First detection of mycoviruses in Gnomoniopsis castaneae suggests a putative horizontal gene transfer event between negative-sense and double-strand RNA viruses.

Gnomoniopsis castaneae is an ascomycetous fungus mainly known as a major pathogen of chestnut causing nut rots, although it is often found as an endophyte in chestnut tissues. To date, no virus has been reported as associated with to this fungus. Here, a collection of G. castaneae isolates from several European countries was screened to detect mycoviruses infecting the fungus: for the first time we report the identification and prevalence of mitovirus Gnomoniopsis castaneae mitovirus 1 (GcMV1) and the chrysovirus Gnomoniopsis castaneae chrysovirus 1 (GcCV1). Interestingly, we provide evidence supporting a putative horizontal gene transfer between members of the phyla Negarnaviricota and Duplornaviricota: a small putative protein of unknown function encoded on the RNA3 of GcCV1 (Chrysoviridae) has homologs in the genome of viruses of the family Mymonaviridae.

Host-associated Intraspecific Phenotypic Variation in the Saprobic Fungus Phlebiopsis gigantea.

Whether intraspecific phenotypic variation in saprobic fungi may be driven by the host of origin has received little attention. We addressed this issue by testing hypotheses using the model system Phlebiopsis gigantea, a wood destroying fungus associated with Picea abies and Pinus sylvestris, among others, and widely employed in practical forestry as a biological control agent. By examining approximately 60 sympatric P. gigantea isolates from both P. abies and P. sylvestris, we showed that the former grew in vitro significantly (P < 0.05) slower than the latter (average 5.56 mm/day vs. 6.84) while producing 1.8-fold significantly higher number of mitospores. An overall significant trade-off between these two phenotypic traits was detected, in particular for isolates originating from P. abies. Comparative inoculation experiments of a subsample of isolates and the assessment of mycelial growth in logs of both hosts allowed to reject the hypothesis that isolates are equally fit in terms of growth rate in wood of both hosts regardless of the host of origin. Tree models revealed that the growth rate of isolates was associated not only with the wood species in which the isolates were inoculated (P < 0.001), P. sylvestris being more rapidly colonized than P. abies, but also with the host of origin of isolates (P < 0.001). Results showed that P. gigantea isolates originating from different hosts differ phenotypically in terms of some key phenotypic traits demonstrating that a host-driven intraspecific phenotypic variation may occur in saprobic fungi.

Pyoluteorin Produced by the Biocontrol Agent Pseudomonas protegens Is Involved in the Inhibition of Heterobasidion Species Present in Europe.

Pseudomonas protegens (strain DSMZ 13134) is a biocontrol agent with promising antagonistic activity hinging on antibiosis against the fungal forest pathogens Heterobasidion spp. Here, by using High-Performance Liquid Chromatography coupled to Mass Spectrometry (HPLC-MS), we assessed whether monocultures of P. protegens (strain DSMZ 13134) produce the three major determinants of biocontrol activity known for the genus Pseudomonas: 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin (PLT), and pyrrolnitrin (PRN). At the tested culture conditions, we observed the production of PLT at concentrations ranging from 0.01 to 10.21 mg/L and 2,4-DAPG at a concentration not exceeding 0.5 mg/L. Variations of culture conditions involving culture medium, incubation temperature, and incubation period had no consistent influence on PLT production by the bacterium. Assays using culture medium amended with PLT at the same concentration of that present in cell-free filtrate of the bacterium, i.e., 3.77 mg/L, previously documented as effective against Heterobasidion spp., showed a remarkable activity of PLT against genotypes of all the four Heterobasidion species present in Europe, including the non-native invasive H. irregulare. However, such antifungal activity decreased over time, and this may be a constraint for using this molecule as a pesticide against Heterobasidion spp. When the bacterium was co-cultured in liquid medium with genotypes of the different Heterobasidion species, an increased production of PLT was observed at 4 °C, suggesting the bacterium may perform better as a PLT producer in field applications under similar environmental conditions, i.e., at low temperatures. Our results demonstrated the role of PLT in the inhibition of Heterobasidion spp., although all lines of evidence suggest that antibiosis does not rely on a single constitutively produced metabolite, but rather on a plethora of secondary metabolites. Findings presented in this study will help to optimize treatments based on Pseudomonas protegens (strain DSMZ 13134) against Heterobasidion spp.

