# Sensitivity to demethylation‐inhibiting fungicides and induced expression of CYP51 associated with tebuconazole resistance in Alternaria species on mandarin in Brazil

## TL;DR

This study shows that some Alternaria fungi causing citrus disease have become resistant to tebuconazole, a fungicide, due to increased CYP51 gene activity, and suggests alternative fungicides for better control.

## Contribution

The study identifies CYP51 overexpression as a mechanism of tebuconazole resistance in Alternaria and evaluates fungicide efficacy for resistance management.

## Key findings

- Tebuconazole-resistant isolates overexpress the CYP51 gene, which is linked to resistance.
- Difenoconazole and mefentrifluconazole remain effective against resistant isolates.
- Cross-resistance among DMI fungicides may limit disease management options.

## Abstract

Alternaria brown spot (ABS), caused by Alternaria spp., is an important disease affecting mandarin production in several citrus‐growing regions worldwide. In Brazil, ABS has become increasingly problematic in Paraná State, where high disease pressure and reports of control failures have raised concerns about the long‐term efficacy of fungicides. Demethylation inhibitor (DMI) fungicides are commonly used in ABS spray programs; however, the rising frequency of resistant Alternaria phenotypes may compromise their efficacy and sustainability. This study aimed to (i) assess DMI sensitivity and cross‐resistance in Alternaria spp., (ii) investigate CYP51 expression differences between tebuconazole‐sensitive and tebuconazole‐resistant isolates, and (iii) assess the practical efficacy of tebuconazole in detached‐leaf assays.

Fifty‐four isolates were grouped into three DMI phenotypes for resistance (R) or sensitivity (S): DIFSTEBS, DIFSTEBR, and DIFRTEBR. A. longipes was the most and A. arborescens the least sensitive species. Mean EC50 values for difenoconazole, tebuconazole, and mefentrifluconazole ranged from 0.29 to 4.00, 2.70 to 58.30, and 0.14 to 2.30 μg mL−1, respectively. Cross‐resistance was observed among the three DMIs. CYP51 was significantly overexpressed in tebuconazole‐resistant (TEB‐resistant) isolates. Correlated CYP51 expression with the EC50 values demonstrated the association between up‐regulation and resistance intensity in TEB‐resistant isolates. Tebuconazole reduced ABS severity caused by sensitive Alternaria isolates, but this fungicide was ineffective to control ABS on leaves inoculated with resistant isolate, indicating practical resistance under bioassay conditions.

CYP51 overexpression contributes to DMI resistance in Alternaria spp., and practical and cross‐resistance among fungicides may limit ABS management strategies. Mefentrifluconazole and difenoconazole are effective fungicides for resistance management in Brazilian mandarin orchards. © 2025 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Tebuconazole‐resistant isolates overexpress CYP51 and, when tested at field rates, did not reduce ABS severity, whereas difenoconazole and mefentrifluconazole remained effective.