▪ Abstract Most hypovirulence in the chestnut blight fungus, Cryphonectria parasitica, is associated with infection by fungal viruses in the family Hypoviridae. Hypovirulence has controlled chestnut blight well in some locations in Europe and in Michigan in the United States. In contrast, with few exceptions, biological control has failed almost completely in eastern North America. Therapeutic treatment of individual cankers is successful in most cases, but the success of hypovirulence at the population level depends on the natural spread of viruses. Characteristics of three interacting trophic levels (virus, fungus, and tree), plus the environment, determine the success or failure of hypovirulence. Vegetative incompatibility restricts virus transmission, but this factor alone is a poor predictor of biological control. Any factor reducing the rate of chestnut blight epidemics enhances hypovirus invasion. Overall, however, not enough is understood about the epidemiological dynamics of this system to determine the crucial factors regulating the establishment of hypovirulence in chestnut forests.

Programmed cell death (PCD) is an essential part of the defence response in plants and animals against pathogens. Here, we report that PCD is also involved in defence against pathogens of fungi. Vegetative incompatibility is a self/non–self recognition system in fungi that results in PCD when cells of incompatible strains fuse. We quantified the frequency of cell death associated with six vegetative incompatibility (vic) genes in the filamentous ascomycete fungus Cryphonectria parasitica. Cell death frequencies were compared with the effects of vic genes on transmission of viruses between the same strains. We found a significant negative correlation between cell death and virus transmission. We also show that asymmetry in cell death correlates with asymmetry in virus transmission; greater transmission occurs into vic genotypes that exhibit delayed or infrequent PCD after fusion with an incompatible strain. Furthermore, we found that virus infection can have a significant, strain–specific, positive or negative effect on PCD. Specific interactions between vic gene function and viruses, along with correlations between cell death and transmission, strongly implicate PCD as a host–mediated pathogen defence strategy in fungi.

Vegetative incompatibility is a self/nonself-recognition system in fungi that has often been used for describing phenotypic diversity in fungal populations. A common hypothesis is that vegetative incompatibility polymorphisms are maintained by balancing selection. However, understanding the evolutionary significance of vegetative incompatibility and the factors that maintain these polymorphisms has been limited by a lack of knowledge of the underlying genetics of vegetative compatibility (vc) types. Genotypes of 64 vc types, controlled by six unlinked vegetative incompatibility ( vic ) loci, have been identified in the chestnut blight fungus, Cryphonectria parasitica . By interpreting vc type survey data in terms of vic genotypes, we estimated vic -allele frequencies and analyzed the multilocus genetic structure of 13 populations in Europe and 3 populations in the U.S. European populations have less vc type diversity than the US populations because of a combination of lower vic -allele diversity and limited recombination. Genotypic diversity of 10 populations in Italy correlated to the abundance of sexual structures; however, significant deviations from random mating suggest that either sexual reproduction may not contribute many offspring in these populations or that vic genes (or vic genotypes) are under selection. Most vic -allele frequencies deviated from 0.5, the equilibrium frequency predicted under frequency-dependent selection, providing no evidence for selection acting on these loci.

In France, chestnut blight, caused by Cryphonectria parasitica, has been controlled since 1974 in orchards, but never in coppice forests, by releasing hypovirulent strains infected with CHV1 hypovirus. We tested the hypothesis that this biological control (BC) has lead to a decrease in blight severity, spread of hypovirulence, and change in C. parasitica populations. The low severity of chestnut blight was confirmed in the six regions studied (subdivided into zones). The remission of cankers was associated with the presence of white isolates presumed to be hypovirulent. These two parameters were also correlated, at the zonal level, to the frequency of sites where BC was used. However, the estimates of the natural background level of hypovirulence, independent of BC, ranged from 4% in forests in Dordogne to 60% in orchards in Lozère. Differences in the rate of hypovirulent isolates among regions were consistent with the diversity of vegetative compatibility (VC) types in populations of C. parasitica. The highest VC-type diversity and mean allelic diversity for known vegetative incompatibility (vic) genes were observed in Dordogne. We showed that the current diversity of VC types in populations of C. parasitica was lower than in 1981. We found 30 VC types among 1,113 isolates of C. parasitica. Ten VC types were incompatible with known EU testers, suggesting that one additional vic gene or allele at one of the six vic loci known should be present in Europe.

