Multiple and multidrug resistance in Botrytis cinerea: molecular mechanisms of MLR/MDR strains in Greece and effects of co-existence of different resistance mechanisms on fungicide sensitivity.

Botrytis cinerea is a high-risk pathogen for fungicide resistance development. Within the fungal populations, strains have developed multiple mutations in different target genes leading to multiple resistance (MLR) or mutations associated with overexpression of efflux transporters leading to multidrug resistance (MDR). These types of resistance are a major threat, and their successful management is a major challenge. The current study was initiated to a) determine frequencies of MLR/MDR strains in populations originating from several crops, b) identify the types of MDR that occur in Greece, and c) determine interactions between MLR and MDR at the level of sensitivity to botryticides. The frequencies of MLR/MDR phenotypes were determined in 515 isolates subjected to bioassays using discriminatory concentrations of thiophanate-methyl, iprodione, cyprodinil, fenhexamid, boscalid, fluopyram, fludioxonil, pyraclostrobin, and tolnaftate. Interestingly, 7.8% and 31.3% of isolates from strawberry and rootstock seedlings were resistant to every single fungicide class, while MDR phenotypes from strawberries, rootstocks, and tomatoes accounted for 26%, 87%, and 13.4%, respectively. The MLR and MDR isolates were further molecularly analyzed regarding genes erg27, sdhB, Bcpos5, and Mrr1, responsible for resistance to fenhexamid, boscalid and fluopyram, cyprodinil, and MDR, respectively. The different mutations' presence was determined along with a new mutation in Mrr1 leading to MDR. MDR isolates were characterized as MDR1 or MDR1h based on the presence of a 3-bp deletion in Mrr1. MDR1h was predominant in isolates from rootstocks and MDR1 from tomatoes and strawberries, whereas the most frequent target-site mutations were F412S (erg27), H272R (sdhB), and L412F (Bcpos5). To determine whether the accumulation of target-site mutations along with MDR mutations exhibits an additive effect concerning fungicide resistance, the sensitivity of isolates possessing the predominant target-site mutations was calculated in both the presence and the absence of MDR-associated mutations. EC50 in cyprodinil and boscalid increased to about twofold in the presence of MDR mutations, while there was no difference for fenhexamid. In conclusion, MLR/MDR frequencies are notably high in heavily treated crops in Greece, and the combination of MLR and MDR mutations leads to even higher fungicide resistance levels, highlighting the importance of resistance management.

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.

Under Pressure: A Comparative Study of Botrytis cinerea Populations from Conventional and Organic Farms in Cyprus and Greece.

The highly heterogeneous nature of Botrytis cinerea provides adaptive benefits to variable environmental regimes. Disentangling pathogen population structure in anthropogenic agroecosystems is crucial to designing more effective management schemes. Herein, we studied how evolutionary forces exerted in different farming systems, in terms of agrochemicals-input, shape B. cinerea populations. In total, 360 B. cinerea isolates were collected from conventional and organic, strawberry and tomato farms in Cyprus and Greece. The occurrence and frequency of sensitivities to seven botryticides were estimated. Results highlighted widespread fungicide resistance in conventional farms since only 15.5% of the isolates were sensitive. A considerable frequency of fungicide-resistant isolates was also detected in the organic farms (14.9%). High resistance frequencies were observed for boscalid (67.7%), pyraclostrobin (67.3%), cyprodinil (65.9%), and thiophanate-methyl (61.4%) in conventional farms, while high levels of multiple fungicide resistance were also evident. Furthermore, B. cinerea isolates were genotyped using a set of seven microsatellite markers (simple sequence repeat [SSR] markers). Index of association analyses (Ia and rBarD) suggest asexual reproduction of the populations, even though the mating-type idiomorphs were equally distributed, indicating frequency-dependent selection. Fungicide resistance was correlated with farming systems across countries and crops, while SSRs were able to detect population structure associated with resistance to thiophanate-methyl, pyraclostrobin, boscalid, and cyprodinil. The expected heterozygosity in organic farms was significantly higher than in conventional, suggesting the absence of selective pressure that may change the allelic abundance in organic farms. However, genetic variance among strawberry and tomato populations was high, ranking host specificity higher than other selection forces studied.

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.

Whole Genome Sequencing and Root Colonization Studies Reveal Novel Insights in the Biocontrol Potential and Growth Promotion by Bacillus subtilis MBI 600 on Cucumber.

Bacillus spp. MBI 600 is a gram-positive bacterium and is characterized as a PGPR strain involved in plant growth promotion and control of various plant pathogens which has recently been introduced into the agricultural practice. In this study we performed a Next Generation Sequencing analysis, to analyze the full genome of this microorganism and to characterize it taxonomically. Results showed that MBI 600 strain was phylogenetically close to other Bacillus spp. strains used as biocontrol agents and identified as B. subtilis. GOG analysis showed clusters contributed to secondary metabolites production such as fengycin and surfactin. In addition, various genes which annotated according to other plant-associated strains, showed that play a main role in nutrient availability from soil. The root colonization ability of MBI 600 strain was analyzed in vivo with a yellow fluorescence protein (yfp) tag. Confocal laser scanning microscopy of cucumber roots treated with yfp-tagged MBI 600 cells, revealed that the strain exhibits a strong colonization ability of cucumber roots, although it is affected significantly by the growth substrate of the roots. In vitro and in planta experiments with MBI 600 strain and F. oxysporum f.sp. radicis cucumerinum and P. aphanidernatum, showed a high control ability against these soilborne pathogens. Overall, our study demonstrates the effectiveness of MBI 600 in plant growth promotion and antagonism against different pathogens, highlighting the use of this microorganism as a biocontrol agent.