The Cytochrome P450 Lanosterol 14α-Demethylase Gene Is a Demethylation Inhibitor Fungicide Resistance Determinant in Monilinia fructicola Field Isolates from Georgia

Resistance in Monilinia fructicola to demethylation inhibitor (DMI) fungicides is beginning to emerge in North America, but its molecular basis is unknown. Two potential genetic determinants of DMI fungicide resistance including the 14α-demethylase gene (MfCYP51) and the ATP-binding cassette transporter gene MfABC1, were investigated in six resistant (DMI-R) and six sensitive (DMI-S) field isolates. No point mutations leading to an amino acid change were found in the MfCYP51 gene. The constitutive expression of the MfCYP51 gene in DMI-R isolates was significantly higher compared to DMI-S isolates. Gene expression was not induced in mycelium of DMI-R or DMI-S isolates treated with 0.3 μg of propiconazole/ml. A slightly higher average MfCYP51 copy number value was detected in DMI-R isolates (1.35) compared to DMI-S isolates (1.13); however, this difference could not be verified in Southern hybridization experiments or explain the up to 11-fold-increased MfCYP51 mRNA levels in DMI-R isolates. Analysis of the upstream nucleotide sequence of the MfCYP51 gene revealed a unique 65-bp repetitive element at base pair position −117 from the translational start site in DMI-R isolates but not in DMI-S isolates. This repetitive element contained a putative promoter and was named Mona. The link between Mona and the DMI resistance phenotype became even more apparent after studying the genetic diversity between the isolates. In contrast to DMI-S isolates, DMI-R isolates contained an MfCYP51 gene of identical nucleotide sequence associated with Mona. Still, DMI-R isolates were not genetically identical as revealed by Microsatellite-PCR analysis. Also, real-time PCR analysis of genomic DNA indicated that the relative copy number of Mona among DMI-S and DMI-R isolates varied, suggesting its potential for mobility. Interestingly, constitutive expression of the MfABC1 gene in DMI-R isolates was slightly lower than that of DMI-S isolates, but expression of the MfABC1 gene in DMI-R isolates was induced in mycelium after propiconazole treatment. Therefore, the MfABC1 gene may play a minor role in DMI fungicide resistance in M. fructicola. Our results strongly suggest that overexpression of the MfCYP51 gene is an important mechanism in conferring DMI fungicide resistance in M. fructicola field isolates from Georgia and that this overexpression is correlated with Mona located upstream of the MfCYP51 gene.     

Sensitivity of Magnaporthe grisea to the Sterol Demethylation Inhibitor Fungicide Propiconazole

Baseline sensitivity of Magnaporthe grisea to a sterol demethylation inhibitor (DMI) propiconazole was determined using 52 wild-type single-spore isolates. The 50% effective concentrations of these 52 isolates to propiconazole ranged from 0.145 to 1.446  μ g/ml. Among the 52 isolates, two (07–82 and 04–006) were hypersensitive to propiconazole. The propiconazole-hypersensitive (PHS) isolates were also hypersensitive to another DMI fungicide triadimefon, but not to a benzimidazole fungicide carbendazim. Compared with the propiconazole-sensitive (PS) isolates, the PHS isolates retained normal pathogenicity. Real-time PCR analysis showed that expression of cyp51 gene in the PHS isolates was not significantly different from that in the PS isolates. Analysis of DNA sequence of cyp51 gene showed that the PHS isolates 07–82 and 04–006 had an amino acid substitution at the codon position 234 and 450, respectively, where the amino acids were conserved in the CYP51 of other fungi, which indicated that the substitutions in CYP51 might be related to hypersensitivity of M. grisea to DMI fungicides.     

