Functional analysis of the copy 1 of the fixNOQP operon of Ensifer meliloti under free‐living micro‐oxic and symbiotic conditions

Aim

In this work, phenotypic analyses of a Ensifer meliloti fixN1 mutant under free‐living and symbiotic conditions have been carried out.

Methods and Results

Ensifer meliloti fixN1 mutant showed a defect in growth as well as in TMPD‐dependent oxidase activity when cells were incubated under micro‐oxic conditions. Furthermore, haem c staining analyses of a fixN1 and a fixP1 mutant identified two membrane‐bound c‐type cytochromes of 27 and 32 kDa, present in microaerobically grown cells and in bacteroids, as the FixO and FixP components of the E. meliloti cbb₃ oxidase. Under symbiotic conditions, fixN1 mutant showed a clear nitrogen fixation defect in alfalfa plants that were grown in an N‐free nutrient solution during 3 weeks. However, in plants grown for a longer period, fixNOQP1 copy was not indispensable for symbiotic nitrogen fixation.

Conclusions

The copy 1 of the fixNOQP operon is involved in E. meliloti respiration and growth under micro‐oxic conditions as well as in the expression of the FixO and FixP components of the cbb₃ oxidase present in free‐living microaerobic cultures and in bacteroids. This copy is important for nitrogen fixation during the early steps of the symbiosis.

Significance and Impact of the Study

It is the first time that a functional analysis of the E. meliloti copy 1 of the fixNOQP operon is performed. In this work, the cytochromes c that constitute the cbb₃ oxidase operating in free‐living micro‐oxic cultures and in bacteroids of E. meliloti have been identified.     

Investigation of Mannheimia haemolytica bacteriophages relative to host diversity

Aims

This study aimed to characterize the impact of lytic and temperate bacteriophages on the genetic and phenotypic diversity of Mannheimia haemolytica from feedlot cattle.

Methods and Results

Strictly lytic phages were not detected from bovine nasopharyngeal (n = 689) or water trough (n = 30) samples, but Myoviridae‐ or Siphoviridae‐like phages were induced from 54 of 72 M. haemolytica strains by mitomycin C, occasionally from the same strain. Phages with similar restriction fragment length polymorphism profiles (RFLP ≥70% relatedness) shared common host serotypes 1 or 2 (P < 0·000 1). Likewise, phages with similar RFLP tended to occur in genetically related host bacteria (70–79% similarity). Host range assays showed that seven phages from host serotypes 1, 2 and 6 lysed representative strains of serotypes 1, 2 or 8. The genome of vB_MhM_1152AP from serotype 6 was found to be collinear with P2‐like phage φMhaA1‐PHL101.

Conclusions

Prophages are a significant component of the genome of M. haemolytica and contribute significantly to host diversity. Further characterization of the role of prophage in virulence and persistence of M. haemolytica in cattle could provide insight into approaches to control this potential respiratory pathogen.

Significance and Impact of the Study

This study demonstrated that prophages are widespread within the genome of M. haemolytica isolates and emphasized the challenge of isolating lytic phage as a therapeutic against this pathogen.     

A single sequence context cannot satisfy all non-AUG initiator codons in yeast<Superscript>†</Superscript>

Background  

Previous studies in Saccharomyces cerevisiae showed that ALA1 (encoding alanyl-tRNA synthetase) and GRS1 (encoding glycyl-tRNA synthetase) respectively use ACG and TTG as their alternative translation initiator codons. To explore if any other non-ATG triplets can act as initiator codons in yeast, ALA1 was used as a reporter for screening.     

A meta‐analysis of the association between Helicobacter pylori (H. pylori) infection and hyperemesis gravidarum

Background

Hyperemesis gravidarum remains a common, distressing, and significant yet poorly understood disorder during pregnancy. The association between maternal Helicobacter pylori (H. pylori) infection and hyperemesis gravidarum has been increasingly recognized and investigated. This study thus aimed to provide an updated review and meta‐analysis of the topic.

Methods

Using the search terms (H. pyloriOR Helicobacter ORHelicobacter pyloriOR infection) AND (pregnancy OR emesis OR hyperemesis gravidarum OR nausea OR vomiting), a preliminary search on the PubMed, Ovid, Web of Science, Google Scholar, and WanFang database yielded 372 papers published in English between January 1st, 1960 and June 1st, 2017.

