Laccase Activity in Fungus Cryphonectria parasitica Is Affected by Growth Conditions and Fungal–Viral Genotypic Interactions

Laccase activity reduction in the chestnut blight fungus Cryphonectria parasitica usually accompanies the hypovirulence caused by the infection of fungus with Cryphonectria hypovirus 1 (CHV1). However, the different methods utilized for assessing this phenomenon has produced varied and often conflicting results. Furthermore, the majority of experimental setups included only one prototypic system, further confounding the results. Considering the diversity of fungal isolates, viral strains, and variability of their effects on the phytopathogenic process observed in nature, our goal was to ascertain if laccase activity variability is affected by (1) different C. parasitica isolates infected with several CHV1 strains, and (2) growth conditions. We have demonstrated that some CHV1 strains, contrary to previous assumptions, increase the activity of C. parasitica laccases. The specific fungal isolates used in the experiments and culture conditions also affected the results. Furthermore, we showed that two commonly used laccase substrates, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and 2,4-dimethoxyphenol, cannot be used interchangeably in C. parasitica laccase activity measurements. Our results illustrate the importance of conducting this type of study in experimental systems and culture conditions that resemble natural conditions as much as possible to be able to infer the most relevant conclusions applicable to natural populations.

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Mixed mating in natural populations of the chestnut blight fungus, Cryphonectria parasitica

Heredity ◽

10.1038/sj.hdy.6800492 ◽

2004 ◽

Vol 93 (2) ◽

pp. 189-195 ◽

Cited By ~ 24

Author(s):

R E Marra ◽

P Cortesi ◽

M Bissegger ◽

M G Milgroom

Keyword(s):

Natural Populations ◽

Cryphonectria Parasitica ◽

Chestnut Blight ◽

Mixed Mating ◽

Chestnut Blight Fungus

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Characterization of the ERK homologue CpMK2 from the chestnut blight fungus Cryphonectria parasitica

Microbiology ◽

10.1099/mic.0.27796-0 ◽

2005 ◽

Vol 151 (5) ◽

pp. 1349-1358 ◽

Cited By ~ 28

Author(s):

Eun-Sil Choi ◽

Hea-Jong Chung ◽

Myoung-Ju Kim ◽

Seung-Moon Park ◽

Byeong-Jin Cha ◽

...

Keyword(s):

Growth Conditions ◽

Cryphonectria Parasitica ◽

Mitogen Activated Protein Kinase ◽

Wild Type ◽

Growth Defects ◽

Chestnut Blight Fungus ◽

Phosphorylation Level ◽

Solid Media ◽

Mitogen Activated Protein ◽

Hypovirulent Strain

The Cryphonectria parasitica gene cpmk2, which encodes a mitogen-activated protein kinase belonging to the yeast extracellular signalling-regulated kinase (YERK1) subfamily, was isolated and its biological function was examined. Disruption of cpmk2 resulted in impaired pigmentation and abolished conidiation. Growth defects were observed in the cpmk2 mutant grown on solid plates, but growth of the mutant appeared normal in liquid media, including EP complete and PD broth, suggesting that the cpmk2 gene is involved in sensing and responding to growth conditions. The mutant's production of laccase, as measured by the size of the coloured area produced on tannic-acid-supplemented plates, was significantly reduced compared with the wild-type, but the intensity of the coloured area was unchanged, suggesting that the reduced laccase activity was owing to reduced growth on solid media rather than transcriptional downregulation. A dramatic reduction observed in the canker area produced by the cpmk2 mutant compared with the wild-type, even more severe than that of a hypovirulent strain, can also be ascribed to defective growth on solid surfaces rather than to impairments in a virulence factor(s). Downregulation of the pheromone gene Mf2/1 was also observed in the mutant, indicating a possible explanation for the regulation of the pheromone precursor gene in filamentous fungi and suggesting the presence of the yeast-like pheromone-responsive pathway in C. parasitica. Immunoblot analyses revealed that the phosphorylation level of CpMK2 increased in both virus-free and virus-containing strains in liquid cultures of up to 5 days old and decreased in older cultures. Moreover, the CpMK2 phosphorylation level increased in both strains after transfer from liquid to solid medium. However, levels of phosphorylated CpMK2 were similar in the two strains, suggesting that CpMK2, unlike CpMK1, is not under the direct control of a hypovirus.

