Perlerou
Hungary 1938 18 EU-1, EU-6,
EU-12, EU-13 + L. Radócz
Italy 1938 20 EU-2, EU-1, EU-5 + CORTESI et al., 1998
Macedonia 1974 5? EU-12 + K. Sotirovski
Portugal 1989 4 ? N. Santos
Slovak Republic 1976 8 EU-12 + G. Juhásová
Spain 1947 13 EU-2 ? C. Colinas
Switzerland 1948 28 EU-2, EU-1, EU-5 + U. Heiniger, D. Rigling
Turkey 1968 EU-1 + M. Gurer, M. Çeliker
5 Chestnut blight epidemiology
Except for some regional occurrence of perithecia in the field (Table 2, CORTESI et al. 1996,
BISSEGGER et al. 1997), we do not know much about how and when C. parasitica completes
its biological cycle in the different countries with their various climatic conditions. However,
knowledge of C. parasitica biology is essential to understand and predict disease develop-
ment, i.e. C. parasitica and CHV1 spread. Since C. parasitica is heterothallic but with a low
outcrossing rate, it has a clonal and a sexual cycle. This may have consequences for canker
spread, vc type diversity, CHV1 spread and thus disease severity (MILGROOM 1995).
In the USA, where vc type diversity is much higher than in Europe, most of the ascopores
are discharged in autumn and are the main source of primary inoculum responsible for
establishing new cankers (HEALD and GARDNER 1913; ANAGNOSTAKIS et al. 1998).
Ascospore production and dispersal have been investigated in one orchard in France
(GUÉRIN et al. 1998). Results showed that ascospores were discharged from spring until
autumn, with a peak of spore trapping in spring, after rainfall. The coincidence of the
discharge of ascospores in air with the highest chestnut receptivity to blight (in spring)
suggested a high impact of these propagules on blight incidence and distribution (L. Guérin
and C. Robin, unpublished results).
Very few data exist in Europe on the genetic structure of C. parasitica populations
assessed with molecular and neutral markers other than vc types. The latter are not poly-
morphic enough to differentiate genotypes. In southern Switzerland, vc type and DNA
fingerprint diversities were high and did not correlate. In contrast, fingerprint and vc type
diversity were very low and found to correlate in two populations with recently introduced
C. parasitica and one mating type clearly dominating. This suggests that these populations
were clonal and that sexual reproduction was not effective (HOEGGER et al. 2000).
366 Cécile Robin, Ursula Heiniger
6 Conclusion
With the exception of the UK and northern France, almost all the European chestnut grow-
ing areas have been infected by the blight caused by C. parasitica. However, chestnut blight
distribution is not an indicator of chestnut blight severity unless it is associated with the
presence of the hypovirus CHV1, the causal agent of hypovirulence. Disease management
should be applied according to the presence or absence of CHV1. In Europe there are chest-
nut sites where the disease was introduced more than 30 years ago and where the CHV1 is
well established and thus the blight severity low. But there are also sites where the introduc-
tion of the blight is recent and has not yet been followed by CHV1. Here the blight epidemic
may still turn out to be devastating.
As the success of biocontrol with the hypovirus CHV1 is negatively correlated to the
number of vc types present, an increase in vc type diversity may jeopardize biocontrol
efforts. Movement of chestnut plants and wood should be restricted and mycelial mixtures
of CHV1 infested C. parasitica for biocontrol should only contain the local vc and mating
types.
More epidemiological studies of the natural spread of the hypovirus, as well as the epi-
demiology of C. parasitica are required in order to predict disease development better and
to improve biological control measures.
Acknowledgements
We would like to thank the members of the COST G4 action that submitted information about
the chestnut blight situation in their country: C. Abreu, C. Colinas, P. Cortesi, S. Diamandis, D.
Floarea, G. Juhásová, U. Kudera, B. Mal Lushaj, S. Novak Agbaba, C. Perlerou, L. Radócz, A.
Solar, D. Seemann, K. Sotirovski, M. Uscuplic, E. Wilhelm.
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Accepted 28.1.02
... sativa (sweet chestnut) in most of continental Europe, where the fungus was first introduced via Italy in 1938 (Robin & Heiniger, 2001). ...