Results

A total of 38 cross‐sectional and case‐control studies, with a total of 10 289 patients were eligible for review. Meta‐analysis revealed a significant association between H. pylori infection and hyperemesis gravidarum during pregnancy, with a pooled odds ratio of 1.348 (95% CI: 1.156‐1.539, P < .001). Subgroup analysis found that serologic and stool antigen tests were comparable methods of detecting H. pylori as they yielded similar odds ratios.

Limitations

Although the studies did not have high heterogeneity (I² = 28%), publication bias was observed, and interstudy discrepancies in the diagnostic criteria adopted for hyperemesis gravidarum limit the reliability of findings. Also, 15 of the included studies were from the same country (Turkey), which could limit the generalizability of current findings. The prevalence of H. pylori infection varies throughout the world, and there may also be pathogenic differences as most strains of H. pylori in East Asia carry the cytotoxin‐associated gene A gene.

Conclusion

H. pylori infection was associated with an increased likelihood of hyperemesis gravidarum during pregnancy. Given the high prevalence of H. pylori infections worldwide, detecting H. pylori infection and the eradication of maternal H. pylori infection could be part of maternal hyperemesis gravidarum management. Further confirmation with robust longitudinal studies and mechanistic investigations are needed.     

Past vegetation dynamics in the Yellowstone region highlight the vulnerability of mountain systems to climate change

Aim

Reconstruct the long‐term ecosystem dynamics of the region across an elevational gradient as they relate to climate and local controls. In particular, we (1) describe the dominant conifers' history; (2) assess changes in vegetation composition and distribution; and (3) note periods of abrupt change versus stability as means of better understanding vegetation responses to environmental variability.

Location

Greater Yellowstone Ecosystem (GYE; USA).

Time period

16.5 ka bp ‐present.

Major taxa studied

Juniperus, Picea, Abies, Pinus, Pseudotsuga.

Methods

The vegetation reconstruction was developed from 15 pollen records. Results were interpreted based on modern pollen–vegetation relationships estimated from a suite of regression‐based approaches.

Results

Calibrated pollen data suggest that late‐glacial vegetation, dominated by shrubs and Juniperus, lacks a modern counterpart in the area. Picea, Abies and Pinus expanded at 16 ka bp in association with postglacial warming and co‐occurred in mixed‐conifer parkland/forest after 12 ka bp . This association along with Pinus contorta forest, which was present after 9 ka bp , has persisted with little change at middle and high elevations to the present day. This stability contrasts with the dynamic history of plant communities at low elevations, where shifts between parkland, steppe and forest over the last 8,000 years were likely driven by variations in effective moisture and fire.

Main conclusions

The postglacial vegetation history of the GYE highlights the dynamic nature of mountain ecosystems and informs on their vulnerability to future climate change: (1) most of the conifers have been present in the area for >12,000 years and survived climate change by adjusting their elevational ranges; (2) some plant associations have exhibited stability over millennia as a result of nonclimatic controls; and (3) present‐day forest cover is elevationally more compressed than at any time in history, probably due to the legacy of the Medieval Climate Anomaly and the Little Ice Age.     

Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples

Aims

Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10‐fold serial dilutions of Bacillus anthracis spores using quantitative real‐time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6·5 × 10¹ and 1·3 × 10² CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS).

Methods and Results

The MasterPure kit effectively and consistently isolated DNA from low amounts of B. anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 ± 14, 68 ± 8, 66 ± 4 and 52 ± 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors.

Conclusions

Detection of B. anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit.

Significance and Impact of the Study

The extraction protocol identified herein combined with IMS is novel for B. anthracis and allows detection of low levels of B. anthracis spores from contaminated food samples.     

Biochanin A improves fibre fermentation by cellulolytic bacteria

Aims

The objective was to determine the effect of the isoflavone biochanin A (BCA) on rumen cellulolytic bacteria and consequent fermentative activity.

Methods and Results

When bovine microbial rumen cell suspensions (n = 3) were incubated (24 h, 39°C) with ground hay, cellulolytic bacteria proliferated, short‐chain fatty acids were produced and pH declined. BCA (30 μg ml^?1) had no effect on the number of cellulolytic bacteria or pH, but increased acetate, propionate and total SCFA production. Addition of BCA improved total digestibility when cell suspensions (n = 3) were incubated (48 h, 39°C) with ground hay, Avicel, or filter paper. Fibrobacter succinogenes S85, Ruminococcus flavefaciens 8 and Ruminococcus albus 8 were directly inhibited by BCA. Synergistic antimicrobial activity was observed with BCA and heat killed cultures of cellulolytic bacteria, but the effects were species dependent.