Comparative Efficacy of State-of-the-Art and New Biological Stump Treatments in Forests Infested by the Native and the Alien Invasive Heterobasidion Species Present in Europe.

The Heterobasidion annosum species complex includes major fungal pathogens of conifers worldwide. State-of-the-art preventative stump treatments with urea or with commercial formulations of the fungal biological control agent Phlebiopsis gigantea (i.e., Rotstop®) may become no longer available or are not approved for use in many areas of Europe infested by the three native Heterobasidion species and by the North American invasive H. irregulare, making the development of new treatments timely. The efficacy of Proradix® (based on Pseudomonas protegens strain DSMZ 13134), the cell-free filtrate (CFF) of the same bacterium, a strain of P. gigantea (MUT 6212) collected in the invasion area of H. irregulare in Italy, Rotstop®, and urea was comparatively investigated on a total of 542 stumps of Abies alba, Picea abies, Pinus pinea, and P. sylvestris in forest stands infested by the host-associated Heterobasidion species. Additionally, 139 logs of P. pinea were also treated. Results support the good performances of Rotstop®, and especially of urea against the native Heterobasidion species on stumps of their preferential hosts and, for the first time, towards the invasive North American H. irregulare on stumps of P. pinea. In some experiments, the effectiveness of Proradix® and of the strain of P. gigantea was weak, whereas the CFF of P. protegens strain DSMZ 13134 performed as a valid alternative to urea and Rotstop®. The mechanism of action of this treatment hinges on antibiosis; therefore, further improvements could be possible by identifying the active molecules and/or by optimizing their production. Generally, the performance of the tested treatments is not correlated with the stump size.

Castanea spp. Agrobiodiversity Conservation: Genotype Influence on Chemical and Sensorial Traits of Cultivars Grown on the Same Clonal Rootstock.

A large species diversity characterises the wide distribution of chestnuts in Asia, North America, and Europe, hence reflecting not only the adaptation of the genus Castanea to diverse environmental conditions, but also to different management strategies encompassing orchards. The characterisation and description of chestnut populations and cultivars are crucial to develop effective conservation strategies of one of the most important Italian and European fruit and wood species. Chestnut cultivars grown in the same pedoclimatic conditions and on the same clonal rootstock were characterised with sensory, spectrophotometric, and chromatographic analysis to determine the phytochemical composition and nutraceutical properties. A multivariate approach, including principal component analysis and conditional inference tree models, was also performed. The ease of peeling, seed colour, and intensity of sweetness were the sensory descriptors that allowed us to differentiate C. sativa cultivars. Antioxidant capacity ranged from 9.30 ± 0.39 mmol Fe+2 kg-1 DW ('Bouche de Bètizac') to 19.96 ± 1.89 mmol Fe+2 kg-1 DW ('Garrone Rosso'). Monoterpenes represented the main component, reaching 88% for hybrids, followed by polyphenols (10-25% for hybrids and chestnuts, respectively). A multivariate approach showed that phenolic acids and tannins were the bioactive classes with the highest discriminating power among different genotypes, and that genotype is a significant variable (p < 0.05). In addition, most of the analysed chestnut cultivars showed a content of bioactive compounds similar to or higher than the main hazelnut, walnut, and almond varieties. Chestnut agrobiodiversity could be intended as strictly associated to the genotype effect and underlines the large variability within the genus Castanea, and therefore, the importance of in farm and ex situ conservation of local germplasm is part of a global strategy aimed at increasing the levels of agrobiodiversity.