... Some of these VCGs occur frequently across Europe. The VCG EU10 is dominant in Bulgaria (Milgroom et al., 2008) but subdominant in Bosnia, Greece, Italy, Macedonia and Switzerland (Perlerou & Diamandis, 2006;Radócz, 1998;Robin & Heiniger, 2001;Sotirovski et al., 2004), while EU2 and EU1 dominate in Croatia, France, northern Italy, eastern Spain and southern Switzerland (Krstin et al., 2008;Robin & Heiniger, 2001). ...
... Some of these VCGs occur frequently across Europe. The VCG EU10 is dominant in Bulgaria (Milgroom et al., 2008) but subdominant in Bosnia, Greece, Italy, Macedonia and Switzerland (Perlerou & Diamandis, 2006;Radócz, 1998;Robin & Heiniger, 2001;Sotirovski et al., 2004), while EU2 and EU1 dominate in Croatia, France, northern Italy, eastern Spain and southern Switzerland (Krstin et al., 2008;Robin & Heiniger, 2001). ...
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Cryphonectria parasitica was detected for the first time in UK in 2011. A 2017-2018 survey identified the disease at different sites in Berkshire, Derbyshire, Devon, Dorset, and London, while the present study comprises the results of the 2019-2020 survey with new findings and additional sites in Berkshire, Buckinghamshire, Cornwall, Derbyshire, Devon, London, West Sussex, and the island of Jersey, reflecting the progressive diagnosis of more infected trees. A total of 189 samples were collected from 52 surveyed sites, and 123 samples tested positive both by real-time PCR and/or isolation from 43 sites. A total of 115 isolates were tested for mating type, vegetative compatibility group (VCG) and Cryphonectria hypovirus 1 (CHV-1). Twelve VCGs were identified, with four of them being first records in UK. The highest diversity of VCGs was found in Devon followed by West Sussex while London and Derbyshire presented the lowest. Both mating types were present (41% MAT-1 and 59% MAT-2), and no heterokaryons were observed. Only one mating type was present per site and VCG, with the exceptions of two sites in West Sussex, two in London, one in Derbyshire, and one in Devon, where both mating types for EU2 and EU33, EU10 and EU37, EU2, and EU9 were detected respectively in different trees. Perithecia of C. parasitica were not observed at any site during this survey. Cryphonectria hypovirus 1 (CHV-1) was infecting three isolates in very low concentration (1.9 to 48.1 ng/µl) from three different locations in London and was always non-mutated subtype-I haplotype E-5. A greater diversity of VCGs at outbreak sites compared with previous surveys combined with their scattered distribution and the slow spread of the pathogen, supported the hypothesis that this disease has been introduced through imports over time from Europe.
... worldwide. The disease in Europe was first detected in Italy in 1938 (EPPO current status: Present) [1,2] from where it spread to the neighboring countries of Switzerland (1948; widespread), Slovenia (1950; restricted distribution), and France (1956; restricted distribution) [3,4]. The disease is currently present in Spain (1947; restricted distribution), Croatia (1955; widespread), Albania (1967; widespread), Serbia (1975; widespread), Portugal (1989; widespread) and Germany (1992; restricted distribution), Azerbaijan (2004; present), United Kingdom (2011; restricted distribution), and Belgium (2014; restricted distribution) [1,5]. ...
... The disease in Europe was first detected in Italy in 1938 (EPPO current status: Present) [1,2] from where it spread to the neighboring countries of Switzerland (1948; widespread), Slovenia (1950; restricted distribution), and France (1956; restricted distribution) [3,4]. The disease is currently present in Spain (1947; restricted distribution), Croatia (1955; widespread), Albania (1967; widespread), Serbia (1975; widespread), Portugal (1989; widespread) and Germany (1992; restricted distribution), Azerbaijan (2004; present), United Kingdom (2011; restricted distribution), and Belgium (2014; restricted distribution) [1,5]. Most of the European C. parasitica populations seem to have originated from single introductions since they have reduced genetic diversity [6]. ...