Conclusions

These results indicate that BCA improves fibre degradation by influencing cellulolytic bacteria competition and guild composition.

Significance and Impact of the Study

BCA could serve as a feed additive to improve cellulosis when cattle are consuming high‐fibre diets. Future research is needed to evaluate the effect of BCA on fibre degradation and utilization in vivo.     

Screening of white‐rot fungi for bioprocessing of wheat straw into ruminant feed

Aim

In this study, the biological variation for improvement of the nutritive value of wheat straw by 12 Ceriporiopsis subvermispora, 10 Pleurotus eryngii and 10 Lentinula edodes strains was assessed. Screening of the best performing strains within each species was made based on the in vitro degradability of fungal‐treated wheat straw.

Methods and Results

Wheat straw was inoculated with each strain for 7 weeks of solid state fermentation. Weekly samples were evaluated for in vitro gas production (IVGP) in buffered rumen fluid for 72 h. Out of the 32 fungal strains studied, 17 strains showed a significantly higher (P < 0·05) IVGP compared to the control after 7 weeks (227·7 ml g^?1 OM). The three best Ceriporiopsis subvermispora strains showed a mean IVGP of 297·0 ml g^?1 OM, while the three best P. eryngii and L. edodes strains showed a mean IVGP of 257·8 and 291·5 ml g^?1 OM, respectively.

Conclusion

Ceriporiopsis subvermispora strains show an overall high potential to improve the ruminal degradability of wheat straw, followed by L. edodes and P. eryngii strains.

Significance and Impact of the Study

Large variation exists within and among different fungal species in the valorization of wheat straw, which offers opportunities to improve the fungal genotype by breeding.     

Biocontrol potential of phylloplane bacterium Ochrobactrum anthropi BMO‐111 against blister blight disease of tea

Aims

The present study was carried out to screen the phylloplane bacteria from tea for antagonism against grey blight caused by Pestalotiopsis theae and blister bight caused by Exobasidium vexans and to further evaluate the efficient isolates for disease control potential under field condition.

Methods and Results

A total of 316 morphologically different phylloplane bacteria were isolated. Among the antagonists, the isolates designated as BMO‐075, BMO‐111 and BMO‐147 exhibited maximum inhibitory activity against both the pathogens under in vitro conditions and hence were selected for further evaluation under microplot field trial. Foliar application of 36‐h‐old culture of BMO‐111 (1 × 10⁸ colony‐forming units ml^?1) significantly reduced the blister blight disease incidence than the other isolates. The culture of BMO‐111 as well as its culture filtrate effectively inhibited the mycelial growth of various fungal plant pathogens. The isolate BMO‐111 was identified as Ochrobactrum anthropi based on the morphological and 16S rDNA sequence analyses.

Conclusions

It could be concluded that the biocontrol agent O. anthropi BMO‐111 was effective against blister blight disease of tea.

Significance and Impact of the Study

Further study is required to demonstrate the mechanism of its action and formulation for the biocontrol potential against blister blight disease of tea.     

Enhanced inhibition of Aspergillus niger on sedge (Lepironia articulata) treated with heat‐cured lime oil

Aims

This study aimed to examine heat curing effect (30–100°C) on antifungal activities of lime oil and its components (limonene, p‐cymene, β‐pinene and α‐pinene) at concentrations ranging from 100 to 300 μl ml^?1 against Aspergillus niger in microbiological medium and to optimize heat curing of lime oil for efficient mould control on sedge (Lepironia articulata).

Methods and Results

Broth dilution method was employed to determine lime oil minimum inhibitory concentration, which was at 90 μl ml^?1 with heat curing at 70°C. Limonene, a main component of lime oil, was an agent responsible for temperature dependencies of lime oil activities observed. Response surface methodology was used to construct the mathematical model describing a time period of zero mould growth on sedge as functions of heat curing temperature and lime oil concentration. Heat curing of 90 μl ml^?1 lime oil at 70°C extended a period of zero mould growth on sedge to 18 weeks under moist conditions.

Conclusions

Heat curing at 70°C best enhanced antifungal activity of lime oil against A. niger both in medium and on sedge.

Significance and Impact of the Study

Heat curing of lime oil has potential to be used to enhance the antifungal safety of sedge products.     

Herd prevalence of Enterobacteriaceae producing CTX‐M‐type and CMY‐2 β‐lactamases among Japanese dairy farms

Aims

To determine the herd prevalence of Enterobacteriaceae producing CTX‐M‐type extended‐spectrum β‐lactamases (ESBLs) among 381 dairy farms in Japan.

