Resistance of Black Aspergilli Species from Grape Vineyards to SDHI, QoI, DMI, and Phenylpyrrole Fungicides.

Fungicide applications constitute a management practice that reduces the size of fungal populations and by acting as a genetic drift factor, may affect pathogen evolution. In a previous study, we showed that the farming system influenced the population structure of the Aspergillus section Nigri species in Greek vineyards. The current study aimed to test the hypothesis that the differences in the population structure may be associated with the selection of fungicide-resistant strains within the black aspergilli populations. To achieve this, we determined the sensitivity of 102, 151, 19, and 22 for the A. uvarum, A. tubingensis, A. niger, and A. carbonarious isolates, respectively, originating either from conventionally-treated or organic vineyards to the fungicides fluxapyroxad-SDHIs, pyraclostrobin-QoIs, tebuconazole-DMIs, and fludioxonil-phenylpyrroles. The results showed widespread resistance to all four fungicides tested in the A. uvarum isolates originating mostly from conventional vineyards. In contrast, all the A. tubingensis isolates tested were sensitive to pyraclostrobin, while moderate frequencies of only lowly resistant isolates were identified for tebuconazole, fludioxonil, and fluxapyroxad. Sequencing analysis of the corresponding fungicide target encoding genes revealed the presence of H270Y, H65Q/S66P, and G143A mutations in the sdhB, sdhD, and cytb genes of A. uvarum resistant isolates, respectively. No mutations in the Cyp51A and Cyp51B genes were detected in either the A. uvarum or A. tubingensis isolates exhibiting high or low resistance levels to DMIs, suggesting that other resistance mechanisms are responsible for the observed phenotype. Our results support the initial hypothesis for the contribution of fungicide resistance in the black aspergilli population structure in conventional and organic vineyards, while this is the first report of A. uvarum resistance to SDHIs and the first documentation of H270Y or H65Q/S66P mutations in sdhB, sdhD, and of the G143A mutation in the cytb gene of this fungal species.

Grapevine Trunk Diseases in Greece: Disease Incidence and Fungi Involved in Discrete Geographical Zones and Varieties.

A three-year survey was conducted to estimate the incidence of grapevine trunk diseases (GTDs) in Greece and identify fungi associated with the disease complex. In total, 310 vineyards in different geographical regions in northern, central, and southern Greece were surveyed, and 533 fungal strains were isolated from diseased vines. Morphological, physiological and molecular (5.8S rRNA gene-ITS sequencing) analyses revealed that isolates belonged to 35 distinct fungal genera, including well-known (e.g., Botryosphaeria sp., Diaporthe spp., Eutypa sp., Diplodia sp., Fomitiporia sp., Phaeoacremonium spp., Phaeomoniella sp.) and lesser-known (e.g., Neosetophoma sp., Seimatosporium sp., Didymosphaeria sp., Kalmusia sp.) grapevine wood inhabitants. The GTDs-inducing population structure differed significantly among the discrete geographical zones. Phaeomoniella chlamydospora (26.62%, n = 70), Diaporthe spp. (18.25%, n = 48) and F. mediterranea (10.27%, n = 27) were the most prevalent in Heraklion, whereas D. seriata, Alternaria spp., P. chlamydospora and Fusarium spp. were predominant in Nemea (central Greece). In Amyntaio and Kavala (northern Greece), D. seriata was the most frequently isolated species (>50% frequency). Multi-genes (rDNA-ITS, LSU, tef1-α, tub2, act) sequencing of selected isolates, followed by pathogenicity tests, revealed that Neosetophoma italica, Seimatosporium vitis, Didymosphaeria variabile and Kalmusia variispora caused wood infection, with the former being the most virulent. To the best of our knowledge, this is the first report of N. italica associated with GTDs worldwide. This is also the first record of K. variispora, S. vitis and D. variabile associated with wood infection of grapevine in Greece. The potential associations of disease indices with vine age, cultivar, GTD-associated population structure and the prevailing meteorological conditions in different viticultural zones in Greece are presented and discussed.

