Pathotype Characterization of Plasmodiophora brassicae, the Cause of Clubroot in Central Europe and Sweden (2016-2020).

Clubroot, caused by Plasmodiophora brassicae, is a crucial oilseed rape disease worldwide. Information on the virulence of P. brassicae populations is essential to apply disease control with proper clubroot-resistant cultivars. In 2016-2020, 84 isolates of P. brassicae were collected in the Czech Republic (CZ), Germany (DE), Poland (PL), and Sweden (SW). Pathotypes were designated using 17 Brassica hosts, including the European Clubroot Differentials (ECD), Somé set, and clubroot-resistant oilseed rape cv. Mendel. According to the ECD set, virulence analyses differentiated the isolates into 42 pathotypes. The most common pathotypes were 16/31/31 (in DE, PL, and SW) and 16/06/12 (in CZ, DE, and PL). Six pathotypes were found according to the Somé set, including 1-4 pathotypes per country. P1 was most prevalent in DE, PL, and SW, while P3 was abundant in CZ, DE, and PL. The current study provides clear evidence for a shift towards increased virulence in P. brassicae populations compared to previous studies. Several isolates overcame the resistance of cv. Mendel and of Brassica rapa genotypes ECD 01 to ECD 04. Considering all investigated samples, significant negative correlations were found between clubroot incidence and the frequency of oilseed rape in crop rotation, as for clubroot incidence and soil pH.

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.

Laimaphelenchus africanus n. sp. (Tylenchomorpha: Aphelenchoididae) from South Africa, a morphological and molecular phylogenetic study, with an update to the diagnostics of the genus.

A newly recovered population of the genus Laimaphelenchus from a dead maritime pine wood sample in Potchefstroom, South Africa, representing a new species, named L. africanus n. sp., is herein described and illustrated based on morphological and molecular data. The new species is mainly characterized by the following: 750-987 µm long females; a cephalic region with no disc and six cephalic lobs not divided by ribs; a 10.0-12.5 µm long stylet; four incisures in the lateral field; secretory-excretory pore (SE-pore) at slightly posterior to the nerve ring; vulva with a well-developed anterior flap, vagina with two well-developed sclerotized pieces; post-vulval uterine sac (PUS) 63-125 µm long; tail conical, 30-44 µm long, ventrally curved with a subventral stalk in terminus, lacking tubercles, with six to nine small projections at the tip in scanning electron microscopy (SEM); and rare males with 17 µm long spicules. The new species was morphologically compared to those species of the genus with a stalk in tail terminus, lacking tubercles, a vulval flap and four incisures in the lateral field viz., L. liaoningensis, L. preissii, L. simlaensis, L. sinensis, L. spiflatus, and L. unituberculus. Phylogenetically, the new species was placed into the major Laimaphelenchus clade using partial large subunit ribosomal DNA (LSU rDNA D2-D3) sequences. An overall literature review corroborated the presence of the stalk (currently with two main groups) at the tail end is the main characteristic trait delimiting the genus. A compendium based on the characters of the stalk, presence/absence of a vulval flap in females and number of the lateral lines was also established.

First record of the ectoparasitic nematode Amplimerlinius macrurus (Nematoda: Tylenchida) on the perennial grass Miscanthus × giganteus (Angiosperms: Poaceae) in Ukraine.

The growing interest in biomass production of Miscanthus × giganteus (M × g) (Poaceae) on agricultural and marginal lands has prompted researches to identify plant pathogens and diseases affecting this crop which has a great potential for production of biofuels and different bioproducts. A soil survey of nematodes in the M × g rhizosphere and a report on the collection of the plant-parasitic nematode Amplimerlinius macrurus (Belonolaimidae) were accomplished in two locations in Ukraine. It is known that this family of nematodes can reduce the root system and biomass of Poaceae family plants. Both molecular and morphological characters were used to identify the nematodes; measurements and photomicrographs of the species were presented. This is the first documentation and description of A. macrurus in Ukraine to the best of our knowledge. Further investigation is underway to confirm the pathogenicity of this species on perennial grasses plantations.

Mixed-Mating Model of Reproduction Revealed in European Phytophthora cactorum by ddRADseq and Effector Gene Sequence Data.