In the search for new natural resources for crop protection, streptomycetes are gaining interest in agriculture as plant growth promoting bacteria and/or biological control agents. Because of their peculiar life cycle, in which the production of secondary metabolites is synchronized with the development of aerial hyphae and sporulation, the commonly used methods to screen for bacterial antagonists need to be adapted. The dual culture assay was standardized in terms of inoculation timing of Streptomyces antagonist and pathogen, and growth rate of different fungal pathogens. In case of fast-growing fungi, inoculation of the antagonist 2 or 3 days prior to the pathogen resulted in significantly stronger inhibition of mycelium growth. One hundred and thirty Streptomyces strains were evaluated against six destructive soil borne pathogens. The activity of strains varied from broad-spectrum to highly specific inhibition of individual pathogens. All strains inhibited at least one tested pathogen. Three strains, which combined the largest broad-spectrum with the highest inhibition activity, were selected for further characterization with four vegetable species. All of them were able to colonize seed surface of all tested vegetable crops. They mostly improved radicle and hypocotyl growth in vitro, although no statistically significant enhancement of biomass weight was observed in vivo. Occasionally, transient negative effects on germination and plant growth were observed. The adapted dual culture assay allowed us to compare the inhibition of individual Streptomyces strains against six fungal soil borne pathogens. The best selected strains were able to colonize the four vegetable crops and have a potential to be developed into biocontrol products. Although they occasionally negatively influenced plant growth, these effects did not persist during the further development. Additional in vivo studies are needed to confirm their potential as biological control or plant growth promoting agents.

Chestnut blight, caused by the fungusCryphonectria parasitica, has been effectively controlled with double-stranded RNA hypoviruses in Europe for over 40 years. The marked reduction in the virulence ofC. parasiticaby hypoviruses is a phenomenon known as hypovirulence. This virus-fungus pathosystem has become a model system for the study of biological control of fungi with viruses. We studied variation in tolerance to hypoviruses in fungal hosts and variation in virulence among virus isolates from a local population in Italy. Tolerance is defined as the relative fitness of a fungal individual when infected with hypoviruses (compared to being uninfected); virulence is defined for each hypovirus as the reduction in fitness of fungal hosts relative to virus-free hosts. Six hypovirus-infected isolates ofC. parasiticawere sampled from the population, and each hypovirus was transferred into six hypovirus-free recipient isolates. The resulting 36 hypovirus-fungus combinations were used to estimate genetic variation in tolerance to hypoviruses, in hypovirus virulence, and in virus-fungus interactions. Four phenotypes were evaluated for each virus-fungus combination to estimate relative fitness: (i) sporulation, i.e., the number of asexual spores (conidia) produced; (ii) canker area on field-inoculated chestnut trees, (iii) vertical transmission of hypoviruses into conidia, and (iv) conidial germination. Two-way analysis of variance (ANOVA) revealed significant interactions (P< 0.001) between viruses and fungal isolates for sporulation and canker area but not for conidial germination or transmission. One-way ANOVA among hypoviruses (within each fungal isolate) and among fungal isolates (within each hypovirus) revealed significant genetic variation (P< 0.01) in hypovirus virulence and fungal tolerance within several fungal isolates, and hypoviruses, respectively. These interactions and the significant genetic variation in several fitness characters indicate the potential for future evolution of these characters. However, biological control is unlikely to break down due to evolution of tolerance to hypoviruses in the fungus because the magnitudes of tolerance and interactions were relatively small.