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Recombination and Migration of Cryphonectria hypovirus 1 as Inferred From Gene Genealogies and the Coalescent

Genetics ◽

10.1093/genetics/166.4.1611 ◽

2004 ◽

Vol 166 (4) ◽

pp. 1611-1629

Author(s):

Ignazio Carbone ◽

Yir-Chung Liu ◽

Bradley I Hillman ◽

Michael G Milgroom

Keyword(s):

Horizontal Transmission ◽

Natural Populations ◽

Recognition System ◽

Cryphonectria Parasitica ◽

Pathogen Transmission ◽

Vegetative Compatibility ◽

Common Mutation ◽

Transmission Rates ◽

And Migration ◽

Cryphonectria Hypovirus

Abstract Genealogy-based methods were used to estimate migration of the fungal virus Cryphonectria hypovirus 1 between vegetative compatibility types of the host fungus, Cryphonectria parasitica, as a means of estimating horizontal transmission within two host populations. Vegetative incompatibility is a self/non-self recognition system that inhibits virus transmission under laboratory conditions but its effect on transmission in nature has not been clearly demonstrated. Recombination within and among different loci in the virus genome restricted the genealogical analyses to haplotypes with common mutation and recombinational histories. The existence of recombination necessitated that we also use genealogical approaches that can take advantage of both the mutation and recombinational histories of the sample. Virus migration between populations was significantly restricted. In contrast, estimates of migration between vegetative compatibility types were relatively high within populations despite previous evidence that transmission in the laboratory was restricted. The discordance between laboratory estimates and migration estimates from natural populations highlights the challenges in estimating pathogen transmission rates. Genealogical analyses inferred migration patterns throughout the entire coalescent history of one viral region in natural populations and not just recent patterns of migration or laboratory transmission. This application of genealogical analyses provides markedly stronger inferences on overall transmission rates than laboratory estimates do.

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Hypovirus Virulence and Vegetative Incompatibility in Populations of the Chestnut Blight Fungus

Phytopathology ◽

10.1094/phyto-01-12-0013-r ◽

2012 ◽

Vol 102 (12) ◽

pp. 1161-1167 ◽

Cited By ~ 18

Author(s):

Sarah Franziska Bryner ◽

Daniel Rigling

Keyword(s):

Biocontrol Agent ◽

Cryphonectria Parasitica ◽

Vegetative Compatibility ◽

Chestnut Blight ◽

Vegetative Incompatibility ◽

Tree Disease ◽

Chestnut Blight Fungus ◽

Fungal Populations ◽

Cryphonectria Hypovirus ◽

High Diversity

Cryphonectria hypovirus 1 hyperparasitizes the chestnut blight fungus Cryphonectria parasitica and acts as a biocontrol agent for this serious tree disease. The virus is transmitted cytoplasmatically between fungal individuals. However, highly virulent viruses strongly debilitate their host and, thus, reduce their own transmission probability. Furthermore, vegetative incompatibility between fungi is an important transmission barrier. Therefore, virulent viruses are expected to be strongly selected against in fungal populations with high levels of vegetative incompatibility, eventually leading to the erosion of biocontrol. To test this prediction, we assessed the virulence of the virus in four European C. parasitica populations with high diversity of vegetative compatibility types and in four populations with low diversity. We expected the degree of virus virulence to be lower in fungal populations with high levels of vegetative incompatibility. However, our results did not reveal such a trend. No significant differences in virus virulence between populations with low versus high diversity of vegetative compatibility types were observed. There was no evidence for an erosion of disease control due to the presence of these transmission barriers. Thus, the findings of this study are promising for the sustainability of Cryphonectria hypovirus 1 as a biocontrol agent for chestnut blight in Europe.

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Role of Fresh Dead Wood in the Epidemiology and the Biological Control of the Chestnut Blight Fungus

Plant Disease ◽

10.1094/pdis-05-18-0796-re ◽

2019 ◽

Vol 103 (3) ◽

pp. 430-438 ◽

Cited By ~ 2

Author(s):

Joana Beatrice Meyer ◽

Loïc Chalmandrier ◽

Fabio Fässler ◽

Christopher Schefer ◽

Daniel Rigling ◽

...