... To find a way out of chestnut blight, two main strategies are being considered: biocontrol through virus-induced fungus hypovirulence, and prevention by breeding disease-resistant genotypes. The use of RNA mycoviruses (i.e., Cryphonectria hypovirus 1; Hypoviridae) has resulted in highly effective in Europe [1], thus becoming one of the most successful alternatives for controlling the disease in natural stands. This biocontrol method is based on inoculation of virus-infected fungal strains belonging to the same vegetative compatibility group/s (vcg) than those causing disease in the target stand [15,16]. ...
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Cryphonectria parasitica (Murrill) M.E. Barr (Sordariomycetes, Valsaceae) is the causal agent of chestnut blight. This disease is a major concern for chestnut cultivation in Europe. The fungus colonizes vascular tissues and evolves generating cankers causing severe dieback and the death of the tree. Excised and debarked well-lignified shoots of 28 C. sativa genotypes (assay A) and of 10 progenies (assay B) were inoculated with C. parasitica strain FMT3bc2 (vcg: EU2). Fungal growth was measured along the longitudinal axis on the 3rd and 6th days after inoculation. Results indicated the inoculation methodology works and the results were clear after 6 days. Differences in susceptibility to chestnut blight among C. sativa trees of Montseny have been detected both at the individual genotype level and at the progeny level. Nineteen genotypes and four progenies showed a susceptibility to Blight not significantly different from C. mollissima. The methodology was easy to apply in extensive/preliminary selection screenings to assess the susceptibility of C. sativa materials to the Blight.
... In 2008, a severe needle disease of Pinus radiata caused by a new and undescribed Phytophthora was reported in New Zealand [6]. In 2013, P. pluvialis was isolated from stream, soil and canopy drip in tanoak-Douglas fir forest in Oregon (USA) and described as a new pathogen species within also associated with an increase in the survival time of host plants, this mycovirus was used as a biocontrol agent of chestnut blight in Europe [21]. Additionally, study revealed PiRV-2 affects the sporulation in P. infestans [23]. ...
... Some mycoviruses affect the host virulence and may play an important role in the development of the controlling methods for plant diseases. For example, CHV1 decreases the pathogenicity of its host C. parasitica which was used as a biocontrol agent of chestnut blight in Europe [21]; Conversely, PiRV-2 stimulates the sporangia production in P. infestans and could impact the ecological fitness of P. infestans [23]. Since P. pluvialis results in severe red needle cast disease in New Zealand, future research on the virus transmission and whether PplRV1 affects host virulence (i.e., hyper-or hypo-virulence) will be important for the development of a potential biocontrol for red needle cast disease. ...
Article
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A new dsRNA virus from the oomycete Phytophthora pluvialis has been characterized and designated as Phytophthora pluvialis RNA virus 1 (PplRV1). The genome of the PplRV1 reference genome is 6742 bp that encodes two predicted open reading frames (ORFs). ORF1 and ORF2 overlap by a 47 nt “slippery” frameshift sequence. ORF1 encodes a putative protein of unknown function. ORF2 shows high similarity to the RNA-dependent RNA polymerase (RdRp) of other dsRNA viruses. Phylogenetic analysis of the putative PplRV1 RdRp and its most closely related viruses showed PplRV1 is distinct from other known viruses (below 33% amino acid similarity), which indicates this virus may belong to a new virus family. Analyses of the geographical distribution of PplRV1 in relation to two genetically distinct classes of its host revealed two corresponding genotypes of the PplRV1 (termed a and b), which share 92.3% nt identity. The reference genome for the second genotype is 6760 bp long and a prediction of its genetic organization shows three ORFs, with ORF2 being split into two ORFs, ORF2a and ORF2b, that is conserved in seven of eleven genotype b isolates. Additionally, a quick and simple diagnostic method using qPCR has been developed, which is suitable for large scale screens to identify PplRV1 in Phytophthora.
... This genomic region would either be exchanged very regularly between these lineages by an undetermined genetic mechanism or would sometimes be exchanged and maintained in the lineages by regular sexual reproduction within the lineages during European colonization. Second, we expected negative values of Tajima's D in asexually spread lineages (Gladieux et al., 2017), and in the case of population expansion (Aris-Brosou & Excoffier, 1996) as for C. parasitica populations in Western Europe (Robin & Heiniger, 2001). ...
