PdCYP51B, a new putative sterol 14α-demethylase gene of Penicillium digitatum involved in resistance to imazalil and other fungicides inhibiting ergosterol synthesis

Abstract

Penicillium digitatum, causing green mold decay, is the most destructive postharvest pathogen of citrus fruits worldwide. The phenotypes and genotypes of 403 isolates of P. digitatum, collected from packing houses and supermarkets in Zhejiang, China, during 2000 to 2010, were characterized in terms of their imazalil sensitivity. The frequency of detected imazalil-resistant (IMZ-R) isolates increased from 2.1% in 2000 to 60-84% during 2005-2010. Only 6.5% and 4.5% of the collected IMZ-R isolates belong to the previously described IMZ-R1 and IMZ-R2 genotypes, respectively. To determine the resistance mechanism of the predominant and novel IMZ-R isolates of P. digitatum (termed IMZ-R3), genes PdCYP51B and PdCYP51C, homologous to the sterol 14α-demethylase encoded gene PdCYP51, were cloned from six IMZ-R3 and eight imazalil-sensitive (IMZ-S) isolates of P. digitatum. A unique 199-bp insertion was observed in the promoter region of PdCYP51B in all IMZ-R3 isolates examined but in none of the tested IMZ-S isolates. Further analysis by PCR confirmed that this insertion was present in all IMZ-R3 isolates but absent in IMZ-S, IMZ-R1, and IMZ-R2 isolates. Transcription levels of PdCYP51B in three IMZ-R3 isolates were found to be 7.5- to 13.6-fold higher than that in two IMZ-S isolates of P. digitatum. Introduction of another copy of PdCYP51B ( s ) (from IMZ-S) into an IMZ-S isolate decreased the sensitivity of P. digitatum to 14α-demethylation inhibitors (DMIs) only to a small extent, but introduction of a copy of PdCYP51B ( R ) (from IMZ-R3) dramatically increased the resistance level of P. digitatum to DMIs. Regarding PdCYP51C, no consistent changes in either nucleotide sequence or expression level were correlated with imazalil resistance among IMZ-R and IMZ-S isolates. Based on these results, we concluded that (1) the CYP51 family of P. digitatum contains the PdCYP51B and PdCYP51C genes, in addition to the known gene PdCYP51A (previously PdCYP51); (2) PdCYP51B is involved in DMI fungicide resistance; and (3) overexpression of PdCYP51B resulting from a 199-bp insertion mutation in the promoter region of PdCYP51B is responsible for the IMZ-R3 type of DMI resistance in P. digitatum.

Novel mutations in CYP51B from Penicillium digitatum involved in prochloraz resistance

“…Another report described that an insertion of a 199-bp sequence was found in the 126-bp transcriptional enhancer unit of Pdcyp51A in some of the IMZ-resistant strains, but none were found in the sensitive ones (Ghosoph et al, 2007). Furthermore, a 199-bp insertion (PdMLE1) was also found in the promoter region of Pdcyp51B, which may confer DMI resistance in P. digitatum (Sun et al, 2011). Blast searching and southern blot analyses showed that this 199 bp element was unique to P. digitatum.…”

“…To detect insertions in the Pdcyp51A and Pdcyp51B promoter regions, two pairs of specific primers were designed for PCR amplification, P1/P2 for Pdcyp51A upstream and P3/P4 for Pdcyp51B upstream (Table 1; Sun et al, 2011). The PCR conditions consisted of an initial denaturation at 95°C for 5 min, followed by 35 cycles of 94°C for 1 min, 58°C for 1 min and 72°C for 2 min, with a final elongation step at 72°C for 10 min.…”

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance

“…This provided a quick and easy method to detect DMIresistant strains of P. digitatum (Hamamoto et al, 2001). Insertions in the promoter were also found in Blumeriella jaapii (Ma et al, 2006), Venturia inaequalis (Schnabel & Jones 2001) and in Monilinia fructicola (Luo et al, 2008) and recently another insertion of 199-bp was found in P. digitatum CYP51A gene (Ghosoph et al, 2007) and PdCYP51B gene (Sun et al, 2011;Sánchez-Torres et al, submitted) leading to resistant phenotypes.…”

“…In Penicillium digitatum, the DMI resistance resulted from over-expression of the PdCYP51 genes driven by a tandem repeat of five copies of a 126 transcriptional enhancer (Hamamoto et al, 2000), or insertion of 199-bp in the promoter region of PdCYP51A (Gosoph et al, 2007) or by the presence of 199-bp enhancer in the promoter region of PdCYP51B (Sun et al, 2011). Based on the DNA sequence of the PdCYP51-genes, PCR primers have been developed which are able to distinguish not only between sensitive and resistant DMI strains but allow identification of molecular mechanism that takes place (Hamamoto et al, 2001;Gosoph et al, 2007;Sánchez-Torres & Tuset, 2011;Sun et al, 2011).…”

An -Omics Insight Into the Pathogenicity of Penicillium Digitatum: An Overview

“…In the resistant strain there is a 199 bp insertion in the promoter region, which is not present in PHI26. The relationship between imazalil resistance and this 199 bp insertion has been previously shown in P. digitatum (Sun et al, 2011). It is noteworthy that this element is present in several copies dispersed throughout the genomes of both strains and has been described as a new active transposon with a strong promoter activity (Sun et al, 2012).…”