Resistance of Sclerotinia minor Isolates to Cyclic Imides in Lettuce Field Soils of Roussillon, France

Lettuce drop caused by Sclerotinia minor Jagger is the most common disease affecting lettuce crops during the fall and winter in Roussillon. Spraying of cyclic imide fungicides (iprodione, vinclozolin, and less commonly procymidone) generally provides satisfactory protection. However, since about 1985, the mean field efficacy of iprodione and vinclozolin has significantly decreased, whereas that of procymidone is currently not in question. The appearance of fungicide-resistant isolates of S.minor could explain the reduced efficacy of these fungicides. We thus looked for iprodione and procymidone-resistant isolates of S.minor in these problem fields. The investigations were earned on for over 3 years. Most of the isolates showed fungicide sensitivity (iprodione IC 50 = 0.17 mg 1⁻¹), about one quarter showed average resistance (iprodione IC 50 = 0.59 mg 1⁻¹) and a few were found to be highly resistant. Resistant isolates were also obtained in vitro when using high fungicide concentrations. Moreover, isolates resistant to iprodione were found to be resistant to procymidone. The most highly resistant isolates produced fewer and larger-sized sclerotia than the sensitive isolates, they were also more quickly inhibited by high osmotic pressures. Field variability was quite low: 73 of 74 isolates obtained from one field were found to be sensitive, with only one showing mild resistance. There did not seem to be any correlation between the observed decrease in the field efficacy of iprodione and the presence of resistant isolates.     

Overexpression of the 14α-Demethylase Target Gene (CYP51) Mediates Fungicide Resistance in Blumeriella jaapii

Sterol demethylation inhibitor (DMI) fungicides are widely used to control fungi pathogenic to humans and plants. Resistance to DMIs is mediated either through alterations in the structure of the target enzyme CYP51 (encoding 14α-demethylase), through increased expression of the CYP51 gene, or through increased expression of efflux pumps. We found that CYP51 expression in DMI-resistant (DMI^R) isolates of the cherry leaf spot pathogen Blumeriella jaapii was increased 5- to 12-fold compared to that in DMI-sensitive (DMI^S) isolates. Analysis of sequences upstream of CYP51 in 59 DMI^R isolates revealed that various forms of a truncated non-long terminal direct repeat long interspersed nuclear element retrotransposon were present in all instances. Similar inserts upstream of CYP51 were not present in any of 22 DMI^S isolates examined.     

Overexpression of Three Glucosinolate Biosynthesis Genes in Brassica napus Identifies Enhanced Resistance to Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are notorious plant pathogenic fungi with an extensive host range including Brassica crops. Glucosinolates (GSLs) are an important group of secondary metabolites characteristic of the Brassicales order, whose degradation products are proving to be increasingly important in plant protection. Enhancing the defense effect of GSL and their associated degradation products is an attractive strategy to strengthen the resistance of plants by transgenic approaches. We generated the lines of Brassica napus with three biosynthesis genes involved in GSL metabolic pathway (BnMAM1, BnCYP83A1 and BnUGT74B1), respectively. We then measured the foliar GSLs of each transgenic lines and inoculated them with S. sclerotiorum and B. cinerea. Compared with the wild type control, over-expressing BnUGT74B1 in B. napus increased the aliphatic and indolic GSL levels by 1.7 and 1.5 folds in leaves respectively; while over-expressing BnMAM1 or BnCYP83A1 resulted in an approximate 1.5-fold higher only in the aliphatic GSL level in leaves. The results of plant inoculation demonstrated that BnUGT74B1-overexpressing lines showed less severe disease symptoms and tissue damage compared with the wild type control, but BnMAM1 or BnCYP83A1-overexpressing lines showed no significant difference in comparison to the controls. These results suggest that the resistance to S. sclerotiorum and B. cinerea in B. napus could be enhanced through tailoring the GSL profiles by transgenic approaches or molecular breeding, which provides useful information to assist plant breeders to design improved breeding strategies.     