... Les explications à ces épidémies moins importantes seraient que les châtaigniers européens sont moins sensibles qu les châtaigniers nord-américains, et qu'un virus (Cryphonectria hypovirus1, CHV-1) qui induit l'hypovirulence en diminuant la fitness des souches de C. parasitica infectées est présent en Europe, probablement dû à l'introduction de souches hypovirulentes directement depuis l'Asie(Nuss, 1992 ; Milgroom & Cortesi, 2004). Des souches de C. parasititica hypovirulentes sont notamment utilisées comme moyen en de lutte biologique car leur inoculation sur des châtaigniers infectés par C. parasitica peut mener à la transmission du virus vers la souche présente par fusion des deux mycéliums(Robin & Heiniger, 2001). Néanmoins, la reproduction sexuée, empêchant la transmission du virus à la descendance, etFigure 4 extraite de Robin & Heiniger (2001) : Carte de l'Europe montrant la présence de Cryphonectria parasitica et l'année de sa première observation par pays. ...
Thesis
Les invasions biologiques sont une composante majeure des changements globaux, causant de nombreuses perturbations dans le fonctionnement des écosystèmes et les activités humaines. Leur fréquence augmente depuis la fin du XXème siècle, notamment dans le cas des champignons pathogènes des arbres forestiers. Dans ce contexte d'introduction, l’adaptation des agents pathogènes à de nouveaux environnements et de nouveaux hôtes constitue un paradoxe évolutif, du fait d'une faible diversité génétique généralement introduite. Des mécanismes évolutifs permettant l’adaptation de ces organismes ont été proposés, mais à l’exception de quelques espèces modèles, souvent agents pathogènes de plantes cultivées, ces mécanismes restent peu étudiés chez les champignons.Cette thèse a pour but d’étudier l’évolution des populations de Cryphonectria parasitica, l’agent causal du chancre du châtaignier, dans le contexte d’une double introduction en Europe. Originaire d’Asie, il a été introduit en Amérique du Nord à la fin du XIXème siècle, entraînant la disparition quasi-totale des populations naturelles de châtaigniers américains. Il a ensuite été introduit en Europe depuis ces populations nord-américaines et des populations asiatiques au début du XXème siècle. En Europe, les populations sont majoritairement structurées en lignées clonales contrairement aux populations d’origine. Ceci suggère un changement de mode de reproduction pouvant être impliqué dans le succès invasif de ces populations. Pourtant, les études précédentes ont montré que plusieurs génotypes n’appartenant pas aux principales lignées clonales se sont maintenus lors de la colonisation et que des croisements entre ces lignées clonales existent, même s’ils semblent limités. Les objectifs de cette thèse ont été de mieux décrire les croisements entre les lignées clonales et d’identifier de possibles barrières aux croisements entre celles-ci.Dans une première partie, le génome de 50 isolats français, nord-américains et asiatiques ont permis de confirmer la forte similarité des isolats appartenant à une même lignée clonale européenne, à l’exception de petites régions divergentes échangées entre ces génomes, plus fréquemment observées entre lignées introduites d’une même origine. Par ailleurs, 5 des 6 lignées étudiées portent une signature d’échange récent de la région portant le gène du type sexuel, conférant aux lignées clonales la capacité de s’autoféconder (haploid-selfing). Ces résultats soulignent que le maintien de lignées clonales chez un champignon hétérothallique comme C. parasitica (cad que les croisements impliquent des génotypes avec des types sexuels différents) n’implique pas toujours et uniquement la reproduction asexuée.Dans une seconde partie, l’assemblage de génomes d’isolats provenant des aires d’origine et introduites, utilisant des méthodes de séquençage long brin, a permis de comparer leur structure et composition chromosomique, et d’identifier de possibles barrières à la recombinaison. Aucun réarrangement majeur n’a cependant été détecté à l’exception d’une région d’1Mb, adjacente au locus du type sexuel, très riche en éléments transposables et variable entre les génomes. Ces résultats semblent infirmer l’hypothèse d’isolement reproducteur par réarrangement du génome. En revanche, les zones contenant de nombreux éléments mobiles pourraient permettre une évolution rapide de C. parasitica lors de l’introduction.Une dernière partie aborde l’étude des génotypes semblant provenir de croisements entre les lignées clonales majoritaires afin d’explorer les processus de recombinaisons entre les pools génétiques des deux introductions et d’identifier de possible barrières à l’hybridation potentiellement associées à des combinaisons génétiques défavorables.Ce travail souligne l'apport des données génomiques dans la compréhension des processus de recombinaison et la détection des variations génétiques d'un champignon pathogène invasif affectant ses capacités évolutives.