Grapevine wood microbiome analysis identifies key fungal pathogens and potential interactions with the bacterial community implicated in grapevine trunk disease appearance.

BACKGROUND: Grapevine trunk diseases (GTDs) is a disease complex caused by wood pathogenic fungi belonging to genera like Phaeomoniella, Phaeoacremonium, Fomitiporia, Eutypa and members of the family Botryosphaeriaceae. However, the co-occurrence of these fungi in symptomatic and asymptomatic vines at equivalent abundances has questioned their role in GTDs. Hence, we still lack a good understanding of the fungi involved in GTDs, their interactions and the factors controlling their assemblage in vines. We determined the fungal and bacterial microbiome in wood tissues of asymptomatic and symptomatic vines of three main Greek cultivars (Agiorgitiko, Xinomavro, Vidiano), each cultivated in geographically distinct viticultural zones, using amplicon sequencing. RESULTS: We noted that cultivar/biogeography (lumped factor) was the strongest determinant of the wood fungal microbiome (p < 0.001, 22.7%), while GTD symptoms condition had a weaker but still significant effect (p < 0.001, 3.5%), being prominent only in the cultivar Xinomavro. Several fungal Amplicon Sequence Variants (ASVs), reported as GTD-associated pathogens like Kalmusia variispora, Fomitiporia spp., and Phaemoniella chlamydosporα (most dominant in our study), were positively correlated with symptomatic vines in a cultivar/viticultural zone dependent manner. Random Forest analysis pointed to P. chlamydosporα, K. variispora, A. alternata and Cladosporium sp., as highly accurate predictors of symptomatic vines (0% error rate). The wood bacterial microbiome showed similar patterns, with biogeography/cultivar being the main determinant (p < 0.001, 25.5%) of its composition, followed by the GTD status of vines (p < 0.001, 5.2%). Differential abundance analysis revealed a universal positive correlation (p < 0.001) of Bacillus and Streptomyces ASVs with asymptomatic vines. Network analysis identified a significant negative co-occurrence network between these bacterial genera and Phaemoniella, Phaeoacrominum and Seimatosporium. These results point to a plant beneficial interaction between Bacillus/Streptomyces and GTD pathogens. CONCLUSIONS: Our study (a) provides evidence that GTD symptomatic plants support a wood fungal microbiome, showing cultivar and biogeography-dependent patterns, that could be used as a proxy to distinguish between healthy and diseased vines, (b) points to strong interactions between the bacterial and fungal wood microbiome in asymptomatic vines that should be further pursued in the quest for discovery of novel biocontrol agents.

Unraveling Interactions of the Necrotrophic Fungal Species Botrytis cinerea With 1-Methylcyclopropene or Ozone-Treated Apple Fruit Using Proteomic Analysis.

Gray mold caused by the necrotrophic fungus Botrytis cinerea is one of the major postharvest diseases of apple fruit. The exogenous application of 1-methylcyclopropene (1-MCP) and gaseous ozone (O 3) is commonly used to ensure postharvest fruit quality. However, the effect of these treatments on the susceptibility of apple fruit to postharvest pathogens remains largely unknown. Herein, the effect of O 3 and 1-MCP treatments on the development of gray mold on apple fruit (cv. "Granny Smith") was investigated. Artificially inoculated apple fruits, treated or not with 1-MCP, were subjected for 2 months to cold storage [0°C, relative humidity (RH) 95%] either in an O3-enriched atmosphere or in a conventional cold chamber. Minor differences between 1-MCP-treated and control fruits were found in terms of disease expression; however, exposure to ozone resulted in a decrease of disease severity by more than 50% compared with 1-MCP-treated and untreated fruits. Proteomic analysis was conducted to determine proteome changes in the mesocarp tissue of control and 1-MCP- or O3-treated fruits in the absence or in the presence of inoculation with B. cinerea. In the non-inoculated fruits, 26 proteins were affected by 1-MCP, while 51 proteins were altered by ozone. Dynamic changes in fruit proteome were also observed in response to B. cinerea. In O3-treated fruits, a significant number of disease/defense-related proteins were increased in comparison with control fruit. Among these proteins, higher accumulation levels were observed for allergen, major allergen, ACC oxidase, putative NBS-LRR disease resistance protein, major latex protein (MLP)-like protein, or 2-Cys peroxiredoxin. In contrast, most of these proteins were down-accumulated in 1-MCP-treated fruits that were challenged with B. cinerea. These results suggest that ozone exposure may contribute to the reduction of gray mold in apple fruits, while 1-MCP was not effective in affecting this disease. This is the first study deciphering differential regulations of apple fruit proteome upon B. cinerea infection and postharvest storage treatments, underlying aspects of host response related to the gray mold disease.

