Fungal community and toxigenic taxa in chestnut fruits in postharvest conditioning process and storage.

BACKGROUND: Chestnut fruit quality is affected by fungal contamination. The study of the patterns of contamination in the postharvest is crucial to individuate the critical phases and propose solutions. To understand how fungal colonization varies on fruits, the composition of mycobiota was investigated in postharvest handling and in between tissues (shell and kernel). RESULTS: Fungal sequences were clustered into 308 operational taxonomic units (OTUs). Biodiversity was higher in shell than kernel tissues. Results evidenced the risk of new contamination in specific phases such as the 'cold bath' and storage. Genera known as mycotoxin producers were detected in all phases. Specifically, 47 OTUs belonging to Penicillium, eight to Fusarium and two to Aspergillus genera were identified. While Fusarium spp. was sensitive to 'warm bath' phase, Penicillium spp. was largely insensitive and accumulated in storage conditions. Surprisingly, Aspergillus spp. was poorly represented. Aflatoxin, ochratoxin A, fumonisins and T-2/HT-2 detection was performed for shell and kernel, and process phases. Higher contamination was observed on shell than in kernel samples. While aflatoxins were within the European Union (EU) limits for dry fruits, Ochratoxin exceeded the EU limits. The present study represents the first report of fumonisins and T-2/HT-2 detection in chestnuts. CONCLUSION: Fungal contamination taxa is high in chestnut fruits following postharvest handling and storage. A parametrization of process phases such as the 'warm bath' is functional to reduce the risk for some taxa. For other spoilage and mycotoxigenic genera strict sanitation procedures of equipment and water must be individuated and implemented to reduce their impact. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Novel soil-less potting mixes for the mycorrhization of Quercus pubescens Willd. seedlings with Tuber melanosporum Vittad.

Truffle cultivation has quickly grown in Europe and elsewhere as a consequence of the increase in the demand of the market. Thus the optimization of the protocols for the production of elite mycorrhized plants are also needed, keeping in consideration the economic and environmental sustainability. The suitability of two compost-based potting mixes to produce Quercus pubescens Willd. plants mycorrhized with the black Périgord truffle T. melanosporum Vittad. was tested as an alternative to the traditional potting mix used. The effects on mycorrhizal development and the morphometric assessment of the root and shoot system of the Q. pubescens seedlings were investigated eight months after the spore slurry inoculation in a glasshouse experiment. From the results obtained, the compost mix containing green organic residues from pruning and mowing (Mix 2) achieved better performance than the control and the potting mix based on composted municipal organic wastes, showing significantly higher mycorrhization percentage, root length, number of root tips, and root forks. In conclusion, a potting mix containing recycled green organic matter, which is readily available, cheap, and environmentally sustainable, can offer excellent mycorrhization performances and may be included in the mycorrhization process of downy oak seedlings with T. melanosporum under controlled conditions.

Hybrid de novo genome assembly and comparative genomics of three different isolates of Gnomoniopsis castaneae.

The first genome assemblies of Gnomoniopsis castaneae (syn. G. smithogilvyi), the causal agent of chestnut brown rot of kernels, shoot blight and cankers, are provided here. Specifically, the complete genome of the Italian ex-type MUT401 isolate was compared to the draft genome of a second Italian isolate (GN01) and to the ICMP 14040 isolate from New Zealand. The three genome sequences were obtained through a hybrid assembly using both short Illumina reads and long Nanopore reads, their coding sequences were annotated and compared with each other and with other Diaporthales. The information offered by the genome assembly of the three isolates represents the base of data for further application related to -omics strategies of the fungus and to develop markers for population studies at a local and global scale.

Impact of 'brown rot' caused by Gnomoniopsis castanea on chestnut fruits during the post-harvest process: critical phases and proposed solutions.