... (and partially EU-06), which are the most frequent vc types in central Europe (Robin & Heiniger, 2001). A relative stability of vc type diversity over time was also recently reported by Ježić et al. (2021) for in severe inbreeding within a few generations (Carlson et al., 2017). ...
Article
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Hyperparasites can affect the evolution of pathosystems by influencing the stability of both pathogen and host populations. However, how pathogens of perennial hosts evolve in the presence of a hyperparasite has rarely been studied. Here, we investigated temporal changes in genetic diversity of the invasive chestnut blight pathogen Cryphonectria parasitica in presence of its parasitic mycovirus Cryphonectria hypovirus 1 (CHV1). The virus reduces fungal virulence and represents an effective natural biocontrol agent against chestnut blight in Europe. We analyzed genome‐wide diversity and CHV1 prevalence in C. parasitica populations in southern Switzerland that were sampled twice at a time interval of about 30 years. Overall, we found that both pathogen population structure and CHV1 prevalence were retained over time. The results suggest though that recent bottlenecks have influenced the structure of C. parasitica populations in southern Switzerland. Strong balancing selection signals were found at a single vegetative incompatibility (vic) locus, consistent with negative frequency dependent selection imposed by the vegetative incompatibility system. High levels of mating among related individuals (i.e. inbreeding) and genetic drift are likely at the origin of imbalanced allele ratios at vic loci and subsequently low vc type diversity. Virus infection rates were stable at ~30% over the study period and we found no significant impact of the virus on fungal population diversity. Consequently, the efficacy of CHV1 mediated biocontrol was likely retained.
... Anagnostakis (1987) reported that in Italy, virulent strains of C. parasitica could travel at an approximate rate of 30 km/ year. Consequently, it is possible that new vc types arrived in South Tyrol from more diverse neighboring populations, such as those in northern Italy (Robin & Heiniger, 2001). Prospero and Rigling (2012) found that the migration of C. parasitica from divergent genetic pools might have increased the genetic diversity (including vc type diversity) in Switzerland. ...
Article
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European chestnut ( Castanea sativa ) is threatened by the invasive fungus Cryphonectria parasitica, which causes chestnut blight. The virulence of the fungus can be reduced by a group of mycoviruses that can spread among vegetatively compatible strains through hyphal anastomosis. Hypovirulent isolates are used as biocontrol agents, but their efficiency can be diminished by restricted hyphal anastomosis if the variability of vegetative compatibility (vc) types in a population is high. Sexual reproduction could increase the vc type diversity and further complicate biocontrol in a region. Therefore, knowledge of genetic diversity of C. parasitica is important to assess the effectiveness of a biological control program. The present study was performed in the Autonomous Province of Bozen-Bolzano (South Tyrol) in northern Italy, where chestnut cultivation provides an additional income to farmers. The genetic characterization of C. parasitica isolates from 35 chestnut stands and one forest population in different districts of South Tyrol was performed based on the analysis of vegetative incompatibility loci, the mating type locus, and the internal transcribed spacer (ITS) region. In this study, a total of 23 different vc types were found all over South Tyrol with a Shannon diversity index of 1.86. EU-2, EU-1, and EU-13 were the most widespread vc types comprising 51%, 13%, and 9% of the fungal isolates, respectively. Both mating types were present in the region with a ratio close to 1:1. Three different haplotypes were identified based on ITS sequence analysis, which pointed to two introduction events of the fungus to the region and allowed placing C. parasitica from South Tyrol into a larger phylogeographic context.
... Borkh), така и при европейския кестен (C. sativa) (anagnostakis, 1987;Robin, Heiniger, 2001). Гъбата C. parasitica произхожда от източна Азия, където заболяването не причинява сериозни повреди по азиатските видове кестен Castanea mollissima Blume и Castanea crenata Sieb & Zucc. ...