A population study of Phytophthora cactorum was performed using ddRADseq sequence variation analysis completed by the analysis of effector genes-RXLR6, RXLR7 and SCR113. The population structure was described by F-statistics, heterozygosity, nucleotide diversity, number of private alleles, number of polymorphic sites, kinship coefficient and structure analysis. The population of P. cactorum in Europe seems to be structured into host-associated groups. The isolates from woody hosts are structured into four groups described previously, while isolates from strawberry form another group. The groups are diverse in effector gene composition and the frequency of outbreeding. When populations from strawberry were analysed, both asexual reproduction and occasional outbreeding confirmed by gene flow among distinct populations were detected. Therefore, distinct P. cactorum populations differ in the level of heterozygosity. The data support the theory of the mixed-mating model for P. cactorum, comprising frequent asexual behaviour and inbreeding alternating with occasional outbreeding. Because P. cactorum is not indigenous to Europe, such variability is probably caused by multiple introductions of different lineages from the area of its original distribution, and the different histories of sexual recombination and host adaptation of particular populations.

Insecticidal and Behavioral Effect of Microparticles of Pimpinella anisum Essential Oil on Larvae of Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The Colorado potato beetle ranks as one of the most important potato pests, mainly due to its high feeding rate during all developmental stages, particularly third and fourth larval instar, and high fecundity. The effect of essential oil (EO) from anise (Pimpinella anisum L. [Apiales: Apiaceae]) prepared as conventional and encapsulated (EN) formulations on the mortality and antifeedant responses of young larvae of Colorado potato beetles was studied to evaluate the insecticidal and antifeedant effects of five concentrations of this EO and to assess the persistence of both formulations on potato plants. The EN formulation had a significantly higher residual amount compared with that of the conventionally formulated EO. Significantly different values of LC50 and LC90 (ppm) were established for the EO (LC50 = 1,700 and LC90 = 9500) and EN (LC50 = 3,100 and LC90 = 14,300) formulations. The effects of both P. anisum formulations (EO and EN) applied topically to Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) larvae were distinctly different from those observed with the contact treatment. At the highest concentration of 20,000 ppm, the mortality of the second instars of the L. decemlineata larvae did not exceed 25%. On the other hand, both tested formulations of P. anisum were highly effective when administered orally. The encapsulated EO formulation achieved a distinctly higher biological activity. Our results confirm that the EO from P. anisum, especially the encapsulated formulation, has high insecticidal properties that may lead to the development of new organic products for the control of Colorado potato beetles.

The immunophilin repertoire of Plasmodiophora brassicae and functional analysis of PbCYP3 cyclophilin.

Plasmodiophora brassicae is a soil-borne pathogen that belongs to Rhizaria, an almost unexplored eukaryotic organism group. This pathogen requires a living host for growth and multiplication, which makes molecular analysis further complicated. To broaden our understanding of a plasmodiophorid such as P. brassicae, we here chose to study immunophilins, a group of proteins known to have various cellular functions, including involvement in plant defense and pathogen virulence. Searches in the P. brassicae genome resulted in 20 putative immunophilins comprising of 11 cyclophilins (CYPs), 7 FK506-binding proteins (FKBPs) and 2 parvulin-like proteins. RNAseq data showed that immunophilins were differentially regulated in enriched life stages such as germinating spores, maturing spores, and plasmodia, and infected Brassica hosts (B. rapa, B. napus and B. oleracea). PbCYP3 was highly induced in all studied life stages and during infection of all three Brassica hosts, and hence was selected for further analysis. PbCYP3 was heterologously expressed in Magnaporthe oryzae gene-inactivated ΔCyp1 strain. The new strain ΔCyp1+ overexpressing PbCYP3 showed increased virulence on rice compared to the ΔCyp1 strain. These results suggest that the predicted immunophilins and particularly PbCYP3 are activated during plant infection. M. oryzae is a well-studied fungal pathogen and could be a valuable tool for future functional studies of P. brassicae genes, particularly elucidating their role during various infection phases.

Cyclophilins: Less Studied Proteins with Critical Roles in Pathogenesis.

