The rapid emergence of resistance in plant pathogens to the limited number of chemical classes of fungicides challenges sustainability and profitability of crop production worldwide. Understanding mechanisms underlying fungicide resistance facilitates monitoring of resistant populations at large-scale, and can guide and accelerate the development of novel fungicides. A majority of modern fungicides act to disrupt a biochemical function via binding a specific target protein in the pathway. While target-site based mechanisms such as alternation and overexpression of target genes have been commonly found to confer resistance across many fungal species, it is not uncommon to encounter resistant phenotypes without altered or overexpressed target sites. However, such non-target site mechanisms are relatively understudied, due in part to the complexity of the fungal genome network. This type of resistance can oftentimes be transient and noninheritable, further hindering research efforts. In this review, we focused on crop pathogens and summarized reported mechanisms of resistance that are otherwise related to target-sites, including increased activity of efflux pumps, metabolic circumvention, detoxification, standing genetic variations, regulation of stress response pathways, and single nucleotide polymorphisms (SNPs) or mutations. In addition, novel mechanisms of drug resistance recently characterized in human pathogens are reviewed in the context of nontarget-directed resistance.

Few fungicides are effective against anthracnose, caused by Colletotrichum spp., and emerging resistance makes the search for chemical alternatives more relevant. Isolates of the Colletotrichum acutatum species complex were collected from South Carolina and Georgia peach orchards and phylogenetic analysis of the combined internal transcribed spacer region, glyceraldehyde-3-phosphate dehydrogenase, and β-tubulin gene sequences separated the isolates into C. nymphaeae and C. fioriniae. The sensitivity of these and three other previously reported Colletotrichum spp. from peach, including C. fructicola, C. siamense, and C. truncatum, to demethylation inhibitor (DMI) fungicides difenoconazole, propiconazole, tebuconazole, metconazole, flutriafol, and fenbuconazole was determined based upon mycelial growth inhibition. C. truncatum was resistant to tebuconazole, metconazole, flutriafol, and fenbuconazole and C. nymphaeae was resistant to flutriafol and fenbuconazole based on 50% effective concentration (EC₅₀) values >100 μg/ml. C. fructicola and C. siamense were sensitive to all DMI fungicides (EC₅₀values of 0.2 to 13.1 μg/ml). C. fioriniae subgroup 2 isolates were less sensitive to DMI fungicides (EC₅₀values of 0.5 to 16.2 μg/ml) compared with C. fioriniae subgroup 1 (EC₅₀values of 0.03 to 2.1 μg/ml). Difenoconazole and propiconazole provided the best control efficacy in vitro to all five species, with EC₅₀values of 0.2 to 2.7 μg/ml. Tebuconazole and metconazole were effective against all Colletotrichum spp., except for C. truncatum. The strong in vitro activity of some DMI fungicides against Colletotrichum spp. may be exploited for improved anthracnose disease management of peach.

Resistance to multiple chemical classes of fungicides in Botrytis cinerea isolates from eastern United States strawberry fields is common and strategies to control them are needed. In this study, we compared fitness and competitive ability of eight sensitive isolates (S), eight isolates resistant to five or six chemical classes of fungicides but not to phenylpyrroles (5CCR), and eight isolates resistant to six or seven chemical classes including phenylpyrroles (6CCR/MDR1h). The latter included the MDR1h phenotype due to overexpression of atrB based on Δ497V/L in mrr1. The 6CCR/MDR1h isolates grew more slowly at 4°C on potato dextrose agar, and both 5CCR and 6CCR/MDR1h isolates were hypersensitive to osmotic stress compared with S isolates. In contrast, no differences were found in oxidative sensitivity, aggressiveness, and spore production in vivo, and sclerotia production and viability in vitro. In competition experiments, the 5CCR and 6CCR/MDR1h isolates were both outcompeted by S isolates and 6CCR/MDR1h isolates were outcompeted by 5CCR isolates in the absence of fungicide pressure. Under selective pressure of a fludioxonil/pyraclostrobin rotation, the 6CCR/MDR1h isolates dominated after coinoculation with 5CCR and S isolates. The competitive disadvantage of 5CCR and especially 6CCR/MDR1h isolates suggest that, in the absence of fungicide selection pressure, S isolates may reduce inoculum potential of multifungicide-resistant isolates under field conditions.

Anthracnose disease, caused by Colletotrichum truncatum, affects marketable yield during preharvest production and postharvest storage of fruits and vegetables worldwide. Demethylation inhibitor (DMI) fungicides are among the very few chemical classes of single-site mode of action fungicides that are effective in controlling anthracnose disease. However, some species are inherently resistant to DMIs and more information is needed to understand this phenomenon. Isolates of C. truncatum were collected from the United States and China from peach, soybean, citrus, and begonia and sensitivity to six DMIs (difenoconazole, propiconazole, metconazole, tebuconazole, flutriafol, and fenbuconazole) was determined. Compared with DMI sensitive isolates of C. fructicola, C. siamense, and C. fioriniae (EC₅₀ value ranging from 0.03 to 16.2 µg/ml to six DMIs), C. truncatum and C. nymphaeae were resistant to flutriafol and fenbuconazole (with EC₅₀ values of more 50 µg/ml). Moreover, C. truncatum was resistant to tebuconazole and metconazole (with resistance factors of 27.4 and 96.0) and displayed reduced sensitivity to difenoconazole and propiconazole (with resistance factors of 5.1 and 5.2). Analysis of the Colletotrichum spp. genome revealed two potential DMI targets, CYP51A and CYP51B, that putatively encode P450 sterol 14α-demethylases. Both genes were identified and sequenced from C. truncatum and other species and no correlation between CYP51 gene expression levels and fungicide sensitivity was found. Four amino acid variations L208Y, H238R, S302A, and I366L in CYP51A, and three variations H373 N, M376L, and S511T in CYP51B correlated with the DMI resistance phenotype. CYP51A structure model analysis suggested the four alterations may reduce azole affinity. Likewise, CYP51B structure analysis suggested the H373 N and M376L variants may change the conformation of the DMI binding pocket, thereby causing differential sensitivity to DMI fungicides in C. truncatum.

In total, 112 Monilinia spp. single-spore isolates were collected from plum fruit (Prunus salicina) symptomatic for brown rot disease from Yunnan, Hubei, and Zhejiang provinces and Chongqing municipality, China between 2012 and 2014. Three distinct colony morphologies (phenotypes) were observed on potato dextrose agar and two isolates per phenotype were selected for further analysis. Colony color, colony shape, conidia size, number of germ tubes per conidia, and pathogenicity on plum were investigated. The ribosomal internal transcribed spacer regions 1 and 2 as well as a polymerase chain reaction-based method that amplified fragments of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) and β-tubulin (TUB2) genes were used to identify the isolates to the species level. The three phenotypes were identified to be three different species: Monilinia fructicola, Monilia mumecola, and Monilia yunnanensis. Phylogenetic analysis based on G3PDH and TUB2 nucleotide sequences revealed that isolates within species clustered together regardless of host or geographical origin, suggesting that these factors did not play an important role for the evolutionary separation of the described species.