Article
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Deterioration of the health of chestnut (Castanea sativa) in Bulgaria is caused by the negative impact of local and introduced fungal pathogens and insect pests. During the period 2018-2020, studies were conducted on the processes that caused irreversible damage to chestnut plantations as a result of prolonged pathological processes from the development of the virulent strain of the fungal pathogen Cryphonectria parasitica. The sample areas are located on the territory of seven State Forestries, at an altitude between 481 and 1138 m. The pathogen C. parasitica has caused necrotic disease, damaging non-renewable and difficult-torenew tissues, resulting in the death of infected trees. In the worst condition are the test areas at lower altitudes – between 400 and 600 m a.s.l. in the area of Brezhani (Pirin), Dupnitsa (Rila), Krumovgrad (Eastern Rhodopes) and Belasitsa, and in the higher parts of the mountains Belasitsa and Ograzhden, the health of the trees is satisfactory due to the better conditions for the development of the host and negative for pathogen development. In 96% of the isolated cultures, the mycelium of the fungus is virulent with orange color, and in the other samples it is whitish in color, which suggests the presence of hypovirulence of the fungus.
Article
Introduction: Cryphonectria parasitica, the causal agent of chestnut blight, causes necrotic lesions (so-called cankers) on the bark of stems and branches of susceptible host trees. Cryphonectria hypovirus 1 (CHV1) infects C. parasitica and reduces the fungus virulence (hypovirulence) and alters the fungus morphology in culture (pigmentation and sporulation capacity). By these characteristics, the strains with hypovirus CHV1 are used in Europe as a biological control agent of chestnut blight. Objectives: The aim of this work is to understand the effect of hypovirus on fungi virulence by comparing the production of some lignin-degrading enzymes and the metabolic profiles of some isogenic virulent and hypovirulent (converted and original) strains. Methods: The virulence of each strain was evaluated by mycelial inoculation on apple fruits (cv. Golden Delicious) and on detached, one year old, chestnut branches. To detect the activity of ligninolytic enzymes (laccases, peroxidases and cellulases), various substrates and indicator compounds were used. The metabolic profile of C. parasitica was evaluated by the Biolog FF system using 95 different carbon sources. Results: Virulent strains were found to cause more significant necrotic lesions in chestnut branches (p<0.05) and to produce larger lignin-degrading enzymes. The use of Biolog FF MicroPlates indicated that the use of 95 carbon sources five isolates of C. parasitica were significantly different (p <0.001), when the substrates were grouped into six types of chemical compounds. The highest AWCD values were obtained for carbohydrates, carboxylic acids and polymers, and the lowest values for amines/amides, amino acids and miscellaneous. Conclusions: Virulence evaluation of C. parasitica strains is important to study the hypovirulence processes mediated by the hypovirus CHV1. The detached branches of chestnut were, in our study, more suitable than apple fruits test in discriminating hypovirulent from virulent strains of C. parasitica. Virulent strains showed higher activity of acid-tannic inducible laccase (Lac3) and other lignin-degrading enzymes (LiP, MnP, and cellulase) when compared with hypovirulent ones. The results of the metabolic profiles studies may lead to new perspectives for understanding the biological process used by the hypovirus therefore, this may suggest a method for discriminating hypovirulent strains and study ecology and field fitness in this fungal strains.
Article
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The most important disease of European chestnut (Castanea sativa Mill.) is chestnut blight caused by the fungus Cryphonectria parasitica (Murrill) Barr which induces yield reduction in Europe and North America. This study aimed to investigate the impacts of C. parasitica infection on the physiological and biochemical characteristics of European chestnut at two different growth stages, 3 and 6 weeks after the infection. The amount of photosynthetic pigments (chlorophyll-a, chlorophyll-b, and carotenoids), the relative chlorophyll content, and the photochemical efficiency of the photosystem II (PSII) were measured in the leaves above and below the virulent and hypovirulent C. parasitica infections. The highest values were measured in the control leaves, the lowest values were in the leaves of the upper part of virulent necrosis. Antioxidant enzyme activities such as ascorbate peroxidase (APX), guaiacol peroxidase (POD), and superoxide dismutase (SOD), proline, and malondialdehyde concentrations were also investigated. In each of these measured values, the lowest level was measured in the control leaves, while the highest was in leaves infected with the virulent fungal strain. By measuring all of these stress indicator parameters the responses of chestnut to C. parasitica infection can be monitored and determined. The results of this study showed that the virulent strain caused more pronounced defense responses of chestnut's defense system. The measured parameter above the infection was more exposed to the blight fungus disease relative to the leaves below the infection.
