Jana Mazáková

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.

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.

In the Czech Republic, demethylation inhibitors (DMIs) are used both as fungicides in controlling phoma stem canker and as growth regulators. This heavy use can result in the development of resistant isolates. A total of 45 and 286 Leptosphaeria maculans isolates were tested in vitro, using the mycelial growth and microtiter plate assays, respectively. The objective was to determine the sensitivity of L. maculans isolates collected in the Czech Republic to the fungicides tetraconazole, metconazole, and prochloraz. The mean EC₅₀ values with the mycelial growth plate method were 1.33, 0.78, and 0.40 µg mL⁻¹ for tetraconazole, metconazole, and prochloraz, respectively. The mean EC₅₀ values for the microtiter plate assay were 3.01, 0.44, and 0.19 µg mL⁻¹ for tetraconazole, metconazole, and prochloraz, respectively. All three fungicides also had high variation factors that may be due to inserts in the ERG11 promoter region. In addition, cross sensitivity among the three fungicides was observed. Overall, the high variation factors and the PCR (polymerase chain reaction) results observed in this study could signify the presence of resistant isolates in L. maculans Czech populations, especially in isolates tested for sensitivity to tetraconazole.

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.

Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis, the scientific journal of the Mendel University in Brno, Czech Republic

A total of 235 Phytophthora infestans isolates were collected from five regions of the Czech Republic during the growing seasons 2012–2014 and 2016 and examined using the in vitro amended agar method for their sensitivity to metalaxyl-M (MFX), propamocarb-HCl (PCH), and dimethomorph (DMM). A majority of the isolates (50%) were sensitive to MFX. Resistant isolates were found in all four years of the survey; they represented 30% of the samples. The EC ₅₀ values of PCH in inhibiting mycelial growth of 65% of the overall isolates were higher than 100 μg ml ⁻¹ , which indicates the occurrence of insensitivity to PCH in the Czech P. infestans populations. DMM was very effective, and the mycelial growth of all isolates tested was completely suppressed at the concentration of 0.1 μg ml ⁻¹ . Furthermore, the efficacy of 12 plant essential oils was tested against 20 isolates of P. infestans using the in vitro amended agar method. Essential oils of Cymbopogon winterianus , Litsea cubeba , Mentha spicata , Pelargonium graveolens , Syzygium aromaticum , and Thymus vulgaris were observed to have the highest antifungal activity against P. infestans, with minimal inhibitory concentrations less than or equal to 1 μl ml ⁻¹ .

To evaluate the frequency and stability of the occurrence of P. infestans races and mating types in the Czech Republic, 338 monosporic isolates were collected from 31 sites in different potato-growing areas from 2012 to 2014 and in 2016. In total, 142 isolates were evaluated for virulence and race structure using the detached leaflet assay on Black’s differential set, supplemented with cultivar Sarpo Mira and somatic hybrid REG 46F. With the exception of virulence for resistance genes R9 and Rpi-blb-1, all virulence genes were detected among isolates, with a predominance of genes R1, R3, R7, R10, and R11. Most isolates were virulent to five or more R-genes, with a mean virulence complexity of 7.1. Among the 38 races detected, the most commonly occurring races were 1.2.3.4.6.7.10.11 and 1.2.3.4.7.10.11. Of the 338 isolates tested by the pairing test and the cleaved amplified polymorphism sequence (CAPS) marker, 40% were of the A1 mating type and 60% were of the A2 mating type, with an A1 : A2 isolate ratio demonstrating the predominance of the A2 mating type each year of the survey.

In the Czech Republic, increases in the area sown with oilseed rape (Brassica napus L.) and shifts to intensive crop farming systems have contributed to increased incidence and harmfulness of phoma stem canker. This study comprised a large-scale survey of the occurrence of two closely related causal agents of the disease, Leptosphaeria maculans and L. biglobosa, in oilseed rape tissues and the country-wide distribution of each fungal species. In the 2007–11 growing seasons, 1132 leaves with phoma leaf spot symptoms were sampled; from those, 977 L. maculans-type and 477 L. biglobosa-type leaf spots were sampled and analysed by species-specific PCR without pathogen isolation. There were 1159 leaf spots confirmed as infected by Leptosphaeria spp., with 65% of 907 L. maculans-type leaf spots infected by L. maculans only and 35% co-infected by both species; and with 88% of 252 L. biglobosa-type leaf spots infected by L. biglobosa only and 12% co-infected by both species. Furthermore, 217 monopycnidial isolates were collected from selected leaf spots and identified based on pigment production during solid- and liquid-media culture and PCR assay. Most (82%) isolates originating from L. maculans-type leaf lesions were L. maculans, and most (69%) isolates collected from L. biglobosa-type leaf lesions were L. biglobosa. Co-infection by both species was found in both L. maculans-type and L. biglobosa-type leaf lesions. In 2007–12, 708 stems with phoma stem canker symptoms and 2635 plant tissues from upper stem, stem base, root collar and taproot of each stem were sampled for PCR; symptoms on the four parts of each stem were assessed before taking tissue samples. There were 1495 plant tissues confirmed as infected by Leptosphaeria spp., with the proportion of plant tissue in which only L. biglobosa DNA (62%) was amplified greater than that with only L. maculans DNA (11%) or with both L. maculans and L. biglobosa DNA (27%). Although both species were detected in leaf samples in autumn, L. biglobosa was the more frequently detected species in stem samples in summer, suggesting that L. biglobosa is the more successful in colonising oilseed rape tissues in later growth stages of the plant in the Czech Republic.

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 β 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.