In-Tree Behavior of Diverse Viruses Harbored in the Chestnut Blight Fungus, Cryphonectria parasitica.

The ascomycete Cryphonectria parasitica causes destructive chestnut blight. Biological control of the fungus by virus infection (hypovirulence) has been shown to be an effective control strategy against chestnut blight in Europe. To provide biocontrol effects, viruses must be able to induce hypovirulence and spread efficiently in chestnut trees. Field studies using living trees to date have focused on a selected family of viruses called hypoviruses, especially prototypic hypovirus CHV1, but there are now known to be many other viruses that infect C. parasitica Here, we tested seven different viruses for their hypovirulence induction, biocontrol potential, and transmission properties between two vegetatively compatible but molecularly distinguishable fungal strains in trees. The test included cytosolically and mitochondrially replicating viruses with positive-sense single-stranded RNA or double-stranded RNA genomes. The seven viruses showed different in planta behaviors and were classified into four groups. Group I, including CHV1, had great biocontrol potential and could protect trees by efficiently spreading and converting virulent to hypovirulent cankers in the trees. Group II could induce high levels of hypovirulence but showed much smaller biocontrol potential, likely because of inefficient virus transmission. Group III showed poor performance in hypovirulence induction and biocontrol, while efficiently being transmitted in the infected trees. Group IV could induce hypovirulence and spread efficiently but showed poor biocontrol potential. Nuclear and mitochondrial genotyping of fungal isolates obtained from the treated cankers confirmed virus transmission between the two fungal strains in most isolates. These results are discussed in view of dynamic interactions in the tripartite pathosystem.IMPORTANCE The ascomycete Cryphonectria parasitica causes destructive chestnut blight, which is controllable by hypovirulence-conferring viruses infecting the fungus. The tripartite chestnut/C. parasitica/virus pathosystem involves the dynamic interactions of their genetic elements, i.e., virus transmission and lateral transfer of nuclear and mitochondrial genomes between fungal strains via anastomosis occurring in trees. Here, we tested diverse RNA viruses for their hypovirulence induction, biocontrol potential, and transmission properties between two vegetatively compatible but molecularly distinguishable fungal strains in live chestnut trees. The tested viruses, which are different in genome type (single-stranded or double-stranded RNA) and organization, replication site (cytosol or mitochondria), virus form (encapsidated or capsidless) and/or symptomatology, have been unexplored in the aforementioned aspects under controlled conditions. This study showed intriguing different in-tree behaviors of the seven viruses and suggested that to exert significant biocontrol effects, viruses must be able to induce hypovirulence and spread efficiently in the fungus infecting the chestnut trees.

Sixty Years from the First Disease Description, a Novel Badnavirus Associated with Chestnut Mosaic Disease.

Although chestnut mosaic disease (ChMD) was described several decades ago, its etiology is still not clear. Using classical approaches and high-throughput sequencing (HTS) techniques, we identified a novel Badnavirus that is a strong etiological candidate for ChMD. Two disease sources from Italy and France were submitted to HTS-based viral indexing. Total RNAs were extracted, ribodepleted, and sequenced on an Illumina NextSeq500 (2 × 150 nt or 2 × 75 nt). In each source, we identified a single contig of ≈7.2 kb that corresponds to a complete circular viral genome and shares homologies with various badnaviruses. The genomes of the two isolates have an average nucleotide identity of 90.5%, with a typical badnaviral genome organization comprising three open reading frames. Phylogenetic analyses and sequence comparisons showed that this virus is a novel species; we propose the name Chestnut mosaic virus (ChMV). Using a newly developed molecular detection test, we systematically detected the virus in symptomatic graft-inoculated indicator plants (chestnut and American oak) as well in chestnut trees presenting typical ChMD symptoms in the field (100 and 87% in France and Italy surveys, respectively). Datamining of publicly available chestnut sequence read archive transcriptomic data allowed the reconstruction of two additional complete ChMV genomes from two Castanea mollissima sources from the United States as well as ChMV detection in C. dentata from the United States. Preliminary epidemiological studies performed in France and central eastern Italy showed that ChMV has a high incidence in some commercial orchards and low within-orchard genetic diversity.

Biological control of chestnut blight in Croatia: an interaction between host sweet chestnut, its pathogen Cryphonectria parasitica and the biocontrol agent Cryphonectria hypovirus 1.

BACKGROUND: Chestnut blight, caused by the fungus Cryphonectria parasitica, is a severe chestnut disease that can be controlled with naturally occurring hypoviruses in many areas of Europe. The aim of this research was to measure the effect of different Cryphonectria hypovirus 1 (CHV1) strains on the growth of the fungal host and select strains that could potentially be used for human-mediated biocontrol in forests and orchards, and to investigate whether and how chestnut-fungus-virus interactions affect the development and growth of the lesion area on cut stems. RESULTS: Two Croatian CHV1 strains (CR23 and M56/1) were selected as potential biocontrol agents. The sequencing of CHV1/ORF-A showed that both of these virus strains belonged to the Italian subtype of CHV1. In vitro transfection of selected virus strains from hypovirulent to genetically diverse virus-free fungal isolates and subsequent inoculation of all virus/fungus combinations on stems of genetically diverse sweet chestnut trees revealed that Croatian virus strain CR23 had an equally hypovirulent effect on the host as the strong French strain CHV1-EP713, while M56/1 had a weaker effect. Furthermore, it was shown that in some cases the same hypovirus/fungus combinations induced various degrees of canker development on different chestnut genotypes. CONCLUSION: Some CHV1 strains belonging to the Italian subtype have similar hypovirulent effects on C. parasitica to those belonging to the French subtype. Furthermore, chestnut susceptibility and recovery could be influenced by the response of chestnut trees to particular hypovirulent C. parasitica isolates, and virus-fungus-chestnut interactions could have significant implications for the success of chestnut blight biocontrol. © 2016 Society of Chemical Industry.

Dynamics of Cryphonectria hypovirus infection in chestnut blight cankers.

Virulent strains of the chestnut blight fungus Cryphonectria parasitica cause lethal bark cankers on chestnut trees. Infection of C. parasitica with Cryphonectria hypovirus 1 in Europe biologically controls this disease, leading to nonlethal and inactive cankers. Unexpectedly, virus-free C. parasitica strains have been isolated from inactive cankers. In this study, we compared the virulence of virus-infected and virus-free C. parasitica strains isolated from either inactive or active cankers on chestnut seedlings and sprouts. In the seedling experiment, we assessed canker growth and seedling mortality. In the sprout experiment, we also assessed canker growth and made fungal reisolations to determine virus infection and immigration of foreign vegetative compatibility (vc) types over a period of 13 years in a coppice forest. Overall, the virulence of virus-free C. parasitica strains isolated from inactive versus active cankers did not differ. Significant differences were only attributed to virus infection. Virus infection and fungal strain composition in cankers changed over time. Foreign vc types immigrated into cankers and virus-free cankers became virus-infected within a few years. Most of the cankers were callused over time and became inactive. However, we observed that the virus did not always persist in these cankers. This study demonstrates that virus spread occurs effectively in European chestnut forests and that this biocontrol system is highly dynamic.