Epidemiological Studies of Pan-Azole Resistant Aspergillus fumigatus Populations Sampled during Tulip Cultivation Show Clonal Expansion with Acquisition of Multi-Fungicide Resistance as Potential Driver

Pan-azole resistant isolates are found in clinical and environmental Aspergillus fumigatus (Af) populations. Azole resistance can evolve in both settings, with Af directly targeted by antifungals in patients and, in the environment, Af unintendedly exposed to fungicides used for material preservation and plant disease control. Resistance to non-azole fungicides, including methyl benzimidazole carbamates (MBCs), quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs), has recently been reported. These fungicide groups are not used in medicine but can play an important role in the further spread of pan-azole resistant genotypes. We investigated the multi-fungicide resistance status and the genetic diversity of Af populations sampled from tulip field soils, tulip peel waste and flower compost heaps using fungicide sensitivity testing and a range of genotyping tools, including STRAf typing and sequencing of fungicide resistant alleles. Two major clones were present in the tulip bulb population. Comparisons with clinical isolates and literature data revealed that several common clonal lineages of TR34/L98H and TR46/Y121F/T289A strains that have expanded successfully in the environment have also acquired resistance to MBC, QoI and/or SDHI fungicides. Strains carrying multiple fungicide resistant alleles have a competitive advantage in environments where residues of multiple fungicides belonging to different modes of action are present.

Fungicide resistance in Venturia effusa, cause of pecan scab: current status and practical implications

Pecan scab, caused by Venturia effusa, is the most economically damaging disease of pecan in the southeastern U.S. and annual epidemics are most effectively managed through multiple fungicide applications. The fungicide applications can be the single greatest operating cost for commercial growers and the return on that investment is impacted by fungicide resistance. Venturia effusa produces multiple generations of conidia per season, exhibits substantial genetic diversity, overwinters as stromata in the tree, and is under immense selection from the applied fungicides, all of which lead to a high risk for developing fungicide resistance. Since the mid-1970s, resistance or reduced sensitivity has been observed in isolates of V. effusa to the methyl benzimidazole carbamates, demethylation inhibitors, quinone outside inhibitors, organotin compounds and the guanidines. Over the last ten years, several studies have been conducted that have improved both scab management and fungicide resistance management in V. effusa. The aim of this review is to summarize recent developments in our understanding of fungicide resistance in V. effusa in the context of scab management in southeastern pecan orchards. The history, modes of action, general use of the labelled fungicides, and mechanisms and stability of fungicide resistance in V. effusa are discussed; conclusions and future research priorities are also presented.

Sensitivity of the Colletotrichum acutatum Species Complex From Apple Trees in Brazil to Dithiocarbamates, Methyl Benzimidazole Carbamates, and Quinone Outside Inhibitor Fungicides

Glomerella leaf spot (GLS) and bitter rot (BR) on apples are often caused by Colletotrichum acutatum in Paraná State, Brazil. GLS control is difficult because of its rapid development, with an incubation period of only 2 days under favorable conditions. Therefore, producers use successive fungicide applications every season; however, failure to control GLS has been commonly reported. The objectives of this study were to determine the sensitivity of isolates of the C. acutatum species complex obtained from apple orchards in Brazil to mancozeb, thiophanate-methyl, and azoxystrobin fungicides. Isolates from the different parts of the plant (leaves, flowers, buds, and twigs) and cultivars (Gala and Eva) showed different levels of sensitivity to mancozeb, thiophanate-methyl, and azoxystrobin. For mancozeb, the frequencies of isolates were 25% highly resistant, 50% low-resistance, and 25% sensitive. For thiophanate-methyl, the frequencies of isolates were 72.2% highly resistant, 11.1% resistant, and 16.7% moderately resistant. For azoxystrobin, the frequencies of isolates were 11.1% highly resistant, 5.6% resistant, and 83.3% sensitive. Interestingly, no mutations in the β-tubulin and cytochrome b genes were observed in any of the isolates resistant to thiophanate-methyl and azoxystrobin fungicides.

Sensitivity of Monilinia fructicola from Peach Farms in China to Four Fungicides and Characterization of Isolates Resistant to Carbendazim and Azoxystrobin

Brown rot of peach caused by Monilinia fructicola can cause considerable preharvest and postharvest losses in China. Fungicides are increasingly utilized to minimize such losses. Eighty isolates of M. fructicola were collected from commercial peach orchards located in five provinces in China and the sensitivity to carbendazim, azoxystrobin, tebuconazole, and boscalid was determined. Resistance to carbendazim was detected only in the Yunnan province in 15 of 16 isolates. Characterization of carbendazim-resistant isolates revealed stable resistance, no fitness penalty, and negative cross resistance to diethofencarb. Resistant isolates produced disease symptoms on detached fruit sprayed with label rates of formulated carbendazim and possessed the amino acid mutation E198A in β-tubulin. Resistance to azoxystrobin was detected in 3 of 10 isolates from Fujian. In contrast to carbendazim resistance, however, azoxystrobin resistance was unstable, associated with a fitness penalty, and not associated with mutations in the target gene cytochrome b. The concentration at which mycelial growth is inhibited 50% (EC50) values of the azoxystrobin-sensitive isolates were 0.02 to 1.94 μg/ml, with a mean value of 0.54 μg/ml. All isolates were sensitive to tebuconazole, with a mean EC50 value of 0.03 μg/ml. The EC50 values for boscalid were 0.01 to 3.85 μg/ml, with a mean value of 1.02 μg/ml. Our results indicate that methyl benzimidazole carbamates (MBCs), quionon outside inhibitors, demethylation inhibitor fungicides, and succinate dehydrogenase inhibitors are likely to be very effective in controlling brown rot in many peach production areas in China, but that resistance to MBCs is emerging.

Emergence of Prochloraz-Resistant Populations of Calonectria pauciramosa and Calonectria polizzii in Ornamental Nurseries of Southern Italy

Management of Calonectria spp. infections in nurseries requires scheduled fungicide applications, particularly with methyl benzimidazole carbamates (MBCs) and sterol demethylation inhibitors (DMIs). Due to rising concerns about the occurrence of MBC resistance in different Calonectria populations and variability in prochloraz efficacy in controlling these pathogens, a detailed study on prochloraz sensitivity distributions of Calonectria isolates belonging to the Calonectria scoparia complex was carried out. In total, 105 isolates collected in two distinct periods (1993 to 1996 and 2005 to 2009) were analyzed for prochloraz sensitivity. Based on DNA sequencing and phylogenetic analyses of β-tubulin, histone H3, and translation elongation factor-1α gene sequences, 69 and 36 isolates were identified as C. pauciramosa and C. polizzii, respectively. The isolates collected more recently (group B) had a reduced prochloraz sensitivity, as indicated by greater values for the effective dose to reduce growth by 50% than those collected earlier (group A). The reduced sensitivity detected in vitro corresponded to partial loss of fungicide efficacy in controlling infections in red clover and feijoa under controlled and semi-field conditions, respectively. Frequent prochloraz application in nurseries for controlling Calonectria spp. infections is discouraged.