Cyclophilins (EC 5.2.1.8) belong to a subgroup of proteins known as immunophilins, which also include FK506-binding proteins and parvulins. Members of the immunophilins have two main characteristic properties: (i) peptidyl-prolyl cis-trans isomerase activity and (ii) the ability to bind immunosuppressant molecules of fungal origin. Cyclophilins are some of the most conserved proteins present in eukaryotes and prokaryotes, and they have been implicated in diverse cellular processes and responses to multiple biotic and abiotic stresses. Cyclophilins have been exploited in humans and plants extensively, but they have only recently received attention in regard to phytopathogens. In Phellinus sulphurascens and species of the genus Leptosphaeria and Phytophthora, high expression of cyclophilins was found to be related to infection. Moreover, recent studies of cyclophilins in certain phytopathogens, such as Magnaporthe oryzae, Botrytis cinerea, Cryphonectria parasitica, and Puccinia triticina, demonstrated their roles as a pathogenicity factors. In addition to pathogenicity, cyclophilins have high affinity for the immunosuppressive drug cyclosporin A, which is a potent antifungal agent. Although cyclophilins are highly conserved in phytopathogens, because they have been less studied, their role remains largely unknown. In this review, we provide detailed information on the cyclophilins in several phytopathogens, including fungi and oomycetes, as well as their role in suppressing plant immunity.

Suppression of Meloidogyne incognita by the Entomopathogenic Fungus Lecanicillium muscarium.

The entomopathogenic fungus Lecanicillium muscarium (Petch) Zare and Gams is currently being developed as a biocontrol agent against insect pests, as well as some plant-pathogenic fungi and bacteria. Data about its activity against plant-parasitic nematodes exist, but are relatively limited. To expand this understanding, we investigated the biocontrol efficiency of three isolates of L. muscarium (Lm) against the root knot nematode, Meloidogyne incognita, in both in vitro and in vivo conditions. In our experiments, the maximum number of nematode eggs, juveniles (J2s), females, and egg masses that were parasitized were quantified after a 72-h exposure to the fungus. The isolate Lm1 was designated as the best biocontrol agent against nematode eggs as well as J2s. It showed the highest colonization of eggs and significantly decreased egg hatching events. The results from two additional isolates, Lm2 and Lm3, were also significant (P = 0.05) but less pronounced than those observed with Lm1. L. muscarium treatments had significant (P = 0.05) positive effects on plant shoot and root growth compared with the growth of control plants. These results suggest the effectiveness of the fungus may be due to either the infection of eggs and J2s, or the production of secondary metabolites that induced plant defense mechanisms and lead to systemic resistance. Our study demonstrates that L. muscarium could be used as a potential biocontrol agent against root knot nematodes.

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.

Exploitation of FTA cartridges for the sampling, long-term storage, and DNA-based analyses of plant-parasitic nematodes.

The use of DNA-based analyses in molecular plant nematology research has dramatically increased over recent decades. Therefore, the development and adaptation of simple, robust, and cost-effective DNA purification procedures are required to address these contemporary challenges. The solid-phase-based approach developed by Flinders Technology Associates (FTA) has been shown to be a powerful technology for the preparation of DNA from different biological materials, including blood, saliva, plant tissues, and various human and plant microbial pathogens. In this work, we demonstrate, for the first time, that this FTA-based technology is a valuable, low-cost, and time-saving approach for the sampling, long-term archiving, and molecular analysis of plant-parasitic nematodes. Despite the complex structure and anatomical organization of the multicellular bodies of nematodes, we report the successful and reliable DNA-based analysis of nematode high-copy and low-copy genes using the FTA technology. This was achieved by applying nematodes to the FTA cards either in the form of a suspension of individuals, as intact or pestle-crushed nematodes, or by the direct mechanical printing of nematode-infested plant tissues. We further demonstrate that the FTA method is also suitable for the so-called "one-nematode-assay", in which the target DNA is typically analyzed from a single individual nematode. More surprisingly, a time-course experiment showed that nematode DNA can be detected specifically in the FTA-captured samples many years after initial sampling occurs. Collectively, our data clearly demonstrate the applicability and the robustness of this FTA-based approach for molecular research and diagnostics concerning phytonematodes; this research includes economically important species such as the stem nematode (Ditylenchus dipsaci), the sugar beet nematode (Heterodera schachtii), and the Northern root-knot nematode (Meloidogyne hapla).

Trichostrongylus colubriformis rDNA polymorphism associated with arrested development.

Polymerase chain reaction-restriction fragment length polymorphism) was performed on the cistron of rDNA in the two groups of infective larvae Trichostrongylus colubriformis-the population with and without ability to undergo arrested development. General primers designed by Caenorhabditis elegans rDNA were used for the amplification of the rDNA cistron between genes 18S and 28S. Amplified fragments were digested by using a series of restriction endonucleases. Hinc II restriction profiles unique for each T. colubriformis populations were observed, and therefore enzyme Hinc II appears to be useful for the determination of populations with and without the ability to undergo arrested development. Molecular markers of arrested development ability have not been studied on this part of rDNA before.