Evaluation of the Ability of Seven Active Ingredients of Fungicides to Suppress Phytophthora cactorum at Diverse Life Stages, and Variability in Resistance Found among Isolates.

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.

Peronosporales Species Associated with Strawberry Crown Rot in the Czech Republic.

The symptoms of crown rot on strawberry plants are considered typical for the pathogen Phytophthora cactorum, which causes high losses of this crop. However, an unknown number of related species of pathogens of Peronosporales cause symptoms quite similar to those caused by P. cactorum. To determine their spectrum and importance, strawberry plants were sampled from 41 farms in the Czech Republic. The cultures were isolated from the symptomatic plants using the baiting method, with subsequent cultivation on a semiselective medium. Isolates were identified to the species level using nuclear ribosomal internal transcribed spacer (ITS) barcoding after preliminary morphological determination. In total, 175 isolates of 24 species of Phytophthora, Phytopythium, Pythium, and Globisporangium were detected. The most represented was Phytophthora cactorum, with 113 (65%) isolates, which was recorded in 61% of farms, and the Pythium dissotocum complex with 20 (11%) isolates, which was recorded in 27% of farms. Other species were represented in units of percent. Large differences between farms in the species spectra were ascertained. The differences between species in cardinal growth temperatures and different management of the farms are discussed as a main reason for such a diversification. Regarding the dissimilar sensitivity of various species of Peronosporales against fungicides, the proper determination of the cause of disease is of crucial significance in plant protection.

Characterization of the Molecular Mechanisms of Resistance against DMI Fungicides in Cercospora beticola Populations from the Czech Republic.

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.

Genome wide identification of the immunophilin gene family in Leptosphaeria maculans: a causal agent of Blackleg disease in Oilseed Rape (Brassica napus).

Abstract Phoma stem canker (blackleg) is a disease of world-wide importance on oilseed rape (Brassica napus) and can cause serious losses for crops globally. The disease is caused by dothideomycetous fungus, Leptosphaeria maculans, which is highly virulent/aggressive. Cyclophilins (CYPs) and FK506-binding proteins (FKBPs) are ubiquitous proteins belonging to the peptidyl-prolyl cis/trans isomerase (PPIase) family. They are collectively referred to as immunophilins (IMMs). In the present study, IMM genes, CYP and FKBP in haploid strain v23.1.3 of L. maculans genome, were identified and classified. Twelve CYPs and five FKBPs were determined in total. Domain architecture analysis revealed the presence of a conserved cyclophilin-like domain (CLD) in the case of CYPs and FKBP_C in the case of FKBPs. Interestingly, IMMs in L. maculans also subgrouped into single domain (SD) and multidomain (MD) proteins. They were primarily found to be localized in cytoplasm, nuclei, and mitochondria. Homologous and orthologous gene pairs were also determined by comparison with the model organism Saccharomyces cerevisiae. Remarkably, IMMs of L. maculans contain shorter introns in comparison to exons. Moreover, CYPs, in contrast with FKBPs, contain few exons. However, two CYPs were determined as being intronless. The expression profile of IMMs in both mycelium and infected primary leaves of B. napus demonstrated their potential role during infection. Secondary structure analysis revealed the presence of atypical eight ß strands and two α helices fold architecture. Gene ontology analysis of IMMs predicted their significant role in protein folding and PPIase activity. Taken together, our findings for the first time present new prospects of this highly conserved gene family in phytopathogenic fungus.