Characterisation of heteroplasmic status at codon 143 of the <i>Botrytis cinerea</i>
 cytochrome <i>b</i>
 gene in a semi-quantitative AS-PCR assay

Hashimoto, Mitsuru; Aoki, Yoshinao; Saito, Seiya; Suzuki, Shunji

doi:10.1002/ps.3867

Cited by 9 publications

(6 citation statements)

References 15 publications

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“…Differences in resistance levels to QiIs may also be due to heteroplasmy. This phenomenon has been reported for QoI resistance in various species like Botrytis cinerea (Hashimoto et al ., 2015), Venturia inaequalis (Villani and Cox, 2014), or Podosphaera xanthii (Vielba-Fernández et al ., 2018). The existence of a stable heteroplasmic state in Z. tritici has never been described but cannot be excluded.…”

Section: Discussionsupporting

confidence: 73%

Directed evolution predicts cytochrome b G37V target site modification as probable adaptive mechanism towards the QiI fungicide fenpicoxamid in Zymoseptoria tritici

Fouché

Michel

Lalève

et al. 2021

Preprint

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Acquired resistance is a threat for antifungal efficacy in medicine and agriculture. The diversity of possible resistance mechanisms, as well as the highly adaptive traits of pathogens make it difficult to predict evolutionary outcomes of treatments. We used directed evolution as an approach to assess the risk of resistance to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici. Fenpicoxamid inhibits complexIII of the respiratory chain at the ubiquinone reduction site (Qi site) of the mitochondrially encoded cytochrome b, a different site than the widely-used strobilurins which the respiratory complex by binding to the ubiquinol oxidation site (Qo site). We identified the G37V change, within the cytochrome b Qi site, as the most likely resistance mechanism to be selected in Z. tritici. This change triggered high fenpicoxamid resistance and halved the enzymatic activity of cytochrome b, despite no significant penalty for in vitro growth. In addition, we identified a negative cross-resistance between isolates harboring G37V or G143A, a Qo site change previously selected by strobilurins. Moreover, double mutants were less resistant to both QiIs and QoIs compared to single mutants. This work is a proof of concept that experimental evolution can be used to predict adaptation to fungicides, and provides new perspectives for the management of QiIs.

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Section: Discussionsupporting

confidence: 73%

Directed evolution predicts cytochrome b G37V target site modification as probable adaptive mechanism towards the QiI fungicide fenpicoxamid in Zymoseptoria tritici

Fouché

Michel

Lalève

et al. 2021

Preprint

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“…The high levels of heteroplasmy observed for the G143A mutation, suggests that resistance to Q o Is in the fields, if present, is not yet complete but may correlate with the proportion of mitochondria in each strain carrying the mutant A143 versus the wild-type G143 cytb allele (Gisi et al, 2002; Lesemann et al, 2006). Although most fungi exhibit qualitative resistance against Q o Is, similar cases of quantitative resistance responses to Q o Is in conjunction with the heteroplasmic levels for the G143A mutation have been described in a number of plant pathogenic fungi, including the apple powdery mildew fungus P. leucotricha (Lesemann et al, 2006), the cucurbit powdery mildew pathogen Podosphaera xanthii (Vielba-Fernandez et al, 2018), the apple scab pathogen V. inaequalis (Villani and Cox, 2014), and the gray mold disease fungus Botrytis cinerea (Hashimoto et al, 2015). The high levels of heteroplasmy for the G143A mutation along with the low number of field samples in which the A143 cytb allele was fixed (8/104; 7.7%) even after a decade of use of Q o Is against tomato powdery mildew in California, could also imply a fitness penalty associated with the G143A mutation in L. taurica .…”

Section: Discussionmentioning

confidence: 92%

Cloning of the Cytochrome b Gene From the Tomato Powdery Mildew Fungus Leveillula taurica Reveals High Levels of Allelic Variation and Heteroplasmy for the G143A Mutation

Mosquera

Chen

Aegerter³

et al. 2019

Front. Microbiol.

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Leveillula taurica is a major pathogen of tomato and several other crops that can cause substantial yield losses in favorable conditions for the fungus. Quinone outside inhibitor fungicides (Q o Is) are routinely used for the control of the pathogen in tomato fields across California, but their recurrent use could lead to the emergence of resistance against these compounds. Here, we partially cloned the cytochrome b gene from L. taurica ( Lt cytb ) and searched within populations of the fungus collected from tomato fields across California for mutations that confer resistance to Q o Is. A total of 21 single nucleotide polymorphisms (SNPs) were identified within a 704 bp fragment of the Lt cytb gene analyzed, of which five were non-synonymous substitutions. Among the most frequent SNPs encountered within field populations of the pathogen was the G143A substitution that confers high levels of resistance against Q o Is in several fungi. The other four amino acid substitutions were novel mutations, whose effect on Q o I resistance is currently unknown. Sequencing of the Lt cytb gene from individual single-cell conidia of the fungus further revealed that most SNPs, including the one leading to the G143A substitution, were present in a heteroplasmic state, indicating the co-existence of multiple mitotypes in single cells. Analysis of the field samples showed that the G143A substitution is predominantly heteroplasmic also within field populations of L. taurica in California, suggesting that Q o I resistance in this fungus is likely to be quantitative rather than qualitative.