Keyword(s):

Biological Control ◽

Dead Wood ◽

Biocontrol Agent ◽

Castanea Sativa ◽

Cryphonectria Parasitica ◽

Chestnut Blight ◽

Chestnut Blight Fungus ◽

Study Sites ◽

Cryphonectria Hypovirus

The invasive fungus Cryphonectria parasitica, the causal agent of chestnut blight, is able to survive and sporulate on the bark of fresh dead Castanea sativa wood for at least 2 years. Here, we experimentally investigated the role of fresh dead wood in the epidemiology of chestnut blight, specifically in the spread of the hyperparasitic virus Cryphonectria hypovirus 1, which acts as biocontrol agent of C. parasitica. A total of 152 artificially initiated, virulent bark cankers in four chestnut stands were treated with virus-infected asexual spores originating either from sporulating dead wood or from a spore suspension. Molecular markers for both the virus and the fungal carrier were used to examine the spread of the applied biocontrol virus. Fourteen months after treatment, 42 to 76% of the conidial spray-treated cankers and 50 to 60% of the cankers exposed to a sporulating dead stem had been virus infected by the applied hypovirulent conidia in all four study sites. Virus infection reduced canker expansion and promoted canker healing (callusing). Thus, fresh chestnut dead wood may play an important role in supporting the successful spread of natural hypovirulence in chestnut forests. Further, combined with the application of virus-infected conidial suspensions, it may help promote the establishment of artificially released hypoviruses in chestnut stands to control chestnut blight.

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Characterization of a Hypovirus-Regulated Septin Cdc11 Ortholog, CpSep1, from the Chestnut Blight Fungus Cryphonectria parasitica

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-18-0194-r ◽

2019 ◽

Vol 32 (3) ◽

pp. 286-295 ◽

Cited By ~ 1

Author(s):

Myeongjin Jo ◽

Kum-Kang So ◽

Yo-Han Ko ◽

Jeesun Chun ◽

Jung-Mi Kim ◽

...

Keyword(s):

Cryphonectria Parasitica ◽

Chestnut Blight ◽

Null Mutant ◽

Wild Type ◽

Chestnut Blight Fungus ◽

Germination Process ◽

Cryphonectria Hypovirus ◽

Null Mutants

We identified a protein spot showing downregulation in the presence of Cryphonectria hypovirus 1 and tannic acid supplementation as a septin subunit with the highest homology to the Aspergillus nidulans aspA gene, an ortholog of the Saccharomyces cerevisiae Cdc11 gene. To analyze the functional role of this septin component (CpSep1), we constructed its null mutant and obtained a total of eight CpSep1-null mutants from 137 transformants. All CpSep1-null mutants showed retarded growth, with fewer aerial mycelia and intense pigmentation on plates of potato dextrose agar supplemented with L-methionine and biotin. When the marginal hyphae were examined, hyperbranching was observed in contrast to the wild type. The inhibition of colonial growth was partially recovered when the CpSep1-null mutants were cultured in the presence of the osmostabilizing sorbitol. Conidia production of the CpSep1-null mutants was significantly increased by at least 10-fold more. Interestingly, the conidial morphology of the CpSep1-null mutants changed to circular in contrast to the typical rod-shaped spores of the wild type, indicating a role of septin in the spore morphology of Cryphonectria parasitica. However, no differences in the germination process were observed. Virulence assays using excised chestnut bark, stromal pustule formation on chestnut stems, and apple inoculation indicated that the CpSep1 gene is important in pathogenicity.