Investigation of the Sterol Composition and Azole Resistance in Field Isolates of Septoria tritici

We report here a biochemical study of resistance to azole antifungal agents in a field isolate (S-27) of a fungal phytopathogen. Isolates of Septoria tritici were compared in vitro, and their responses reflected that observed in the field, with S-27 exhibiting resistance relative to RL2. In untreated cultures, both RL2 and S-27 contained isomers of ergosterol and ergosta-5,7-dienol, although in differing concentrations. Under azole treatment, this phytopathogen exhibited a response similar to that of other pathogenic fungi, with a reduction in desmethyl sterols and an accumulation of 14(alpha)-methyl sterols, indicative of inhibition of the P450-mediating sterol 14(alpha)-demethylase. Growth arrest was attributed to the reduction of ergosterol combined with an accumulation of nonutilizable sterols. Strain S-27 exhibited an azole-resistant phenotype which was correlated with decreased cellular content of azole.     

Multiple Mutations and Overexpression in the CYP51A and B Genes Lead to Decreased Sensitivity of Venturia effusa to Tebuconazole

Multiple demethylation-inhibiting (DMI) fungicides are used to control pecan scab, caused by Venturia effusa. To compare the efficacy of various DMI fungicides on V. effusa, field trials were conducted at multiple locations applying fungicides to individual pecan terminals. In vitro assays were conducted to test the sensitivity of V. effusa isolates from multiple locations to various concentrations of tebuconazole. Both studies confirmed high levels of resistance to tebuconazole. To investigate the mechanism of resistance, two copies of the CYP51 gene, CYP51A and CYP51B, of resistant and sensitive isolates were sequenced and scanned for mutations. In the CYP51A gene, mutation at codon 444 (G444D), and in the CYP51B gene, mutations at codon 357 (G357H) and 177 (I77T/I77L) were found in resistant isolates. Expression analysis of CYP51A and CYP51B revealed enhanced expression in the resistant isolates compared to the sensitive isolates. There were 3.0- and 1.9-fold increases in gene expression in the resistant isolates compared to the sensitive isolates for the CYP51A and CYP51B genes, respectively. Therefore, two potential mechanisms—multiple point mutations and gene over expression in the CYP51 gene of V. effusa isolates—were revealed as likely reasons for the observed resistance in isolates of V. effusa to tebuconazole.     

Changes in Adhesion of Candida tropicalis Clinical Isolates Exhibiting Switch Phenotypes to Polystyrene and HeLa Cells

Background

Candida tropicalis is an important human pathogen that can undergo multiple forms of phenotypic switching.

Aim

We aimed to evaluate the effect of phenotypic switching on the adhesion ability of C. tropicalis.

Methods

C. tropicalis morphotypes included parental phenotypes (clinical isolates) and switch phenotypes (crepe, revertant of crepe—CR, rough, revertant of rough—RR, irregular center and revertant of irregular center—ICR). Adhesion to polystyrene and HeLa cells was determined by crystal violet assay. The percentage of HeLa cells with adhered yeasts and the number of adhered yeasts per HeLa cell were determined by light microscopy. Filamentation among adhered cells was assessed by direct counting.

Results

On polystyrene, 60% of the switch strains showed difference (p?<?0.05) on adhesion ability compared to their parental counterpart strains, and altered thickness of adhered cells layers. Filamentation was increased among adhered cells of the switched strains compared to parental strains. A positive correlation was observed between adhesion on polystyrene and filamentation for morphotypes of the system 49.07. The majority of the switched strains showed higher adhesion capability to HeLa cells in comparison to the adherence of the clinical strains. All revertant strains showed a higher number of yeast cells per HeLa cell compared to their variant counterparts (p?<?0.05), with exception of the ICR.

Conclusions

Our findings indicate that switching events in C. tropicalis affect adhesion and filamentation of adhered cells on polystyrene and HeLa cells. The rise of switch strains with increased adhesion ability may contribute to the success of infection associated with C. tropicalis.

