Abstract
BackgroundPenicillium italicum (blue mold) is one of citrus pathogens causing undesirable citrus fruit decay even at strictly-controlled low temperatures (< 10 °C) during shipping and storage. P. italicum isolates with considerably high resistance to sterol demethylation inhibitor (DMI) fungicides have emerged; however, mechanism(s) underlying such DMI-resistance remains unclear. In contrast to available elucidation on anti-DMI mechanism for P. digitatum (green mold), how P. italicum DMI-resistance develops has not yet been clarified.ResultsThe present study prepared RNA-sequencing (RNA-seq) libraries for two P. italicum strains (highly resistant (Pi-R) versus highly sensitive (Pi-S) to DMI fungicides), with and without prochloraz treatment, to identify prochloraz-responsive genes facilitating DMI-resistance. After 6 h prochloraz-treatment, comparative transcriptome profiling showed more differentially expressed genes (DEGs) in Pi-R than Pi-S. Functional enrichments identified 15 DEGs in the prochloraz-induced Pi-R transcriptome, simultaneously up-regulated in P. italicum resistance. These included ATP-binding cassette (ABC) transporter-encoding genes, major facilitator superfamily (MFS) transporter-encoding genes, ergosterol (ERG) anabolism component genes ERG2, ERG6 and EGR11 (CYP51A), mitogen-activated protein kinase (MAPK) signaling-inducer genes Mkk1 and Hog1, and Ca2+/calmodulin-dependent kinase (CaMK) signaling-inducer genes CaMK1 and CaMK2. Fragments Per Kilobase per Million mapped reads (FPKM) analysis of Pi-R transcrtiptome showed that prochloraz induced mRNA increase of additional 4 unigenes, including the other two ERG11 isoforms CYP51B and CYP51C and the remaining kinase-encoding genes (i.e., Bck1 and Slt2) required for Slt2-MAPK signaling. The expression patterns of all the 19 prochloraz-responsive genes, obtained in our RNA-seq data sets, have been validated by quantitative real-time PCR (qRT-PCR). These lines of evidence in together draw a general portrait of anti-DMI mechanisms for P. italicum species. Intriguingly, some strategies adopted by the present Pi-R were not observed in the previously documented prochloraz-resistant P. digitatum transcrtiptomes. These included simultaneous induction of all major EGR11 isoforms (CYP51A/B/C), over-expression of ERG2 and ERG6 to modulate ergosterol anabolism, and concurrent mobilization of Slt2-MAPK and CaMK signaling processes to overcome fungicide-induced stresses.ConclusionsThe present findings provided transcriptomic evidence on P. italicum DMI-resistance mechanisms and revealed some diversity in anti-DMI strategies between P. italicum and P. digitatum species, contributing to our knowledge on P. italicum DMI-resistance mechanisms.
Highlights
Penicillium italicum is one of citrus pathogens causing undesirable citrus fruit decay even at strictly-controlled low temperatures (< 10 °C) during shipping and storage
The sterol demethylation inhibitor (DMI) fungicides, such as imazalil and prochloraz, have been widely applied to control citrus molds [2,3,4,5,6]. Resistance to these DMI fungicides has frequently occurred for the Penicillium molds in the past decade, especially for P. digitatum isolates with high DMIresistance [5, 7], considerably reducing the efficacy of the fungicides
We identified 1) 1052 Differentially expressed gene (DEG) between Pi-R-I and Pi-R-NI (614 up-regulated and 438 down-regulated) (Fig. 2a and Additional file 5: Table S3), representing the drug-responsive genes in prochloraz-resistant strain; 2) 298 DEGs between Pi-S-I and Pi-S-NI (63 up-regulated and 235 downregulated) (Fig. 2b and Additional file 6: Table S4), representing the drug-responsive genes in prochloraz-sensitive strain; 3) 1482 DEGs between Pi-R-I and Pi-S-I (811 upregulated and 671 down-regulated) (Fig. 2c and Additional file 7: Table S5), representing difference in drug-induced gene expression between fungicide-resistant and -sensitive P. italicum strains; and 4) 958 DEGs between Pi-R-NI and Pi-S-NI (422 up-regulated and 536 down-regulated) (Fig. 2d and Additional file 8: Table S6), representing different genetic background between the two P. italicum strains
Summary
Penicillium italicum (blue mold) is one of citrus pathogens causing undesirable citrus fruit decay even at strictly-controlled low temperatures (< 10 °C) during shipping and storage. P. italicum isolates with considerably high resistance to sterol demethylation inhibitor (DMI) fungicides have emerged; mechanism(s) underlying such DMI-resistance remains unclear. The sterol demethylation inhibitor (DMI) fungicides, such as imazalil and prochloraz, have been widely applied to control citrus molds [2,3,4,5,6]. Resistance to these DMI fungicides has frequently occurred for the Penicillium molds in the past decade, especially for P. digitatum isolates with high DMIresistance [5, 7], considerably reducing the efficacy of the fungicides.
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