BACKGROUND: The brown rot fungus, Gnomoniopsis castanea, is the main organism responsible for the outbreak of chestnut postharvest decay that is threatening the sustainability of the chestnut market in Europe. Currently, no specific strategy is available to mitigate the impact and remediate the high losses of fruits in postharvest storage. In the present study, the different phases of chestnut handling in a standard facility plant were analyzed by evaluating the amount of fruit rot and infection by G. castanea at each phase. RESULTS: The warm bath (48 °C) was identified as the critical phase, requiring strict parametrization to effectively inactivate G. castanea in fruits. Laboratory tests indicated that maintaining fruits at 50 °C for a maximum of 45 min provided optimal conditions to completely inactivate G. castanea inoculum during postharvest handling. However, the warm bath at 50 °C and over was not effective in inactivating the complex of fungal taxa responsible for contamination and development of molds. Higher temperatures and extended treatment times caused significant losses in fruit quality, as indicated by taste panel evaluation. Upscaling of postharvest facilities is discussed and critically evaluated. CONCLUSION: The warm bath (50 °C for 45 min) is effective in completely inactivating G. castanea in fruits but did not reduce the impacts of the complex of molds responsible for external contamination and mycotoxin production. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The fungal community associated with the ambrosia beetle Xylosandrus compactus invading the mediterranean maquis in central Italy reveals high biodiversity and suggests environmental acquisitions.

In summer 2016 a severe infestation of the alien ambrosia beetle Xylosandrus compactus was recorded from the Mediterranean maquis in the Circeo National Park in Central Italy. Trees and shrubs were infested and displayed wilting and necrosis of terminal branches caused by the combined impact of the insect and associated pathogenic fungi. A preliminary screening carried out on captured adults resulted in the isolation of a discrete number of fungal taxa with different life strategies, ranging from true mutualist (e.g. Ambrosiella xylebori) to plant pathogens (Fusarium spp.). In the present study, high-throughput sequencing was applied to determine the total diversity and functionality of the fungal community associated with X. compactus adults collected in the galleries of three Mediterranean woody hosts, Quercus ilex, Laurus nobilis, and Ceratonia siliqua. The effect of season and host in determining the composition of the associated fungal community was investigated. A total of 206 OTUs composed the fungal community associated with X. compactus. Eighteen OTUs were shared among the three hosts, including A. xylebori and members of the Fusarium solani complex. All but two were previously associated with beetles. Sixty-nine out of 206 OTUs were resolved to species level, identifying 60 different fungal species, 22 of which already reported in the literature as associated with beetles or other insects. Functional guild assigned most of the fungal species to saprotrophs and plant pathogens. Effects of seasonality and host on fungal community assemblage were highlighted suggesting the acquisition by the insect of new fungal taxa during the invasion process. The consequences of enriched fungal community on the risk of the insurgence of novel threatful insect-fungus association are discussed considering direct and indirect effects on the invaded habitat.

Fungal community associated with adults of the chestnut gall wasp Dryocosmus kuriphilus after emergence from galls: Taxonomy and functional ecology.

The diversity of the fungal community associated with adults of Dryocosmus kuriphilus following emergence was examined using HTS analysis. Ascomycota dominated the fungal core-biome community. The functional guilds of the 90 taxa forming the core-biome were assessed, demonstrating three main groups: saprotrophs, plant pathogens and entomopathogens. Twenty-nine OTUs out of 90 were resolved to species level identifying 26 different fungal species. Among these species, many were cosmopolitan or previously recorded in Europe. Ten taxa were previously recorded on chestnut, including some recognized plant pathogens associated with foliage and green tissues such as Epicoccum nigrum, Gnomoniopsis castanea, Colletotrichum acutatum, Stromatoseptoria castaneicola, Ramularia endophylla. Beauveria bassiana; within the core microbiome, Fusarium larvarum represented the most abundant entomopathogenic species. Some of these species are known to impact directly or indirectly the vitality of the insects in the galls. The chestnut blight pathogen, Cryphonectria parasitica, was never found associated with D. kuriphilus. Based on the present study, an active role for D. kuriphilus as a vector of chestnut fungal endophyte/pathogens cannot be demonstrated but neither ruled out.

Assessment of functional and structural changes of soil fungal and oomycete communities in holm oak declined dehesas through metabarcoding analysis.