     

Ophiostoma mitovirus 3a , ascorbic acid, glutathione, and photoperiod affect the development of stromata and apothecia by Sclerotinia homoeocarpa

Hypovirulence in Sclerotinia homoeocarpa is associated with infection by Ophiostoma mitovirus 3a (OMV3a). OMV3a is also present in asymptomatic isolates, with growth and virulence comparable to that of virus-free isolates. Hypovirulent isolates have impaired mitochondrial function resulting in increased activity of the alternative oxidase pathway, which is implicated in the reduction of reactive oxygen species in other fungi. In this study, hypovirulent, asymptomatic, and virus-free isolates were grown on potato dextrose agar amended with ascorbic acid or glutathione and were incubated under various photoperiods to determine the importance of reactive oxygen species, light, and OMV3a infection for differentiation of stromata and apothecia by S. homoeocarpa. Hypovirulent isolates did not form stromata or apothecia. Glutathione and darkness reduced stromata size and apothecia production by virulent and asymptomatic isolates. Apothecia formed under several different photoperiods, and ascorbic acid increased apothecia production. Ascospores were not detected in these apothecia. The results suggest that hypovirulence, light, and the superoxide radical are important factors in the formation of stromata and apothecia by S. homoeocarpa isolates. This is the first report of sterile apothecia production by North American isolates of S. homoeocarpa and provides a starting point for attempts to produce fertile apothecia.     

Tissue Inhibitor of Metalloproteinase 1 Expression Associated with Gene Demethylation Confers Anoikis Resistance in Early Phases of Melanocyte Malignant Transformation

Although anoikis resistance has been considered a hallmark of malignant phenotype, the causal relation between neoplastic transformation and anchorage-independent growth remains undefined. We developed an experimental model of murine melanocyte malignant transformation, where a melanocyte lineage (melan-a) was submitted to sequential cycles of anchorage blockade, resulting in progressive morphologic alterations, and malignant transformation. Throughout this process, cells corresponding to premalignant melanocytes and melanoma cell lines were established and show progressive anoikis resistance and increased expression of Timp1. In melan-a melanocytes, Timp1 expression is suppressed by DNA methylation as indicated by its reexpression after 5-aza-2′-deoxycytidine treatment. Methylation-sensitive single-nucleotide primer extension analysis showed increased demethylation in Timp1 in parallel with its expression along malignant transformation. Interestingly, TIMP1 expression has already been related with negative prognosis in some human cancers. Although described as a MMP inhibitor, this protein has been associated with apoptosis resistance in different cell types. Melan-a cells overexpressing Timp1 showed increased survival in suspension but were unable to form tumors in vivo, whereas Timp1-overexpressing melanoma cells showed reduced latency time for tumor appearance and increased metastatic potential. Here, we demonstrated for the first time an increment in Timp1 expression since the early phases of melanocyte malignant transformation, associated to a progressive gene demethylation, which confers anoikis resistance. In this way, Timp1 might be considered as a valued marker for melanocyte malignant transformation.     

Overexpression of a Defensin Enhances Resistance to a Fruit-Specific Anthracnose Fungus in Pepper

Functional characterization of a defensin, J1-1, was conducted to evaluate its biotechnological potentiality in transgenic pepper plants against the causal agent of anthracnose disease, Colletotrichum gloeosporioides. To determine antifungal activity, J1-1 recombinant protein was generated and tested for the activity against C. gloeosporioides, resulting in 50% inhibition of fungal growth at a protein concentration of 0.1 mg·mL⁻¹. To develop transgenic pepper plants resistant to anthracnose disease, J1-1 cDNA under the control of 35S promoter was introduced into pepper via Agrobacterium-mediated genetic transformation method. Southern and Northern blot analyses confirmed that a single copy of the transgene in selected transgenic plants was normally expressed and also stably transmitted to subsequent generations. The insertion of T-DNA was further analyzed in three independent homozygous lines using inverse PCR, and confirmed the integration of transgene in non-coding region of genomic DNA. Immunoblot results showed that the level of J1-1 proteins, which was not normally accumulated in unripe fruits, accumulated high in transgenic plants but appeared to differ among transgenic lines. Moreover, the expression of jasmonic acid-biosynthetic genes and pathogenesis-related genes were up-regulated in the transgenic lines, which is co-related with the resistance of J1-1 transgenic plants to anthracnose disease. Consequently, the constitutive expression of J1-1 in transgenic pepper plants provided strong resistance to the anthracnose fungus that was associated with highly reduced lesion formation and fungal colonization. These results implied the significance of the antifungal protein, J1-1, as a useful agronomic trait to control fungal disease.     