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Genetic and Phenotypic Characterization of Cryphonectria hypovirus 1 from Eurasian Georgia

Viruses ◽

10.3390/v10120687 ◽

2018 ◽

Vol 10 (12) ◽

pp. 687 ◽

Cited By ~ 3

Author(s):

Daniel Rigling ◽

Nora Borst ◽

Carolina Cornejo ◽

Archil Supatashvili ◽

Simone Prospero

Keyword(s):

Biological Control ◽

Biological Control Agent ◽

Cryphonectria Parasitica ◽

Control Agent ◽

Phenotypic Characterization ◽

Tree Disease ◽

Caucasus Region ◽

Chestnut Blight Fungus ◽

Part Sequencing ◽

Cryphonectria Hypovirus

Cryphonectria hypovirus 1 (CHV-1) infects the chestnut blight fungus Cryphonectria parasitica and acts as a biological control agent against this harmful tree disease. In this study, we screened the recently characterized C. parasitica population in Eurasian Georgia for the presence of CHV-1. We found 62 CHV-1 infected C. parasitica isolates (9.3%) among a total of 664 isolates sampled in 14 locations across Georgia. The prevalence of CHV-1 at the different locations ranged from 0% in the eastern part of the country to 29% in the western part. Sequencing of two specific regions of the viral genome one each in ORFA and ORFB revealed a unique CHV-1 subtype in Georgia. This subtype has a recombinant pattern combining the ORFA region from the subtype F2 and the ORFB region from subtype D. All 62 viral strains belonged to this Georgian CHV-1 subtype (subtype G). The CHV-1 subtype G strongly reduced the parasitic growth of C. parasitica isolates from Georgia, with a more severe effect on the European genepool compared to the Georgian genepool. The CHV-1 subtype detected in Georgia provides a valuable candidate for biological control applications in the Caucasus region.

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Identification of an RNA Silencing Suppressor Encoded by a Symptomless Fungal Hypovirus, Cryphonectria Hypovirus 4

Biology ◽

10.3390/biology10020100 ◽

2021 ◽

Vol 10 (2) ◽

pp. 100

Author(s):

Annisa Aulia ◽

Kiwamu Hyodo ◽

Sakae Hisano ◽

Hideki Kondo ◽

Bradley I. Hillman ◽

...

Keyword(s):

Rna Silencing ◽

Expression System ◽

Cryphonectria Parasitica ◽

Inhibitory Effect ◽

Chestnut Blight ◽

Silencing Suppressor ◽

Terminal Portion ◽

Transgenic Expression ◽

Chestnut Blight Fungus ◽

Cryphonectria Hypovirus

Previously, we have reported the ability of a symptomless hypovirus Cryphonectria hypovirus 4 (CHV4) of the chestnut blight fungus to facilitate stable infection by a co-infecting mycoreovirus 2 (MyRV2)—likely through the inhibitory effect of CHV4 on RNA silencing (Aulia et al., Virology, 2019). In this study, the N-terminal portion of the CHV4 polyprotein, termed p24, is identified as an autocatalytic protease capable of suppressing host antiviral RNA silencing. Using a bacterial expression system, CHV4 p24 is shown to cleave autocatalytically at the di-glycine peptide (Gly214-Gly215) of the polyprotein through its protease activity. Transgenic expression of CHV4 p24 in Cryphonectria parasitica suppresses the induction of one of the key genes of the antiviral RNA silencing, dicer-like 2, and stabilizes the infection of RNA silencing-susceptible virus MyRV2. This study shows functional similarity between CHV4 p24 and its homolog p29, encoded by the symptomatic prototype hypovirus CHV1.

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Genetic Control of Horizontal Virus Transmission in the Chestnut Blight Fungus, Cryphonectria parasitica

Genetics ◽

10.1093/genetics/159.1.107 ◽

2001 ◽

Vol 159 (1) ◽

pp. 107-118 ◽

Cited By ~ 1

Author(s):

Paolo Cortesi ◽

Charles E McCulloch ◽

Haiyue Song ◽

Haiqun Lin ◽

Michael G Milgroom

Keyword(s):

