LAMP detection of the genetic element ‘Mona’ associated with DMI resistance in Monilinia fructicola

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BACKGROUND Peach scab, caused by Venturia carpophila, can significantly reduce both the yield and quality of peach fruit. It is difficult to distinguish peach scab from other peach diseases such as black spot and bacterial spot. An efficient assay is needed to identify V. carpophila in order to develop scientific management strategies. RESULTS A set of loop‐mediated isothermal amplification (LAMP) primers was designed based on the internal transcribed spacer (rDNA‐ITS) sequence to detect V. carpophila. Compared with the conventional polymerase chain reaction (PCR) method, the LAMP method not only exhibited higher sensitivity and specificity in the detection of V. carpophila, but also required simpler equipment and less operational time. The minimum detectable concentration of V. carpophila genomic DNA with the LAMP method was 56.6 fg μl–1, which was 100 times lower than with the conventional PCR method. When eight fungal species including V. carpophila (23 isolates from 14 provinces) and one bacterial species were used with LAMP detection, only V. carpophila showed a color change, from brown to yellowish green, and ladder‐like bands in electrophoresis, indicating successful amplification. Moreover, when crude DNAs of peach fruit samples were used in LAMP detection, amplification was observed only from diseased fruits, and not from healthy fruits and the negative control. CONCLUSION The LAMP assay shows simplicity, rapidity, high sensitivity and specificity, and will be useful in distinguishing scab caused by Venturia carpophila from other diseases with similar symptoms. © 2020 Society of Chemical Industry

Section: Discussionsupporting

confidence: 88%

Section: Methodssupporting

confidence: 89%

Section: Optimization Of Lamp Reactionmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Development of a loop‐mediated isothermal amplification method for the rapid detection of Venturia carpophila on peach

Zhou

Fan

Wang

et al. 2020

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Rapid visual detection of benzimidazole resistance in Botrytis cinerea by recombinase polymerase amplification combined with a lateral flow dipstick

Chen

Yuan

Yan

2021

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BACKGROUND Benzimidazole resistance in Botrytis cinerea is related to point mutations in the target β‐tubulin gene (TUB2). Three mutations (E198A, E198K, E198V) at codon 198 account for most of the resistant strains. A rapid on‐site diagnostic assay would be useful to detect the presence and monitor further spread of this resistance mechanism. RESULTS A recombinase polymerase amplification combined with lateral flow detection (RPA–LFD) method was established for the rapid detection of methyl benzimidazole carbamate (MBC) resistance in B. cinerea. Based on the three mutations at TUB2 codon 198, three sets of RPA–LFD primers were designed, and each of these primer sets was able to specifically amplify the DNA containing its corresponding mutation; no amplification was detected with other mutated or wild‐type DNA. The assay was optimized for specificity and sensitivity and was shown to detect the presence of 2 × 102 copies μl−1 of target DNA per reaction within 10 min. DNA from eight other common fungal species of small fruit did not yield a signal. The system worked well over a wide range of temperatures from 25 to 45°C. Crude DNA obtained from boiled mycelium and conidia of symptomatic fruit could be used as templates, which simplified the assay process. CONCLUSION This study developed a novel assay based on RPA–LFD for the rapid and equipment‐free detection of MBC‐resistant isolates. In combination with a simple DNA extraction method, the assay could detect B. cinerea MBC‐resistant isolates even without specialized equipment within 30 min. Considering its specificity, stability and simplicity, the RPA–LFD assay could be a promising tool for rapid on‐site diagnosis of fungicide‐resistant isolates. © 2021 Society of Chemical Industry

Resistance to the DMI fungicide mefentrifluconazole in Monilinia fructicola: risk assessment and resistance basis analysis

Li,

Zhang,

Wang

et al. 2023

BACKGROUNDBrown rot disease, caused by Monilinia fructicola, poses a significant challenge to peach production in China. The efficacy of mefentrifluconazole, a new triazole fungicide, in controlling brown rot in peaches has been remarkable. However, the resistance risk and mechanism associated with this fungicide remain unclear. This study was designed to assess the resistance risk of M. fructicola to mefentrifluconazole and reveal the potential resistance mechanism.RESULTSThe mean median effective concentration (EC50) of 101 M. fructicola isolates to mefentrifluconazole was 0.003 μg mL−1, and the sensitivity exhibited a unimodal distribution. Seven mefentrifluconazole‐resistant mutants were generated from three parental isolates in the laboratory through fungicide adaption. The biological characteristics of the resistant mutants revealed that three of them exhibited enhanced survival fitness compared to the parental isolates, whereas the remaining four mutants displayed reduced survival fitness. Mefentrifluconazole showed strong positive cross‐resistance with fenbuconazole, whereas no cross‐resistance was observed with pyrimethanil, procymidone or pydiflumetofen. No overexpression of MfCYP51 gene was detected in the resistant mutants. Multiple sequence alignment revealed that three resistant mutants (MXSB2‐2, Mf12‐1 and Mf12‐2) had a point mutation (G461S) in MfCYP51 protein. Molecular docking techniques confirmed the contribution of this point mutation to mefentrifluconazole resistance.CONCLUSIONThe risk of M. fructicola developing resistance to mefentrifluconazole is relatively low‐to‐medium and point mutation G461S in MfCYP51 could confer mefentrifluconazole resistance in M. fructicola. This study provided essential data for monitoring the emergence of resistance and developing resistance management strategies for mefentrifluconazole. © 2023 Society of Chemical Industry.