Virulence Pattern of Venturia inaequalis Field Isolates and Corresponding Differential Resistance in Malus x domestica

All commercially important apple cultivars are susceptible in the field to scab caused by Venturia inaequalis. The scope of this study was to investigate variation in virulence in Venturia inaequalis populations towards commercial apple cultivars. For this purpose, primary lesions were sampled in orchards with different varietal compositions and diversities. The virulence pattern of monosporic isolates, obtained by isolation of single conidia, was assessed by cross inoculations of the cv. Ananas Reinette, Boskoop, Glockenapfel, Golden Delicious, Gravenstein, James Grieve, Jonathan, Maigold, Reinette de Champagne, Spartan and Yellow Transparent. All cultivars were susceptible to some isolates and resistant to others. No cultivar behaved the same way, which suggests the presence of differential resistance in each cultivar and corresponding virulence in some isolates. Isolates from a monoculture of Golden Delicious consisted mainly of a pathotype that was virulent to Golden Delicious but not to other cultivars. In the samples from cultivar mixtures, virulence pattern variation was considerable. The results give further evidence of the existence of a large pool of differential and ephemeral resistances in Malus, which were overcome by the local scab populations during co‐evolution.     

Genetic Control of Resistance to the Piperidine Fungicide Fenpropidin in Ustilago maydis

Mutants of Ustilago maydis (DC.) Corda, resistant to the piperidine fungicide fenpropidin, were isolated in a mutation frequency of 3.2 × 10^–5, after UV‐irradiation and selection on media containing 75 μg/ml fenpropidin. Genetic analysis with 15 such mutant isolates resulted in the identification of two unlinked chromosomal loci, U/fpd‐1 and U/fpd‐2. The U/fpd mutations are responsible for moderate resistance levels to fenpropidin (Rf: 42–56 or 15 based on effective concentration causing a 50% reduction in the growth rate (EC₅₀) or minimal inhibitory concentration (MIC) values, respectively). Haploid strains carrying both U/fpd mutations do not exhibit higher levels of resistance to fenpropidin, indicating no additivity of gene effect between non‐allelic genes. Cross‐resistance studies with other Sterol Biosynthesis inhibitors (SBIs) showed that the U/fpd‐mutant isolates exhibited a positive cross‐resistance to the piperidine piperalin and to the related morpholine fungicides fenpropimorph and tridemorph, but not to the inhibitors of C‐14 demethylase and squalene epoxidase. Crosses between mutants carrying the U/fpd‐genes with compatible isolates carrying the U/fpm or U/tdm mutations, which have been identified in previous genetic studies for resistance to morpholine fungicides fenpropimorph and tridemorph, yielded, with the exception of U/fpd‐2 × U/fpm‐2 crosses, a large number of recombinants with wild‐type sensitivity, indicating that the mutant genes involved were not allelic. Analysis of progeny from crosses between U/fpd‐2 and U/fpm‐2 mutants yielded no recombinants with wild‐type sensitivity, but a 1 : 1 progeny segregation was observed at the MIC for the U/fpd‐2 isolates, indicating that these genes are alleles of the same locus. A study of the fitness of fenpropidin‐resistant isolates showed that the U/fpd mutations do not affect the phytopathogenic fitness‐determining characteristics such as growth in liquid culture and pathogenicity on young corn plants.