Phytophthora cactorum is considered an important plant pathogen which is causing major damage to strawberry plants worldwide. In the current study, the ability of the active ingredients of seven different fungicides, azoxystrobin, cymoxanil, dimethomorph, fenamidone, fluopicolide, metalaxyl and propamocarb, to suppress the mycelial growth, sporangial formation and zoospore release of P. cactorum isolates, was tested. The variation in resistance against various fungicides was found among the isolates. The active ingredients are also unequally efficient against different life stages of P. cactorum, which is probably associated with their different modes of action. A significant level of resistance was recorded against metalaxyl and dimethomorph; however, these were totally inefficient against the zoospore release, while azoxystrobin did not inhibit mycelial growth. The only fungicide efficient against all three P. cactorum life stages tested was fluopicolide, although the calculated resistance factor gives evidence of the rise of resistance in the majority of isolates even against this fungicide. Significant differences were found between responses to fungicides of isolates from strawberry and from other host species. Based on the Mahalanobis distances calculated in the discriminant analysis comprising all of the assays performed, the similarities among isolates were estimated.
Salvia rosmarinus L. (rosemary) is known to have a wide range of pharmacological effects including antidiabetic, anticarcinogenic, and antitumorigenic properties owing to its secondary metabolites. Studies aiming to elevate these metabolites have utilized various elicitors and stresses under in vitro conditions, although underlying molecular mechanisms remain unexplored. Gene expression studies using RT-qPCR might provide valuable information regarding how plant and plant cells interact and perceive various treatments and elicitors. However, despite being able to calculate accurate fold changes, the accuracy of the RT-qPCR data highly depends on the expression of reference genes. To the best of our knowledge, there is no information available on the stable reference genes in rosemary under in vitro conditions. Thus, in this paper, we assessed the stability of seven commonly used reference genes under different elicitor and stress conditions using RT-qPCR. Thereafter, the five most commonly used software and algorithms (comparative ΔCt, BestKeeper, NormFinder, geNorm, and RefFinder) were used to rank the candidates based on their expression stabilities. In conclusion, we recommend using a combination of F1-ATPase, ATP synthase and ACCase to normalize the gene expression experiments in rosemary under in vitro conditions. The selected reference genes were verified using 4-coumarate-CoA ligase, a pharmacologically important gene, whose expression might alter under nanoparticle treatment. Additionally, reference genes for several plant tissues, elicitors, and stresses are also proposed. The conclusions obtained from this current study will accelerate the future molecular work in S. rosmarinus and other related species.
Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most important foliar pathogen of sugar beet worldwide. Extensive reliance on fungicides to manage CLS has resulted in the evolution of fungicide resistance in C. beticola worldwide, including populations in the Czech Republic. One important class of fungicides used to manage CLS is the sterol demethylation inhibitors (DMI). The aim of our study was to assess DMI resistance in C. beticola from the Czech Republic and elucidate the molecular basis of DMI resistance in this population. A total of 50 isolates were collected in 2018 and 2019 from the major sugar beet growing regions of the Czech Republic and assessed for in vitro sensitivity to the DMI fungicides propiconazole, prochloraz, and epoxiconazole. These analyses identified three strains that exhibited 50% effective concentration (EC50) values > 1.0 μg mL–1 against respective fungicides, which were therefore considered resistant. In contrast, strains that exhibited lowest EC50 values were considered sensitive. To explore the molecular basis of resistance in these three strains, the cytochrome P450-dependent sterol 14α-demethylase (Cyp51) gene was sequenced. Sequence analysis identified a Y464S mutation in all three resistant strains. To assess whether Cyp51 gene expression may play a role in DMI resistance, selected strains were grown in vitro with and without fungicide treatment. These analyses indicated that Cyp51 gene expression was significantly induced after fungicide treatment. Thus, we conclude that Y464S point mutation along with induced Cyp51 gene overexpression is likely responsible for resistance against DMI fungicides in C. beticola from the Czech Republic.
Melissa officinalis L. is well known for its lemon-scented aroma and various pharmacological properties. Despite these valuable properties, the genes involved in the biosynthetic pathways in M. officinalis are not yet well-explored when compared to other members of the mint family. For that, gene expression studies using quantitative real-time PCR (qRT-PCR) are an excellent tool. Although qRT-PCR can provide accurate results, its accuracy is highly reliant on the expression and stability of the reference gene used for normalization. Hence, selecting a suitable experiment-specific reference gene is very crucial to obtain accurate results. However, to date, there are no reports for experiment-specific reference genes in M. officinalis. Therefore, in the current study, ten commonly used reference genes were assessed for their suitability as optimal reference genes in M. officinalis under various abiotic stress conditions and different plant organs. The candidate genes were ranked based on BestKeeper, comparative ΔCt, geNorm, NormFinder, and RefFinder. Based on the results, we recommend the combination of EF-1α and GAPDH as the best reference genes to normalize gene expression studies in M. officinalis. On the contrary, HLH71 was identified as the least-performing gene. Thereafter, the reliability of the optimal gene combination was assessed by evaluating the relative gene expression of the phenylalanine ammonia lyase (PAL) gene under two elicitor treatments (gibberellic acid and jasmonic acid). PAL is a crucial gene involved directly or indirectly in the production of various economically important secondary metabolites in plants. Suitable reference genes for each experimental condition are also discussed. The findings of the current study form a basis for current and future gene expression studies in M. officinalis and other related species.
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