Methods and Results

Between 2007 and 2009, we screened 897 faecal samples using BTB lactose agar plates containing cefotaxime (2 μg ml^?1). Positive isolates were tested using ESBL confirmatory tests, PCR and sequencing for CTX‐M, AmpC, TEM and SHV. The incidence of Enterobacteriaceae producing CTX‐M‐15 (n = 7), CTX‐M‐2 (n = 12), CTX‐M‐14 (n = 3), CMY‐2 (n = 2) or CTX‐M‐15/2/14 and CMY‐2 (n = 4) in bovine faeces was 28/897 (3·1%) faecal samples. These genes had spread to Escherichia coli (n = 23) and three genera of Enterobacteriaceae (n = 5). Herd prevalence was found to be 20/381 (5·2%) dairy farms. The 23 E. coli isolates showed clonal diversity, as assessed by multilocus sequence typing and pulsed‐field gel electrophoresis. The pandemic E. coli strain ST131 producing CTX‐M‐15 or CTX‐M‐27 was not detected.

Conclusions

Three clusters of CTX‐M (CTX‐M‐15, CTX‐M‐2, CTX‐M‐14) had spread among Japanese dairy farms.

Significance and Impact of the Study

This is the first report on the prevalence of multidrug‐resistant CTX‐M‐15–producing E. coli among Japanese dairy farms.     

Expression of HPV-11 L1 protein in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

Background  

We have investigated the possibility and feasibility of producing the HPV-11 L1 major capsid protein in transgenic Arabidopsis thaliana ecotype Columbia and Nicotiana tabacum cv. Xanthi as potential sources for an inexpensive subunit vaccine.     

TCF/β-catenin plays an important role in HCCR-1 oncogene expression

Background  

Oncogene HCCR-1 functions as a negative regulator of the p53 and contributes to tumorigenesis of various human tissues. However, it is unknown how HCCR-1 contributes to the cellular and biochemical mechanisms of human tumorigenesis.     

Neuraminidase Inhibitors from marine‐derived actinomycete Streptomyces seoulensis

Aims

This work was performed to characterize new secondary metabolites with neuraminidase (NA) inhibitory activity from marine actinomycete strains.

Methods and Results

An actinomycete strain IFB‐A01, capable of producing new NA inhibitors, was isolated from the gut of shrimp Penasus orientalis and identified as Streptomyces seoulensis according to its 16S rRNA sequence (over 99% homology with that of the standard strain). Repeated chromatography of the methanol extract of the solid‐substrate culture of S. seoulensis IFB‐A01 led to the isolation of streptoseolactone ( 1 ), and limazepines G ( 2 ) and H ( 3 ). The structures of 1 – 3 were determined by a combination of IR, ESI‐MS, 1D (¹H and ¹³C NMR, and DEPT) and 2D NMR experiments (HMQC, HMBC, ¹H‐¹H COSY and NOESY). Compounds 1 – 3 showed significant inhibition on NA in a dose‐dependent manner with IC₅₀ values of 3·92, 7·50 and 7·37 μmol l^?1, respectively.

Conclusions

This is the first report of two new ( 1 and 2 ) and known ( 3 , recovered as a natural product for the first time in the work) NA inhibitors from the marine‐derived actinomycete S. seoulensis IFB‐A01.

Significance and Impact of the Study

The three natural NA inhibitors maybe of value for the development of drug(s) necessitated for the treatment of viral infections.     

Fate of Escherichia coli O145 present naturally in bovine slurry applied to vegetables before harvest,after washing and simulated wholesale and retail distribution

Aims

To determine the fate of Escherichia coli on vegetables that were processed through commercial wash treatments and stored under simulated retail conditions at 4°C or wholesale at fluctuating ambient temperatures (0–25°C, dependent on season).

Methods and Results

Bovine slurry that was naturally contaminated with E. coli O145 was applied without dilution or diluted 1:10 using borehole water to growing potatoes, leeks or carrots. Manure was applied 1 week prior to harvest to simulate a near‐harvest contamination event by manure deposition or an application of contaminated water to simulate a flooding event or irrigation from a contaminated water source. At harvest, crops were contaminated at up to 2 log cfu g^?1. Washing transferred E. coli into the water of a flotation tank used for potato washing and did not completely remove all traces of contamination from the crop. Manure‐contaminated potatoes were observed to contain 0·72 cfu E. coli O145 g^?1 after processing and retail storage. Manure‐contaminated leeks harboured 0·73–1·55 cfu E. coli O145 g^?1 after washing and storage. There was no cross‐contamination when leeks were spray washed. Washing in an abrasive drum resulted in less than perfect decontamination for manure‐contaminated carrots. There were five post‐distribution isolations from carrots irrigated with contaminated water 24 h prior to harvest.