Hail-Induced Infections of the Chestnut Blight Pathogen Cryphonectria parasitica Depend on Wound Size and May Lead to Severe Diebacks.

This study combined phytosanitary surveys, laboratory analyses, and mathematical modeling to show how hail-induced wounds can foster the infections of the blight pathogen Cryphonectria parasitica, locally associated with extensive dieback of chestnut (Castanea sativa). Orchards and coppices located within and outside the assessed dieback area in a single location in the North West of Italy were inspected to appraise the abundance of hail-induced wounds and C. parasitica infections. The incidence of C. parasitica was significantly higher within the dieback area compared with outside (92% versus 60%; P < 0.05). Hail-induced wounds were observed on small branches and shoots of all trees sampled within the dieback area, whereas they were less abundant outside (20% of trees), suggesting either that the dieback was directly associated with the injuries caused by the hailstorms or that those injuries may have facilitated infections of C. parasitica. Isolations conducted on 359 branches and shoots showed that hail-induced wounds served as infection courts for C. parasitica and that infections depended on the size rather than on the number of hail wounds. We fitted a logistic model showing that hail-induced wounds whose perimeter was larger than 66 mm were at particular risk of C. parasitica infection. A newly designed geometrical-based model is proposed to relate hailstones size, hail wound perimeter, and the risk of infection. We established that hail-induced wounds are entry points for virulent and hypovirulent strains of C. parasitica, since 6.5% of isolates were infected by Cryphonectria hypovirus-1.

A nonnative and a native fungal plant pathogen similarly stimulate ectomycorrhizal development but are perceived differently by a fungal symbiont.

The effects of plant symbionts on host defence responses against pathogens have been extensively documented, but little is known about the impact of pathogens on the symbiosis and if such an impact may differ for nonnative and native pathogens. Here, this issue was addressed in a study of the model system comprising Pinus pinea, its ectomycorrhizal symbiont Tuber borchii, and the nonnative and native pathogens Heterobasidion irregulare and Heterobasidion annosum, respectively. In a 6-month inoculation experiment and using both in planta and gene expression analyses, we tested the hypothesis that H. irregulare has greater effects on the symbiosis than H. annosum. Although the two pathogens induced the same morphological reaction in the plant-symbiont complex, with mycorrhizal density increasing exponentially with pathogen colonization of the host, the number of target genes regulated in T. borchii in plants inoculated with the native pathogen (i.e. 67% of tested genes) was more than twice that in plants inoculated with the nonnative pathogen (i.e. 27% of genes). Although the two fungal pathogens did not differentially affect the amount of ectomycorrhizas, the fungal symbiont perceived their presence differently. The results may suggest that the symbiont has the ability to recognize a self/native and a nonself/nonnative pathogen, probably through host plant-mediated signal transduction.

The American forest pathogen Heterobasidion irregulare colonizes unexpected habitats after its introduction in Italy.

Habitat preference of an invasive fungal tree pathogen is here compared with that of a sympatric and native closely related congener to test the hypothesis that the invasive ability of the exotic organism may be linked to its capacity to colonize substrates unavailable to the indigenous relative. We compared the distribution of infectious airspora of the North American Heterobasidion irregulare introduced into Italy with that of the native H. annosum, both regarded to be able to establish only in the presence of conifers. Geostatistical and statistical analyses were employed to test for association between both species and five Mediterranean vegetation types. Results show that H. annosum is positively associated with pines and negatively associated with deciduous oaks. The probability of finding its spores decreases to almost 0 at distances over 500 m from pines, and this species is virtually absent in pure oak forests. Spores of H. irregulare are present irrespective of vegetation type, and this species can be found not only where pines are present, but also in pure oak forests. This knowledge implies that spread of H. irregulare is not limited by the fragmented distribution of pine woodlands in central Italy and is essential to both predict and hinder its progress in Europe.