Chapter
Viruses have been identified in virtually every fungal taxon, but their ecological effects remain poorly understood. Some viruses measurably affect fungal growth, reproduction, virulence, and fitness, though many are symptomless. Fungal viruses are difficult to study in natural settings, so their effects are often assessed in fungi in laboratory culture. Effects of viruses on fungal hosts are many: fungal growth, sporulation, and virulence may be increased or decreased by virus presence, and vegetative incompatibility may be suppressed. The fungus that causes chestnut blight, Cryphonectria parasitica, has been particularly important in studying the biology and ecology of fungal viruses and their potential for biocontrol with hypovirulence. “Killer viruses” of yeasts produce toxins that that kill susceptible fungi, helping their hosts secure ecological niches. Fungal viruses are transmitted horizontally through fusion of fungal cells (hyphal anastomosis) and vertically through asexual of sexual spores. With few exceptions, extracellular transmission of viruses is not known to occur. Such confinement within cells has allowed some fungal viruses to shed their capsid proteins, instead encapsulating their genomes within host‐derived lipid vesicles. Fungal defenses against viruses include excluding them during asexual or sexual reproduction; preventing their horizontal transmission through complex vegetative incompatibility systems; and antiviral RNA silencing. Retrotransposons are abundant in fungi, but other than their effects on genome structure, they do not appear to affect fitness. Infections of multiple viruses in a single fungal host are common and may have measurable synergistic effects, including suppression of antiviral RNA silencing by one virus, allowing infection by another.
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Cryphonectria parasitica, the causal agent of the chestnut blight, has been responsible for con -siderable damage in chestnut ecosystems in several European countries in the last three decades. Vegetative compatibility types (vc types) were characterized in three countries as a tool to assess C. parasitica diversity in Europe and because hypovirulence, caused by a hypovirus, spreads through anastomosis between compatible isolates. In Bosnia-Herzegovina, eastern Spain (Catalonia) and France several areas were investigated. Canker samples were removed from several sites, and vc types determined using the European vc type testers. Vc type diversity was assessed with the Shannon index. C. parasitica populations appeared clearly differentiated inside each country and among the countries. In Bosnia-Herzegovina, dominant vc types were EU-1, EU-2, EU-12 or EU-13, de -pending upon the region. All the other vc types found there belonged to the first 31 EU vc types. In Spain the dominant vc type was EU-2, six isolates were distributed in vc types between EU-65 and EU-74, and one vc type was incompatible with all the European vc type testers. In eastern France, dominant vc types were EU-1 and EU-5, whereas in western France, dominant vc types were EU-33 or EU-72, and 15 vc types were incompatible with all the European vc type testers.
Article
The aim of our work was to prove the efficiency of hypovirulent strains to treat cankers on chestnut. We found out that 4 hypovirulent strains were efficient to convert Slovak virulent isolates. In field conditions the treatment efficiency ranged from 20,3 % to 86,3 %. The number of isolates that were tested for vegetative compatibility ranged from 1 to 15 isolates per site (depending on the number of trees and cankers). The low efficiency of biological control may be caused by the high diversity of C. parasitica. In 1997 and 1998 the number of isolates that were tested for vegetative compatibility increased. If the conversion of virulent isolates by compatible strains is improved, the efficiency of biological control in field conditions will increase.
Article
Tester isolates of 20 vegetative compatibility (vc) types from 11 Italian subpopulations and tester isolates of 26 vc types from five Swiss subpopulations of Cryphonectria parasitica were compared by two different vc test methods. A total of 31 different vc types was identified; 15 vc types were common to both countries, five Italian vc types were not found in Switzerland and 11 Swiss vc types were not found in Italy. These 31 vc types were labelled with the acronym EU, followed by progressive numbers and may constitute the base for a common European nomenclature. The vc type EU-2 was the most common vc type in both countries and dominated in all Swiss and in seven Italian subpopulations. The vc types EU-1 and EU-5 were found primarily in northern Italy and Switzerland, while EU-10 and EU-12 were most common in southern Italy. The diversity of vc types was greater in the subpopulations of Switzerland and northern Italy than in the subpopulations of southern Italy. The geographical distribution of vc types in Europe should therefore be taken into consideration for biological control measures of chestnut blight and quarantine regulations.
