Grapevine trunk diseases (GTDs) are the most widespread fungal diseases, affecting grapevines in all the major growing regions of the world, and their complete eradication is still not possible. Aiming to search alternatives to avoid the spread and high incidence of these diseases, the present work intended to molecularly identify the grapevine endophytic community, the phytopathogenic fungi associated with GTDs in vineyards within the Alentejo region, and to test potential antagonist microorganisms as biological control candidates against GTDs-associated fungi. Grapevine endophytic community showed a wide variety of fungi in GTDs’ asymptomatic and symptomatic plants, nine of them previously described as GTDs-associated fungi. GTDs prevalent fungi identified in symptomatic plants were Diaporthe sp., Neofusicoccum sp., and H. viticola. Almost all these fungi were also detected in asymptomatic plants, which shows the importance of investigating the interactions of fungal communities and confirms the need for early diagnosis of these diseases. Direct inhibition antagonism tests were performed among identified endophytes and GTDs phytopathogenic fungi, and all the endophyte fungi showed potential as biocontrol agents. Our findings suggest that endophytes are promising candidates for their use in biological control due to their antagonistic activity against the mycelia growth of some GTDs-associated fungi.

To evaluate wood colonization and interactions with Vitis spp. of Phaeomoniella chlamydospora, a fungal agent involved in Esca disease, isolate CBS 229.95 was transformed using a pCT74 construct which contained the genetic markers for synthetic green fluorescent protein (sGFP) and hygromycin B phosphotransferase. Nine stable P. chlamydospora fungal transformants (Pch-sGFP lines) were obtained using polyethylene-glycol-mediated transformation of protoplasts. These were characterized for sgfp and hygromycin B phosphotransferase (hph) genome insertions and for sGFP fluorescence emission, using quantitative polymerase chain reaction and fluorimetric systems, respectively. No correlation was observed between sgfp copy number genome insertion and sGFP fluorescence expression. Cuttings of Vitis vinifera ‘Montepulciano’, ‘Verdicchio’, ‘Sangiovese’, ‘Biancame’, and ‘Cabernet Sauvignon’; and the grapevine rootstocks ‘Kober 5BB’, ‘SO4’, ‘420A’, ‘1103P’, and V. rupestris were inoculated by immersion in a conidial suspension of the selected fungal Pch-sGFP71 line and incubated at 4 ± 1 and 25 ± 1°C. Wood colonization was estimated through epifluorescence microscopy and was affected by incubation temperature. After 6 months at 4 ± 1°C, the fungal growth was completely inhibited. At 25 ± 1°C, the highest extent of wood colonization was recorded in Montepulciano and Verdicchio, with the lowest in the rootstocks SO4 and V. rupestris. The expression of the Pch-sGFP71 transformed line was localized in the xylem area, primarily around the vessels. The use of sGFP-transformed P. chlamydospora helped to clarify different aspects associated with the location of this pathogen in grapevine tissue, before disease symptom expression.

Chestnut blight is caused by the fungus Cryphonectria parasitica. As one of the most ecologically important diseases of Castanea spp., C. parasitica can rapidly kill trees. In Europe, mitigation of disease severity took place spontaneously through colonization of C. parasitica by mycoviruses, which reduced the virulence of the fungus. In the framework of a survey, 138 C. parasitica isolates were identified, and virulent/hypovirulent phenotypes were determined through morphological properties and pathogenicity tests. For a pool of four hypovirulent isolates, dsRNA was extracted, cDNA synthesized, and a library subjected to next-generation sequencing. The bioinformatics analysis allowed detecting and reconstructing the complete genome of Cryphonectria hypovirus 1 (CHV-1), denoted as CHV-1 Marche. When compared with the available genomes of other hypoviruses that affected the virulence of C. parasitica, available in databases, CHV-1 Marche showed some nucleotide diversity. The approach used in this study was effective to explore the virome inside a pool of hypovirulent C. parasitica isolates. Next-generation sequencing allowed us to exclude the presence of any other ssRNA and dsRNA viruses infecting the fungus and determine CHV-1 as the only responsible of hypovirulence of C. parasitica in the analyzed samples.

Squash is one of the most important crops of tropical and temperate regions, and it can be affected by several fungal pathogens. Most of these pathogens infect the seeds, which become an efficient vehicle to disperse seedborne pathogens over long distances, with consequent severe crop losses. The main objective of this study was the identification of the principal seedborne fungi in seeds extracted from 66 samples of asymptomatic and symptomatic squash fruit (Cucurbita maxima, Cucurbita moschata) collected in two countries, Tunisia and Italy. The symptoms of fruit decay were identified and classified according to lesion size. Following the blotter test, 14 fungal species were detected from the seeds. Seedborne fungi were identified in all fruit samples tested, including asymptomatic fruit. The most frequent fungi from Tunisian seeds were Alternaria alternata (25.1%), followed by Stagonosporopsis cucurbitacearum (24.6%), Fusarium solani (16.6%), Rhizopus stolonifer (13.3%), F. fujikuroi (7.8%), Albifimbria verrucaria (3.3%), and Stemphylium vesicarium (2.3%). For the fruits from Italy, the most frequently identified fungal species in seed samples were Alternaria alternata (40.0%), followed by F. fujikuroi (20.8%), Stemphylium vesicarium (3.0%), and Curvularia spicifera (2.1%). Morphological identification was confirmed by molecular diagnosis using the available species-specific primers. Furthermore, specific primers were designed to identify Albifimbria verrucaria, Paramyrothecium roridum, and Stemphylium vesicarium. Application of seed-health testing methods, including such conventional and molecular diagnostic tools, will help to improve seed quality and crop yields.

Bois noir (BN) is the most important phytoplasma disease of Euro-Mediterranean area and induces severe loss of production and even the death of vines. Understanding the delicate balance between disease progression and recovery of BN infected plants over space and time is crucial to set up management tools. The data collected and analysed allowed to provide insights into dispersal pattern of the disease, caused by’Candidatus Phytoplasma solani’. Point pattern analysis (PPA) was applied to assess the spatial arrangement of symptomatic plants and the spatial correlation of disease levels in four vineyards. For spatio-temporal patterns of BN, a mark-correlation function was applied. Space-time PPA over multiple years (2011–2015) provided graphical visualisation of grapevines more severely affected by BN along the borders of the vineyards, mainly in 2011 when disease incidence was high. PPA across the symptomatic plants in the four vineyards confirmed this visual trend: an overall aggregated pattern at small (<10 m) spatial scales (2013) that were more evident later at all spatial scales (0–15 m). Application of this innovative spatial approach based on point and surface pattern analyses allowed the spread and severity of BN to be monitored, to define the dispersal routes of the pathogen. Such data will contribute to better understand the distribution of symptomatic plants over space and time and to define a model for preventive strategies to reduce future infections.