Miloslav Zouhar

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 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.

First report about the trapping activity of Stropharia rugosoannulata acanthocytes for Northern Root Knot Nematode

This study summarises the results of in vitro screening of the nematophagous activity of Stropharia rugosoannulata and Arthrobotrys oligospora. The tests were conducted with Meloidogyne hapla plant parasitic nematode juveniles placed into Petri dishes containing cultures of the tested fungal species. Immobilisation of the nematodes was observed after 4 and 24 hours. Both species of fungi showed nematophagous activity, however it was much stronger and faster in the case of S. rugosoannulata.

Botanical pesticides and their human health safety on the example of Citrus sinensis essential oil and Oulema melanopus under laboratory conditions

Biopesticides derived from plants have recently received increasing interest as an alternative to conventional chemicals suitable for integrated pest management and organic farming. In this study, Citrus sinensis (Osbeck) essential oil was tested for its potential for wheat protection against Oulema melanopus (L.). The chemical composition of the essential oil was analyzed by gas chromatography-mass spectrometry (GC-MS). Twenty-four compounds were identified in the oil among which d-limonene was a predominant component, followed by myrcene, α-pinene, linalool, sabinene, decanal, and other minor components. Direct contact toxicity assay showed the effectiveness of the essential oil against O. melanopus larvae causing the mortality of 85% during 48 hours. The persistence study revealed high dissipation rate of the oil from treated plants, whereas concentrations lower than 0.01 g/kg were detected by GC-MS 5 min after the application of the oil, suggesting its environmental and food safety. These results, together with previous literature reports reviewed in this study, indicate the great potential of the C. sinensis essential oil for future use in crop protection against insect pests.

Alternative methods of carrot (Daucus carota) protection against the northern root knot nematode (Meloidogyne hapla)

Abstract The presence of northern root knot nematode (Meloidogyne hapla) has considerably increased in the Czech Republic during the last 10 years. The management of this pest is difficult; thus, the aim of this study was to test two alternative pest management techniques on carrots under field conditions. When Indian mustard (Brassica juncea) was ploughed and the treated area was then covered with polyethylene sheets to prolong effect of the treatment, the root gall numbers decreased; whereas a nitrolime application combined with plastic covering had a positive effect on the experimental plants’ fresh weight.

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.

Molecular and morphological exploration of a mixed population of two potato-parasiting nematode species, Globodera rostochiensis and G. pallida

SummaryIn this work, we report results of molecular and morphological analyses of a potato field population of Globodera (Nematoda: Heteroderidae) species, in Slovakia. Unexpectedly, our data show a mixed occurrence of two potato cyst nematode species, Globodera rostochiensis and G. pallida, in this locality. To our knowledge, this is the first report of mixed occurrence of these economically important plant-parasitic species in the same locality in the Central Europe. In addition, this finding reinforces the possibility of the cross-hybridization between these two nematode species that might result in a generation of new genotypes.

Superoxide dismutase isoelectric focusing patterns as a tool to differentiate pathotypes of Globodera spp.

Isoelectric focusing was used to separate proteins from cyst extracts of potato cyst nematode (PCN) populations. In a first set of assays, cyst extracts from standard populations of Globodera rostochiensis pathotypes Ro1, Ro2, Ro3, Ro2/3, Ro4, and Ro5, and G. pallida pathotypes Pa2 and Pa3, were loaded on isoelectric focusing gels. Gels were stained for superoxide dismutase (SOD), esterase, and glucose-6-phosphate isomerase (GPI). Twelve bands of SOD activity were detected, six (B1-B6) migrating towards the basic zone and the other six (A1-A6) migrating towards the acidic zone, starting from the loading point. A cluster analysis was carried out based on a data matrix that reported the presence or absence of SOD bands on the isozyme electrophoresis patterns (IEPs). Globodera spp. were clearly distinguished and, within G. rostochiensis , Ro2 and Ro4 shared a high level of similarity, respectively, with Ro3 and Ro5; moreover, Ro1 could be clearly distinguished from Ro2/3 and Ro4/5. Globodera pallida Pa2 and Pa3 also shared a high level of similarity. In contrast, esterase and GPI IEPs did not discriminate among G. rostochiensis standard pathotypes. Subsequently, 14 field populations of G. rostochiensis , five from Italy and nine from Venezuela, and three field populations of G. pallida , two from Italy and one from Chile, were assayed to obtain SOD IEPs. Italian populations had previously been identified at pathotype level by bioassays according to the generally accepted international test using different resistant potato cultivars and clones. The cluster analysis carried out on the SOD IEPs of all the populations tested formed four distinct groups within G. rostochiensis and only one within G. pallida . Pathotype identification of Globodera populations by SOD IEPs was not able to discriminate between bioassay standard couples Ro2/Ro3, Ro4/Ro5 and Pa2/Pa3. Therefore, three groups were assigned to Ro1, Ro2/3 and Ro4/5, and a fourth group to Pa2/Pa3. Four Venezuelan populations, not identified at pathotype level by bioassays, formed a distinct fifth group. By means of the method described herein, four additional unknown Venezuelan populations could be assigned to Ro1 group and one to Ro2/Ro3 group; one G. pallida population from Chile was assigned to Pa2/Pa3 group.

First report of the potato cyst nematode (Globodera pallida) in the Czech Republic.

Potato cyst nematode, Globodera pallida, was detected in several soil samples collected from various areas of the Czech Republic. Globodera rostochiensis pathotype Ro1 is known to be widespread in the Czech Republic. G. pallida was reported from the neighboring countries of Austria, Germany, and Poland, and also was suspected to be present in the Czech Republic, but it has never been unambiguously proved (1). Recently, nematode isolates have been recovered that multiply readily on Ro1 resistant potato cultivars. These isolates were identified on the basis of three tests: (i) microscopic observations of cyst vulval area morphology and stylets of second-stage larvae; (ii) enzyme-linked immunosorbent assay using a commercial kit; and (iii) a polymerase chain reaction method (2). A complete series of G. pallida and G. rostochiensis pathotypes from Scotland and Germany served as controls. Results were identical for all three methods used. One sample contained G. pallida only, five samples contained mixtures of G. rostochiensis and G. pallida, and one sample contained G. rostochiensis only. The origin of G. pallida contamination is unknown. Strict quarantine measures have been taken to prevent G. pallida from spreading into neighboring areas. To our knowledge, this is the first evidence of the occurrence of G. pallida in the Czech Republic. References: (1) J. Potocek et al. EPPO Bull. 21:81, 1991. (2) M. Zouhar et al. Plant Prot. Sci. 36:81, 2000.

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.

Sensitivity to Fungicides and Esential Oils in Czech Isolates of Phytophthora infestans

Abstract 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 EC50 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.