Forest ecosystems are highly threatened by the simultaneous effects of climate change and invasive pathogens. Chestnut blight, caused by the invasive phytopathogenic fungus
, has caused severe damage ...to European chestnut groves and catastrophic dieback of American chestnut in North America. Within Europe, the impacts of the fungus are widely mitigated through biological control that utilizes the RNA mycovirus: Cryphonectria hypovirus 1 (CHV1). Viral infections, similarly to abiotic factors, can cause oxidative stress in their hosts leading to physiological attrition through stimulating ROS (reactive oxygen species) and NOx production.
To fully understand the interactions leading to the biocontrol of chestnut blight, it is vital to determine oxidative stress damage arising during CHV1 infection, especially considering that other abiotic factors, like long-term cultivation of model fungal strains, can also impact oxidative stress. Our study compared CHV1-infected
isolates from two Croatian wild populations with CHV1-infected model strains (EP713, Euro7 and CR23) that have experienced long-term laboratory cultivation.
We determined the level of oxidative stress in the samples by measuring stress enzymes' activity and oxidative stress biomarkers. Furthermore, for the wild populations, we studied the activity of fungal laccases, expression of the laccase gene
, and a possible effect of CHV1 intra-host diversity on the observed biochemical responses. Relative to the wild isolates, the long-term model strains had lower enzymatic activities of superoxide dismutase (SOD) and glutathione S-transferase (GST), and higher content of malondialdehyde (MDA) and total non-protein thiols. This indicated generally higher oxidative stress, likely arising from their decades-long history of subculturing and freeze-thaw cycles. When comparing the two wild populations, differences between them in stress resilience and levels of oxidative stress were also observed, as evident from the different MDA content. The intra-host genetic diversity of the CHV1 had no discernible effect on the stress levels of the virus-infected fungal cultures. Our research indicated that an important determinant modulating both
expression and laccase enzyme activity is intrinsic to the fungus itself, possibly related to the vc type of the fungus, i.e., vegetative incompatibility genotype.
Transmissible hypovirulence associated with Cryphonectria hypovirus 1 (CHV1) has been used for biological control of chestnut blight, devastating disease of chestnut caused by the fungus ...Cryphonectria parasitica. The main aims of this study were to provide molecular characterization of CHV1 from Croatia and Slovenia and to reveal its genetic variability, phylogeny, and diversification of populations. Fifty-one CHV1 haplotypes were detected among 54 partially sequenced CHV1 isolates, all belonging to Italian subtype (I). Diversity was mainly generated by point mutations while evidence of recombination was not found. The level of conservation over analyzed parts of ORF-A proteins p29 and p40 varied, but functional sites were highly conserved. Phylogenetic analysis revealed close relatedness and intermixing of Croatian and Slovenian CHV1 populations. Our CHV1 isolates were also related to Swiss and Bosnian hypoviruses supporting previously suggested course of CHV1 invasion in Europe. Overall, this study indicates that phylogeny of CHV1 subtype I in Europe is complex and characterized with frequent point mutations resulting in many closely related variants of the virus. Possible association between variations within CHV1 ORF-A and growth of the hypovirulent fungal isolates is tested and presented.
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BFBNIB, EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NMLJ, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Infection of American and European chestnuts with the chestnut blight fungus Cryphonectria parasitica results in the formation of cankers, lesions caused by the growth of mycelia within bark tissue ...of the host plant. Infection of the fungus with Cryphonectria hypovirus 1 (CHV‐1) results in conversion of the mycelial phenotype from virulent to hypovirulent, thus allowing production of callus around cankers as a reaction by infected trees, rendering active into inactive cankers. In this study, we sampled one USA and six European chestnut stands and assessed frequency of hypovirulent C. parasitica and diversity of vegetative compatibility (vc) types present in calluses and randomly sampled cankers. Callused cankers on C. dentata at West Salem in the USA yielded significantly more hypovirulent C. parasitica isolates compared with four sampled populations on C. sativa, while all six sampled European populations did not show any statistically significant differences among themselves. We observed no correlation between hypovirulence frequencies in randomly sampled cankers and calluses, as well as no correlation of C. parasitica vc type diversity in calluses and residential populations of the fungus. Furthermore, even though we have observed calluses with more than one vc type, they do not occur regularly. Even when present in C. parasitica populations with high vc type diversity, no more than three different vc types were observed in a single callus.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Summary
Epigenetic modifications may play an important role in invasion and adaptation of clonal and invasive populations to different environments. The aim of this study was to analyse epigenetic ...diversity and structure within and among populations of invasive pathogenic fungus Cryphonectria parasitica from south‐eastern Europe, where one haplotype S12 dominates. The highest level of epigenetic diversity was found in haplotype S1, followed by S2, while the lowest level of epigenetic diversity was found in haplotype S12. Similar pattern of epigenetic diversity was detected in the control, genetically diverse Croatian population where S1 haplotype dominates. In four south‐eastern European populations, the highest level of epigenetic diversity was observed in the Italian population, the oldest population in the studied area, while the lowest diversity was found in most recently established Bulgarian population. This relationship between epigenetic diversity and population age implies the important role of epigenetic modifications on the process of invasion. Our data suggest that epigenetic differences might affect the success of expansion of certain haplotype into new regions. Understanding the role of epigenetic processes in expansion and (pre)adaptation of fungal plant pathogens, besides fundamental knowledge, can contribute to development of strategies for control of fungal spread and pathogenesis.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Biotic stress caused by virus infections induces epigenetic changes in infected plants and animals, but this is the first report on methylation pattern changes in a fungus after mycovirus infection. ...As a model pathosystem for mycovirushost interactions, we used Cryphonectria hypovirus 1 (CHV1) and its host fungus Cryphonectria parasitica, in which deregulation of methylation cycle enzymes upon virus infection was observed previously. Six CHV1 strains of different subtypes were transferred into three different C. parasitica isolates in order to assess the effect of different CHV1 strains and/or subtypes on global cytosine methylation level in infected fungus, using methylation-sensitive amplification polymorphism (MSAP). Infection with CHV1 affected the methylation pattern of the C. parasitica genome; it increased the number and diversity of methylated, hemi-methylated, and total MSAP markers found in infected fungal isolates compared to virus-free controls. The increase in methylation levels correlated well with the CHV1-induced reduction of fungal growth in vitro, indicating that C. parasitica genome methylation upon CHV1 infection, rather than being the defensive mechanism of the fungus, is more likely to be the virulence determinant of the virus. Furthermore, the severity of CHV1 effect on methylation levels of infected C. parasitica isolates depended mostly on individual CHV1 strains and on the combination of host and virus genomes, rather than on the virus subtype. These novel findings broaden our knowledge about CHV1 strains which could potentially be used in human-aided biocontrol of chestnut blight, a disease caused by C. parasitica in chestnut forest ecosystems and orchards.