Forest decline is nowadays a major challenge for ecosystem sustainability. Dehesas, which consists of savannah-like mediterranean ecosystems, are threatened by the holm oak decline in the south-west of Iberian Peninsula. Phytophthora cinnamomi is considered the main agent of holm oak root rot, but little is known about the relationship between diversity of soilborne microbial community and the decline syndrome of holm oak. It would be hypothesized that the changes in the structure and functionality of the soil microbiome might influence tree health status through changes in richness and diversity of beneficial organisms such as mycorrhizal species, or fungal plant pathogens such as Fusarium spp. or Alternaria spp. Total DNA of soil samples from declined oak dehesas was extracted and analyzed through metabarcoding techniques, to evaluate the specific composition and diversity of the fungal and oomycete communities and their relationship with the disease symptoms. The fungal community included a wide range of pathogens and abundance of ectomycorrhizal key taxa related with low defoliation degree. Phytophthora cinnamomi and Pythium spiculum did not appear among the most abundant oomycetes, nor were they related directly to defoliation levels. Moreover, a particular taxon belonging to the genus Trichoderma was strongly correlated with the scarcity of pathogenic Phytophthora spp. The diversity and composition of fungal and oomycete communities were related to the severity of the decline symptoms. The metabarcoding study of microbiome represents a powerful tool to develop biocontrol strategies for the management of the holm oak root rot.

High-throughput identification and diagnostics of pathogens and pests: Overview and practical recommendations.

High-throughput identification technologies provide efficient tools for understanding the ecology and functioning of microorganisms. Yet, these methods have been only rarely used for monitoring and testing ecological hypotheses in plant pathogens and pests in spite of their immense importance in agriculture, forestry and plant community dynamics. The main objectives of this manuscript are the following: (a) to provide a comprehensive overview about the state-of-the-art high-throughput quantification and molecular identification methods used to address population dynamics, community ecology and host associations of microorganisms, with a specific focus on antagonists such as pathogens, viruses and pests; (b) to compile available information and provide recommendations about specific protocols and workable primers for bacteria, fungi, oomycetes and insect pests; and (c) to provide examples of novel methods used in other microbiological disciplines that are of great potential use for testing specific biological hypotheses related to pathology. Finally, we evaluate the overall perspectives of the state-of-the-art and still evolving methods for diagnostics and population- and community-level ecological research of pathogens and pests.

Emerging new crown symptoms on Castanea sativa (Mill.): Attempting to model interactions among pests and fungal pathogens.

In the 2015-2016 growing seasons, two novel symptoms were assessed on the crown of trees in orchards and coppices of chestnut groves in Central Italy. The first symptom was flagging of annual shoots with green leaves undergoing sudden wilt and turning brown later in the season. The second symptom consisted of leaves on annual shoots turning yellow before wilting in absence of flagging represented the second symptom. Samples were collected along transects in early summer, late summer and winter, and processed in the laboratory. The flagging symptom was associated in early summer with the presence of C. parasitica in cryptic dried buds on stems from the previous year's growth. The pathogen was also found in dormant buds in winter, suggesting that the infection could take place in summer during the Chinese gall wasp oviposition period. Cryphonectria parasitica was also isolated from abandoned galls in winter supporting the hypothesis that galls are a potential source of inoculum for crown infections. Aetiology of yellowing was not clarified and no fungal taxa were specifically associated with this symptom. Gnomoniopsis castanea, C. parasitica and, in early summer, Colletotrichum acutatum were the most abundant fungal taxa isolated from chestnut shoots and buds.

The sentinel tree nursery as an early warning system for pathway risk assessment: Fungal pathogens associated with Chinese woody plants commonly shipped to Europe.

Introduction of and invasion by alien plant pathogens represents the main cause of emerging infectious diseases affecting domesticated and wild plant species worldwide. The trade in living plants is the most common pathway of introduction. Many of the alien tree pathogens recently introduced into Europe were not previously included on any quarantine lists. To help determine the potential risk of pest introduction through trading of ornamental plants, a sentinel nursery was established in Beijing, China in 2008. The sentinel nursery planting included four of the most common ornamental woody species shipped to Europe including Ilex cornuta var. fortunae, Zelkova schneideriana, Fraxinus chinensis and Buxus microphylla. Symptoms developing on these species within the sentinel nursery were detected in 2013 and consisted of necrotic spots on leaves, canker and stem necrosis, shoot blight and shoot necrosis. Fungi associated with the trees and their symptoms included Alternaria alternata detected from all hosts; Diaporthe liquidambaris and Diaporthe capsici from bark and leaf necrosis of Zelkova schneideriana; Botryosphaeria dothidea and Nothophoma quercina from stem cankers on Fraxinus chinensis and leaf necrosis on Ilex cornuta; and Pseudonectria foliicola from leaf necrosis on Buxus microphylla. Next generation sequencing analysis from asymptomatic tissues detected eighteen OTU's at species level among which some taxa had not been previously recorded in Europe. These results clearly demonstrate that looking at trees of internationally traded species in the region of origin can reveal the presence of potentially harmful organisms of major forestry, landscape or crop trees. Results of this study also provide an indication as to how some disease agents can be introduced using pathways other than the co-generic hosts. Hence, sentinel nurseries represent one potential mechanism to address the current lack of knowledge about pests in the countries from where live plants are shipped and the threats they represent to native flora and crops in importing countries.