Article
The genetic structure of three newly established Cryphonectria parasitica populations (Choëx, Weggis, Murg) was analysed and compared to an older, post-epidemic population (Claro). Vegetative compatibility (vc) type and DNA fingerprint analysis revealed an almost clonal C. parasitica population in Choëx, Weggis and Murg. These stands are all situated in northern Switzerland, outside the main range of European chestnut. Only one vc type and one dominant DNA fingerprint was found in Choëx and Murg. In Weggis, two vc types and two dominating DNA fingerprints were found. The European Cryphonectria hypovirus (CHV1) was not detected in these three populations. In contrast, the population in Claro, situated within the main range of European chestnut in southern Switzerland, had much higher vc type and DNA fingerprint diversity. DNA fingerprints were correlated to vc types in Claro and in Weggis. Mating type determination revealed one strongly dominating mating type in each of the three northern populations, but not in Claro. From these results we conclude that C. parasitica disseminated almost exclusively by means of asexual reproduction in Choëx, Weggis and Murg, whereas in Claro sexual reproduction also played an important role. Additionally, founder effects and restricted gene flow were dominating factors in shaping the genetic structure of the three northern populations.
Article
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.
Article
ABSTRACT Strains of the chestnut blight fungus, Cryphonectria parasitica, have been genetically engineered to contain an integrated full-length cDNA copy of the prototypic virulence-attenuating hypovirus CHV1-EP713. Unlike natural hypovirulent C. parasitica strains, these transgenic hypovirulent strains are able to transmit virus to ascospore progeny under laboratory conditions. This ability provides the potential to circumvent barriers to cytoplasmic virus transmission imposed by the fungal vegetative incompatibility system. During July 1994, transgenic hypovirulent strains were introduced into a Connecticut forest site (Biotechnology Permit 94-010-01). Subsequent analysis of the release site confirmed hypovirus transmission from transgenic hypovirulent strains to ascospore progeny under field conditions. Additionally, it was possible to recover transgenic hypovirulent strains from the test site as long as 2 years after the limited, single-season release. Evidence also was obtained for cytoplasmic transmission of transgenic cDNA-derived hypovirus RNA, including transmission to mycelia of a virulent C. parasitica canker after treatment with conidia of a transgenic strain. Finally, a transgenic hypovirulent strain was recovered from a superficial canker formed on an untreated chestnut tree. Genetic characteristics of the recovered strain suggested that the canker was initiated by an ascospore progeny derived from a cross involving an input transgenic hypovirulent strain. The durability of a molecular marker for field-released cDNA-derived hypovirus RNA is discussed.
Article
Environmental concerns have focused attention on natural forms of disease control as potentially safe and effective alternatives to chemical pesticides. This has led to increased efforts to develop control strategies that rely on natural predators and parasites or that involve genetically engineered microbial pest control agents. This review deals with a natural form of biological control in which the virulence of a fungal pathogen is attenuated by an endogenous viral RNA genetic element: the phenomenon of transmissible hypovirulence in the chestnut blight fungus, Cryphonectria parasitica. Recent progress in the molecular characterization of a hypovirulence-associated viral RNA has provided an emerging view of the genetic organization and basic expression strategy of this class of genetic elements. Several lines of evidence now suggest that specific hypovirulence-associated virus-encoded gene products selectively modulate the expression of subsets of fungal genes and the activity of specific regulatory pathways. The construction of an infectious cDNA clone of a hypovirulence-associated viral RNA represents a major advancement that provides exciting new opportunities for examining the molecular basis of transmissible hypovirulence and for engineering hypovirulent strains for improved biocontrol. These developments have significantly improved the prospects of using this system to identify molecular determinants of virulence and elucidate signal transduction pathways involved in pathogenic responses. In addition, novel approaches are now available for extending the application of transmissible hypovirulence for management of chestnut blight and possibly other fungal diseases.