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BFBNIB, EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NMLJ, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Gene flow between cultivated and wild gene pools is common in the contact zone between agricultural lands and natural habitats and can be used to study the development of adaptations and selection of ...novel varieties. This is likely the case in the northern Adriatic region, where centuries-old cultivated orchards of sweet chestnut (Castanea sativa Mill.) are planted within the natural distribution area of the species. Thus, we investigated the population structure of several orchards of sweet chestnuts. Furthermore, the genetic background of three toponymous clonal varieties was explored. Six genomic simple sequence repeat (gSSR) and nine EST-derived SSR (EST-SSR) loci were utilized in this research, and both grafted and non-grafted individuals were included in this study. Five closely related clones were identified, which represent a singular, polyclonal marron variety, found in all three cultivation areas. Furthermore, many hybrids, a result of breeding between cultivated and wild chestnuts, have been found. Analyzed semi-wild orchards defined by a diverse genetic structure, represent a hotspot for further selection and could result in creation of locally adapted, high-yielding varieties.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Understanding virus evolution is a fundamental goal of virology, evolutionary biology, and disease epidemiology. We provide a detailed analysis of evolution and origin of Cryphonectria hypovirus 1 ...(CHV1) populations in Europe, based on the complete genome sequence of all European subtypes. Phylogenetic analyses divided European strains into two closely related clades. Strains of the subtype I belong to the first, while strains of the subtypes F1, D and E belong to the second clade suggesting that the subtypes F1, D and E are more closely related than previously thought. Strains of the subtype F2 appeared to be recombinant; subtypes F1/D/E contributed a larger fraction of sequence while subtype I contributed a smaller fraction. The p29 was the most variable domain, while the replication-associated large ORF B protein was the most conserved domain within the CHV1. Low sequence similarity, predominant negative selection and frequent recombination characterise the evolution of CHV1.
•Subtypes F1, D and E are more closely related than previously thought.•Strains of the subtype F2 are recombinant originating from subtypes F1/D/E and I.•CHV1 protein domains vary considerably in genetic diversity and selection pressure.•Negative selection and frequent recombination characterise CHV1 evolution.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Chestnut blight has spread throughout Europe since the introduction of its causal agent Cryphonectria parasitica over 70 years ago. In our study, we have analysed diversity of vegetative ...compatibility (vc) and microsatellite genotypes of C. parasitica, as well as sequence diversity of Cryphonectria hypovirus 1 (CHV1) in six populations from Switzerland, Croatia and North Macedonia. Resampling of local populations that were already investigated more than a decade ago allowed us to analyse the spatial and temporal population structure across an invasive range of the pathogen in Europe. Regardless which genetic marker was used, the over 60 year-old Swiss and Croatian populations had a high population diversity, while more recent North Macedonian populations were mostly clonal. These diversity differences between the investigated populations remained stable over time. A high diversity of CHV1 was observed in all three countries, with North Macedonian strains forming a separate cluster from strains obtained in other countries. No correlation between vc diversity and CHV1 prevalence was observed, suggesting a well-established and maintained natural hypovirulence in all countries, further corroborated by an observed increase in genetic diversity of Croatian C. parasitica populations over time, without collapse of CHV1 prevalence.
This paper showcases the development of plant virology in Croatia at the University of Zagreb, Faculty of Science, from its beginning in the 1950s until today, more than 70 years later. The main ...achievements of the previous and current group members are highlighted according to various research topics and fields. Expectedly, some of those accomplishments remained within the field of plant virology, but others make part of a much-extended research spectrum exploring subviral pathogens, prokaryotic plant pathogens, fungi and their viruses, as well as their interactions within ecosystems. Thus, the legacy of plant virology in Croatia continues to contribute to the state of the art of microbiology far beyond virology. Research problems pertinent for directing the future research endeavors are also proposed in this review.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Laccase activity reduction in the chestnut blight fungus
usually accompanies the hypovirulence caused by the infection of fungus with
(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
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
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
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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