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“…In our study, the presence of this intron in an isolate (Bc-111) that also contains mutant (G143A) copies of CytB, could suggest the presence of a "fitness balance", whereby intron and G143A CytB copies are balanced to maintain the lowest possible fitness cost. Mitochondrial heteroplasmy with respect to the G143A mutation has been confirmed in numerous fungal plant pathogens; B. cinerea (Hashimoto et al 2015;Ishii et al 2009), Venturia inaequalis (Michalecka et al 2011;Villani and Cox 2014), Corynespora cassiicola (Ishii et al 2007), Mycovellosiella nattrassii (Ishii et al 2007), Colletotrichum gloeosporioides (Ishii et al 2007), and various powdery mildews (Fraaije et al 2002;Lesemann et al 2006;Mosquera et al 2019;Vielba-Fernández et al 2018). The widespread occurrence of mitochondrial heteroplasmy in fungal plant pathogens could be a mechanism by which some QoI resistant isolates overcome potential fitness costs associated with G143A.…”

Section: Discussionmentioning

confidence: 95%

Fungicide resistance characterised across seven modes of action inBotrytis cinereaisolated from Australian vineyards

Harper

Paton²,

Hall

et al. 2021

Preprint

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Gray mold, caused by Botrytis cinerea, is an economically important disease of grapes in Australia and across grape growing regions worldwide. Control of this disease relies heavily on canopy management and the application of single site fungicides. Fungicide application can lead to the selection of fungicide resistant B. cinerea populations, which has an adverse effect on the chemical control of the disease. Characterising the distribution and severity of resistant B. cinerea populations is needed to inform resistance management strategies. In this study, 725 isolates were sampled from 75 Australian vineyards during 2013 – 2016 and were screened against seven fungicides with different MOAs. The resistance frequencies for azoxystrobin, boscalid, fenhexamid, fludioxonil, iprodione, pyrimethanil and tebuconazole were 5, 2.8, 2.1, 6.2, 11.6, 7.7 and 2.9% respectively. Nearly half of the resistant isolates (43.7%) were resistant to more than one of the fungicides tested. The frequency of vineyards with at least one isolate simultaneously resistant to 1, 2, 3, 4 or 5 fungicides was 19.5, 7.8, 6.5, 10.4 and 2.6%. Resistance was associated with previously published genotypes in CytB (G143A), SdhB (H272R/Y), Erg27 (F412S), Mrr1 (D354Y), Bos1 (I365S, N373S + Q369P, I365S + D757N) and Pos5 (P319A, L412F). Expression analysis was used to characterise fludioxonil resistant isolates exhibiting overexpression (6.3 - 9.6-fold) of the ABC transporter encoded by AtrB (MDR1 phenotype). Novel genotypes were also described in CytB (S611N, D616G) and CytB (P357S). Resistance frequencies were lower when compared to most previously published surveys of both grape and non-grape B. cinerea resistance. Nonetheless, continued monitoring of critical chemical groups used in Australian vineyards is recommended.

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Characterisation of heteroplasmic status at codon 143 of the Botrytis cinerea cytochrome b gene in a semi-quantitative AS-PCR assay

Abstract: It is proposed that the semi-quantitative AS-PCR assay is a reliable tool for the detection of QoI fungicide resistance and the evaluation of homoplasmic/heteroplasmic status at codon 143 of the cytochrome b gene in B. cinerea isolates.

Cited by 9 publications

References 15 publications

Directed evolution predicts cytochrome b G37V target site modification as probable adaptive mechanism towards the QiI fungicide fenpicoxamid in Zymoseptoria tritici

Directed evolution predicts cytochrome b G37V target site modification as probable adaptive mechanism towards the QiI fungicide fenpicoxamid in Zymoseptoria tritici

Cloning of the Cytochrome b Gene From the Tomato Powdery Mildew Fungus Leveillula taurica Reveals High Levels of Allelic Variation and Heteroplasmy for the G143A Mutation

Fungicide resistance characterised across seven modes of action inBotrytis cinereaisolated from Australian vineyards

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