Does Gnomoniopsis castanea contribute to the natural biological control of chestnut gall wasp?

Gnomoniopsis castanea has been reported as the causal agent of necrosis of chestnut wasp (Dryocosmus kuriphilus) galls. The fungus is frequently observed on galls in chestnut stands infested by the insect in Italy. In the present study the impact of gall necrosis and the dynamic of its development have been studied in mature and young Castanea sativa stands in Central Italy during spring and early summer, before the D. kuriphilus adult flies. Results suggest that gall necrosis develops from resident endophytic inoculum of G. castanea. During the 2 y of monitoring, no differences were found in incidence and severity of the disease. Gall necrosis increased exponentially during the season, reaching 75,4% of galls totally necrotized in the investigated site in mid July. Gall necrosis was shown to have a severe impact on D. kuriphilus vitality, mostly impacting the adults inside the galls. Gall necrosis by G. castanea appears to efficiently control gall wasp in chestnut stands, although the high virulence of the fungus to chestnut fruits precludes its use as biocontrol agent in biological control strategies.

Control of Phytophthora nicotianae with Mefenoxam, Fresh Brassica Tissues, and Brassica Pellets.

Sensitivity to mefenoxam, fresh Brassicaceae tissues, and Brassica pellets was evaluated in several isolates of Phytophthora nicotianae recovered from pepper and tomato plants. The isolates of P. nicotianae studied were classified as sensitive to mefenoxam, showing great variability among isolates in the 50 and 90% effective concentrations (EC50 and EC90, respectively). Sensitivity differentiated isolates from the two hosts of origin, being isolates from tomato plants more resistant to fungicide than those from pepper plants. This differentiation also occurred in the case of fresh Brassicaceae tissues assay. The most effective biofumigant in inhibiting mycelial growth of P. nicotianae isolates was Brassica nigra. The effectiveness of B. carinata, Sinapis alba, and B. oleracea varied depending on the dose. Isolates differed in susceptibility to compounds released by the Brassica pellets and then in the EC50 and EC90. No significant difference was found between the isolates depending on the host of origin. Greenhouse tests demonstrated the effectiveness of treatments with mefenoxam and with Brassica pellets to control P. nicotianae in pepper plants. Mefenoxam application could be a solution to the disease caused by P. nicotianae in tomato and pepper crops in this region but its use could increase resistance in populations. Biofumigation is a promising technique which can be further developed to form part of integrated pest management strategies.

In vitro inhibition of mycelial growth of Phytophthora nicotianae Breda de Haan from different hosts by Brassicaceae species. Effect of the developmental stage of the biofumigant plants.

BACKGROUND: The hydrolysis products of glucosinolates in Brassicaceae tissues are potentially useful for the control of fungal pathogens. The in vitro activity of these products against Phytophthora nicotianae Breda de Haan was studied, with the isolates exposed to the volatile products released from the brassica tissues. RESULTS: The four species of Brassicaceae tested inhibited the mycelial growth of P. nicotianae isolates. The most effective developmental stage was different, depending on the species: buds yellowing in Sinapis alba L. and Brassica carinata A. Braun; seeds enlarging and all buds open in Brassica nigra (L.) W.D.J. Koch; cauliflower still covered by leaves in Brassica oleracea L. var. botrytis. At this stage, B. nigra and S. alba were the most effective (53.6 and 52.5% inhibition respectively). With all the biofumigants species tested, isolates from pepper plants were more susceptible to the brassica effect than those from tomato isolates. CONCLUSION: The developmental stage of plants has an influence on the biofumigant potential of Brassicaceae species against P. nicotianae. The isolates differ in susceptibility to compounds released, depending on their host of origin, suggesting the differentiation of populations of P. nicotianae in relation to the host of origin.