Genetic diversity of Cryphonectria parasitica causing chestnut blight in South Tyrol (northern Italy)

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.

Evaluation of Chestnut Susceptibility to Cryphonectria parasitica: Screening under Controlled Conditions

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.

Biocontrol via mycoviruses: a neglected option for bioprotection?

Virocontrol involves biocontrol of pathogens via hypovirulence-causing mycoviruses. This chapter discusses the characteristics of mycoviruses and the conditions that need to be met to make a successful virocontrol agent. The chapter describes one of the success stories so far, that of virocontrol of Cryphonectria parasitica, the chestnut blight pathogen. The chapter ends by reviewing future trends and where to find more information on mycoviruses and virocontrol.

Environmental niche and demographic modeling of American chestnut near its southwestern range limit

The inadvertent introduction and rapid spread of chestnut blight (caused by Cryphonectria parasitica (Murr.) Barr) in the early 20th century resulted in the demise of American chestnut (Castanea dentata (Marsh.) Borkh.; Fagaceae) as a major component of forest canopies and had negative impacts on eastern forest communities. Research efforts over the last century have documented the persistence of occasional trees and root crown/stump sprouts throughout much of the historic range of the species providing the basis for ongoing breeding of blight-resistant stock and restoration efforts. Unfortunately, it remains unclear how much of the historic range remains climatically suitable for remnant trees that may harbor unique genetic variation for successful reintroduction efforts. Here we investigate whether the southwestern portion of the historical range remains environmentally suitable for undiscovered remnant populations of C. dentata using environmental niche modeling. We also use stage-structured matrix projection models to investigate the potential demographic future of C. dentata in W Tennessee, N Mississippi, SW Kentucky, and NW Alabama based upon observations of American chestnut in these areas over the last several decades. We found that suitable habitat associated with higher elevations and areas of high forest canopy cover occurs throughout much of southwestern portion of the historical range and that populations of American chestnut in these areas are predicted to drastically decline over the next ~100-200 years without conservation interventions to mitigate the negative consequences of chestnut blight.

Differing Responses to Cryphonectria parasitica at Two Indiana Locations

Chestnut blight, a disease that has spread rampantly among American (Castanea dentata (Marsh.) Borkh.) and European chestnut (C. sativa Mill.) trees, results from infection by the fungal pathogen Cryphonectria parasitica (Murrill) M.E. Barr (C. parasitica). This fungus was introduced in the early 1900s and has almost functionally eliminated chestnut trees from the North American landscape. In 2017, we collected chestnut blight samples from two sites (Site B, (Fulton Co., IN) and Site C (Marshall Co., IN)). At the Fulton County planting, Site B, cankers had formed, healed over, and the trees were healthy. However, at the second site in Marshall County, (Site C), cankers continued to propagate until all of the chestnut trees had died back to the ground. Research evidence worldwide has indicated that these visual clues likely result from the presence of a hypovirus. Upon closer inspection and the subsequent isolation and reproduction of spores, no hypovirus has been identified from either site. Here, we present a curious coincidence where one site has completely succumbed to the disease, while the other has been able to spring back to health.

Castanea mollissima (hairy chestnut).

C. mollissima has been cultivated for its nuts by the Chinese for at least 2500 years. The tree grows under a wide variety of environmental conditions, is drought- and flood-tolerant, and is very resistant to chestnut blight (Cryphonectria parasitica), which is a serious disease of Castanea species. In recent years, Chinese chestnut has been planted on a large scale in rural areas of China and on a smaller scale in the USA (Wallace, 1995), where Warmund (2011) considered it to have potential as a niche crop in the central states. In 1995, approximately 245,000 t of nuts were produced in China, of which 51,000 t came from Shandong Province and 29,000 t from Hubei Province. Overall, mean nut production in China increased from 225 kg/ha during the 1980s to 335 kg/ha in the 1990s, with yields of up to 1500 kg/ha being recorded widely (Zheng, 1985; Gao, 1997).

Emergence and diversification of a highly invasive chestnut pathogen lineage across southeastern Europe

Invasive microbial species constitute a major threat to biodiversity, agricultural production and human health. Invasions are often dominated by one or a small number of genotypes, yet the underlying factors driving invasions are poorly understood. The chestnut blight fungus Cryphonectria parasitica first decimated the North American chestnut, and a more recent outbreak threatens European chestnut stands. To unravel the chestnut blight invasion of southeastern Europe, we sequenced 230 genomes of predominantly European strains. Genotypes outside of the invasion zone showed high levels of diversity with evidence for frequent and ongoing recombination. The invasive lineage emerged from the highly diverse European genotype pool rather than a secondary introduction from Asia or North America. The expansion across southeastern Europe was mostly clonal and is dominated by a single mating type, suggesting a fitness advantage of asexual reproduction. Our findings show how an intermediary, highly diverse bridgehead population gave rise to an invasive, largely clonally expanding pathogen.

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

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.