Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Nov 15;7:1815.
doi: 10.3389/fmicb.2016.01815. eCollection 2016.

Detection of sdh B Gene Mutations in SDHI-Resistant Isolates of Botrytis cinerea Using High Resolution Melting (HRM) Analysis

Affiliations
Free PMC article

Detection of sdh B Gene Mutations in SDHI-Resistant Isolates of Botrytis cinerea Using High Resolution Melting (HRM) Analysis

Anastasios Samaras et al. Front Microbiol. .
Free PMC article

Abstract

Botrytis cinerea, is a high risk pathogen for fungicide resistance development. Pathogen' resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I, and H272L/R/Y mutations. Based on the sequence of sdhB subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant's DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the four mutations located at codon 225, the N230I mutation, the three mutations located in codon 272, and the non-mutated isolates (isolates of wild-type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdhB mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping-off symptoms. HRM analysis data were compared with a standard PIRA-PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I, and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate, and rapid identification of several sdhB mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of anti-resistance strategies implemented in crops heavily treated with botryticides.

Keywords: boscalid; fungicide resistance; gray mold; strawberry; succinate dehydrogenase gene.

Figures

FIGURE 1
FIGURE 1
Multiple sequence alignment of nucleotides (A) and amino acids (B) of the sdhB gene in SDHIs resistant isolates of Botrytis cinerea with eight different mutations in subunit B and one sensitive isolate.
FIGURE 2
FIGURE 2
High resolution melting (HRM) analysis using the primers SDHB-Fw and SDHB-Rev for the discrimination of SDHI-resistant B. cinerea isolates possessing the sdhB mutations P225H/T/F/L, H272R/Y/L, and N230I. (A) Normalized HRM curves of isolates with different mutations, (B) Difference curves of HRM using a sensitive isolate as the reference baseline curve.
FIGURE 3
FIGURE 3
Melting profile of four fungal pathogens, using the specific primers SDHB-Fw and SDHB-Rev with genomic DNA of B. cinerea, Aspergillus niger, Rhizopus stolonifer, and Alternaria alternata.
FIGURE 4
FIGURE 4
Sensitivity of HRM assay to detect DNA of a B. cinerea isolate possessing the H272R mutation (A) and a wild-type isolate (B). Difference curves were generated using 0.1–10 ng DNA per reaction tube. 0.001 ng of genomic DNA from B. cinerea was used as reference baseline curve.
FIGURE 5
FIGURE 5
Normalized HRM analysis curves of two B. cinerea isolates possessing P225F (A) and H272R (B) mutations, generated from genomic DNA extracted from a fungal culture and from artificially inoculated strawberry fruit tissue.
FIGURE 6
FIGURE 6
Representative HRM analysis curves of four B. cinerea genotypes generated from unknown field samples. (A) Normalized HRM curves of DNA samples with H27R/Y and N230I mutations and one non-mutated DNA sample (wild-type), (B) Difference graph of HRM curves using the non-mutated (wild-type) sample as the reference baseline curve.

Similar articles

See all similar articles

Cited by 2 articles

References

    1. Amiri A., Heath S. M., Peres N. A. (2014). Resistance to fluopyram, fluxapyroxad, and penthiopyrad in Botrytis cinerea from strawberry. Plant Dis. 98 532–539. 10.1094/PDIS-07-13-0753-RE - DOI
    1. Bardas G. A., Veloukas T., Koutita O., Karaoglanidis G. S. (2010). Multiple resistance of Botrytis cinerea from kiwifruit to SDHIs, QoIs and fungicides of other chemical groups. Pest Manag. Sci. 66 966–973. 10.1002/ps.1968 - DOI - PubMed
    1. Bester R., Jooste A. E., Maree H. J., Burger J. T. (2012). Realtime RT-PCR high-resolution melting curve analysis and multiplex RT-PCR to detect and differentiate grapevine leafroll-associated virus 3 variant groups I, II, III and VI. Virol. J. 9:219 10.1186/1743-422x-9-219 - DOI - PMC - PubMed
    1. Cecchini G. (2003). Function and structure of complex II of the respiratorychain. Annu. Rev. Biochem. 72 77–109. 10.1146/annurev.biochem.72.121801.161700 - DOI - PubMed
    1. Chatzidimopoulos M., Papaevagellou D., Pappas A. C. (2013). Detection and characterization of fungicide resistant phenotypes of Botrytis cinerea in lettuce crops in Greece. Eur. J. Plant Pathol. 137 363–376. 10.1007/s10658-013-0248-x - DOI

LinkOut - more resources

Feedback