Logistic Regression ◽

Regression Model ◽

Logistic Regression Model ◽

Horizontal Transmission ◽

Natural Populations ◽

Virus Transmission ◽

Cryphonectria Parasitica ◽

Chestnut Blight ◽

Vegetative Incompatibility ◽

Chestnut Blight Fungus

Abstract Vegetative incompatibility in fungi has long been known to reduce the transmission of viruses between individuals, but the barrier to transmission is incomplete. In replicated laboratory assays, we showed conclusively that the transmission of viruses between individuals of the chestnut blight fungus Cryphonectria parasitica is controlled primarily by vegetative incompatibility (vic) genes. By replicating vic genotypes in independent fungal isolates, we quantified the effect of heteroallelism at each of six vic loci on virus transmission. Transmission occurs with 100% frequency when donor and recipient isolates have the same vic genotypes, but heteroallelism at one or more vic loci generally reduces virus transmission. Transmission was variable among single heteroallelic loci. At the extremes, heteroallelism at vic4 had no effect on virus transmission, but transmission occurred in only 21% of pairings that were heteroallelic at vic2. Intermediate frequencies of transmission were observed when vic3 and vic6 were heteroallelic (76 and 32%, respectively). When vic1, vic2, and vic7 were heteroallelic, the frequency of transmission depended on which alleles were present in the donor and the recipient. The effect of heteroallelism at two vic loci was mostly additive, although small but statistically significant interactions (epistasis) were observed in four pairs of vic loci. A logistic regression model was developed to predict the probability of virus transmission between vic genotypes. Heteroallelism at vic loci, asymmetry, and epistasis were the dominant factors controlling transmission, but host genetic background also was statistically significant, indicating that vic genes alone cannot explain all the variation in virus transmission. Predictions from the logistic regression model were highly correlated to independent transmission tests with field isolates. Our model can be used to estimate horizontal transmission rates as a function of host genetics in natural populations of C. parasitica.

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Incidence and Diversity of Double-Stranded RNAs Occurring in the Chestnut Blight Fungus, Cryphonectria parasitica, in China and Japan

Phytopathology ◽

10.1094/phyto.1998.88.8.811 ◽

1998 ◽

Vol 88 (8) ◽

pp. 811-817 ◽

Cited By ~ 48

Author(s):

Tobin L. Peever ◽

Yir-Chung Liu ◽

Kerong Wang ◽

Bradley I. Hillman ◽

Robert Foglia ◽

...

Keyword(s):

New Jersey ◽

North America ◽

North American ◽

Cryphonectria Parasitica ◽

Liaoning Province ◽

Chestnut Blight ◽

Chestnut Blight Fungus ◽

China And Japan ◽

Hybridization Group ◽

Cryphonectria Hypovirus

Isolates of the chestnut blight fungus, Cryphonectria parasitica, were randomly sampled from 10 subpopulations in China and 8 subpopulations in Japan and screened for the presence of double-stranded (ds) RNA using an immunoblot procedure with a monoclonal antibody specific for dsRNA. The overall incidence of dsRNA in C. parasitica was 2 and 6% in China and Japan, respectively, much lower than the 28% found previously in North American populations. Genetic relatedness of dsRNAs within and among populations in China and Japan was examined using RNA-RNA hybridizations with labeled-dsRNA probes. The majority of Chinese and Japanese dsRNAs were members of a single hybridization group, related to Cryphonectria hypovirus 1 (CHV1) from Europe, and are referred to as CHV1-type dsRNAs. No evidence was obtained for genetic differentiation between CHV1-type dsRNAs sampled in China and Japan. Five Japanese isolates contained two genetically distinct dsRNAs. The larger segments (approximately 12 kilobases [kb]) were members of the CHV1 hybridization group, while the smaller segments (approximately 3 kb) did not hybridize with any known dsRNA from C. parasitica including the 2.7-kb dsRNA from isolate NB631 from New Jersey or dsRNA from isolate RC1 from Michigan. Two small dsRNA segments (approximately 1.8 and 2 kb) from one isolate sampled from Liaoning Province in northeastern China did not hybridize with any of the dsRNA probes tested including several described dsRNAs of similar size from C. parasitica in North America. Three dsRNAs from Anhui Province, China, hybridized to Cryphonectria hypovirus 2 (CHV2)-specific probes and are thus referred to as CHV2-type dsRNAs. Sequence analysis of 1,627 base pairs of these three CHV2-type dsRNAs from Anhui revealed that they were identical to each other in the region sequenced and very closely related to CHV2-NB58, isolated from New Jersey. We speculate that CHV2-NB58 may have been introduced into North America from this part of China. This is the first record of a North American C. parasitica dsRNA that is genetically related to a dsRNA from Asia.

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