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.

Agro-Industrial Wastes: A Substrate for Multi-Enzymes Production by Cryphonectria parasitica

This study aims to produce a mix of enzymes through Solid State Fermentation (SSF) of raw materials. Four different, easily available, agro-industrial wastes were evaluated as SSF substrates for enzymes production by Cryphonectria parasitica (Murr.) Barr. environmental strains named CpA, CpB2, CpC4, and CpC7. Among the tested wastes, organic wheat bran for human use and wheat bran for animal feed better supports C. parasitica growth and protease production without any supplements. SDS-PAGE analyses highlighted the presence of three bands corresponding to an extracellular laccase (77 kDa), to the endothiapepsin (37 kDa), and to a carboxylesterase (60.6 kDa). Protease, laccase, and esterase activities by C. parasitica in SSF were evaluated for 15 days, showing the maximum protease activity at day 9 (3955.6 AU/gsf,). Conversely, the best laccase and esterase production was achieved after 15 days. The C. parasitica hypovirulent CpC4 strain showed the highest laccase and esterase activity (93.8 AU/gsf and 2.5 U/gsf, respectively). These results suggest the feasibility of a large-scale production of industrially relevant enzymes by C. parasitica strains in SSF process on low value materials.

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.

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.

A New Double-Stranded RNA Mycovirus in Cryphonectria naterciae Is Able to Cross the Species Barrier and Is Deleterious to a New Host

Cryphonectria is a fungal genus associated with economically significant disease of trees. Herein we characterized a novel double-stranded RNA virus from the fungal species Cryphonectria naterciae, a species unexplored as a virus host. De novo assembly of RNA-seq data and Sanger sequencing of RACE (rapid amplification of cDNA ends) clones gave the complete, non-segmented genome (10,164 bp) of the virus termed Cryphonectria naterciae fusagravirus (CnFGV1) that was phylogenetically placed within the previously proposed viral family Fusagraviridae. Of 31 field-collected strains of C. naterciae, 40% tested CnFGV1-positive. Cocultivation resulted in within-species transmission of CnFGV1 to virus-free strains of C. naterciae. Comparison of the mycelium phenotype and the growth rate of CnFGV1-infected and virus-free isogenic strains revealed frequent sectoring and growth reduction in C. naterciae upon virus infection. Co-culturing also led to cross-species transmission of CnFGV1 to Cryphonectria carpinicola and Cryphonectria radicalis, but not to Cryphonectria parasitica. The virus-infected C. naterciae and the experimentally infected Cryphonectria spp. readily transmitted CnFGV1 through asexual spores to the next generation. CnFGV1 strongly reduced conidiation and in some cases vegetative growth of C. carpinicola, which is involved in the European hornbeam disease. This study is the first report of a fusagravirus in the family Cryphonectriaceae and lays the groundwork for assessing a hypovirulence effect of CnFGV1 against the hornbeam decline in Europe.

The Physiological and Biochemical Responses of European Chestnut (Castanea sativa L.) to Blight Fungus (Cryphonectria parasitica (Murill) Barr)

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.

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.