Identification and mycotoxigenic capacity of fungi associated with pre- and postharvest fruit rots of pomegranates in Greece and Cyprus.

Pre- and postharvest fruit rots of fungal origin are an important burden for the pomegranate industry worldwide, affecting the produce both quantitatively and qualitatively. During 2013, local orchards were surveyed and 280 fungal isolates from Greece (GR) and Cyprus (CY) were collected from pomegranates exhibiting preharvest rot symptoms, and additional 153 isolates were collected postharvest from cold-stored fruit in GR. Molecular identification revealed that preharvest pomegranate fruit rots were caused predominately by species of the genera Aspergillus (Aspergillus niger and Aspergillus tubingensis) and Alternaria (Alternaria alternata, Alternaria tenuissima, and Alternaria arborescens). By contrast, postharvest fruit rots were caused mainly by Botrytis spp. and to a lesser extent by isolates of Pilidiella granati and Alternaria spp. Considering that a significant quota of the fungal species found in association with pomegranate fruit rots are known for their mycotoxigenic capacity in other crop systems, their mycotoxin potential was examined. Alternariol (AOH), alternariol monomethyl-ether (AME) and tentoxin (TEN) production was estimated among Alternaria isolates, whereas ochratoxin A (OTA) and fumonisin B2 (FB2) production was assessed within the black aspergilli identified. Overall in both countries, 89% of the Alternaria isolates produced AOH and AME in vitro, while TEN was produced only by 43.9%. In vivo production of AOH and AME was restricted to 54.2% and 31.6% of the GR and CY isolates, respectively, while none of the isolates produced TEN in vivo. Among black aspergilli 21.7% of the GR and 17.8% of the CY isolates produced OTA in vitro, while in vivo OTA was detected in 8.8% of the isolates from both countries. FB2 was present in vitro in 42.0% of the GR and 22.2% of the CY isolates, while in vivo the production was limited to 27.5% and 4.5% of the GR and the CY isolates, respectively. Our data imply that mycotoxigenic Alternaria and Aspergillus species not only constitute a significant subset of the fungal population associated with pomegranate fruit rots responsible for fruit deterioration, but also pose a potential health risk factor for consumers of pomegranate-based products.

Determination of mycotoxins in pomegranate fruits and juices using a QuEChERS-based method.

A rapid and accurate analytical method for the determination of three Alternaria mycotoxins (alternariol, alternariol monomethyl ether, and tentoxin) in pomegranate samples (fruits and juices) was developed and validated. The overall average recoveries ranged for 82.0-109.4% and the relative standard deviations were from 1.2% to 10.9%. The optimized and validated method was applied to detect the presence of the target mycotoxins in real samples (fruits and juices) purchased from Greek markets. Mycotoxins were not found in any of the analyzed samples. Also, artificially inoculated pomegranate fruits with six different Alternaria alternata species complex isolates, known to produce the target mycotoxins on pure cultures, were analyzed and alternariol concentrations found ranged from 0.3 to 50.5 µg/g, alternariol monomethyl ether from 0.5 to 32.3 µg/g, while tentoxin was not detected. The developed analytical method can be used for the routine monitoring of the major Alternaria mycotoxins in pomegranates.