Conclusions

Standard commercial washing and distribution conditions may be insufficient to reliably control human pathogenic E. coli on fresh produce.

Significance and Impact

Previous speculation that the cause of a UK foodborne disease outbreak was soil from imperfectly cleaned vegetables is plausible.     

Effects of extracellular DNA and DNA‐binding protein on the development of a Streptococcus intermedius biofilm

Aims

The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone‐like DNA‐binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity.

Methods and Results

Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7‐hydoxyl‐9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one) and anti‐HLP antibody without fixation, respectively. DNase I treatment (200 U ml^?1) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 μg ml^?1) purified from Strep. intermedius, other Gram‐positive bacteria, Gram‐negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild‐type, HLP‐downregulated strain and control strains. In contrast, the addition of eDNA (>1 μg ml^?1) decreased the biofilm mass of all Strep. intermedius strains.

Conclusions

These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity.

Significance and Impact of the Study

eDNA‐ and HLP‐targeting strategies may be applicable to novel treatments for bacterial biofilm‐related infectious diseases.     

Characterization and flocculation properties of biopolymeric flocculant (glycosaminoglycan) produced by Cellulomonas sp. Okoh

Aims

Bioflocculant production potential of an actinobacteria isolated from a freshwater environment was evaluated and the bioflocculant characterized.

Methods and Results

16S rDNA nucleotide sequence and BLAST analysis was used to identify the actinobacteria and fermentation conditions, and nutritional requirements were evaluated for optimal bioflocculant production. Chemical analyses, FTIR, 1H NMR spectrometry and SEM imaging of the purified bioflocculant were carried out. The 16S rDNA nucleotide sequences showed 93% similarities to three Cellulomonas species (strain 794, Cellulomonas flavigena DSM 20109 and Cellulomonas flavigena NCIMB 8073), and the sequences was deposited in GenBank as Cellulomonas sp. Okoh (accession number HQ537132 ). Bioflocculant was optimally produced at an initial pH 7, incubation temperature 30°C, agitation speed of 160 rpm and an inoculum size of 2% (vol/vol) of cell density 1·5 × 10⁸ cfu ml^?1. Glucose (88·09% flocculating activity; yield: 4·04 ± 0·33 g l^?1), (NH₄)₂NO₃ (82·74% flocculating activity; yield: 4·47 ± 0·55 g l^?1) and MgCl₂ (90·40% flocculating activity; yield: 4·41 g l^?1) were the preferred nutritional source. Bioflocculant chemical analyses showed carbohydrate, protein and uronic acids in the proportion of 28·9, 19·3 and 18·7% in CPB and 31·4, 18·7 and 32·1% in PPB, respectively. FTIR and 1H NMR indicated the presence of carboxyl, hydroxyl and amino groups amongst others typical of glycosaminoglycan. SEM imaging revealed horizontal pleats of membranous sheets closely packed.

Conclusion

Cellulomonas sp. produces bioflocculant predominantly composed of glycosaminoglycan polysaccharides with high flocculation activity.

Significance and Impact of the Study

High flocculation activity suggests suitability for industrial applications; hence, it may serve to replace the hazardous flocculant used in water treatment.     

Antibacterial and antifungal activities of pyroligneous acid from wood of Eucalyptus urograndis and Mimosa tenuiflora

Aims

This work aimed to evaluate the antibacterial and antifungal activities of two types of pyroligneous acid (PA) obtained from slow pyrolysis of wood of Mimosa tenuiflora and of a hybrid of Eucalyptus urophylla × Eucalyptus grandis.

Methods and Results

Wood wedges were carbonized on a heating rate of 1·25°C min^?1 until 450°C. Pyrolysis smoke was trapped and condensed to yield liquid products. Crude pyrolysis liquids were bidistilled under 5 mmHg vacuum yielding purified PA. Multi‐antibiotic‐resistant strains of Escherichia coli, Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus aureus (ATCC 25923) had their sensitivity to PA evaluated using agar diffusion test. Two yeasts were evaluated as well, Candida albicans (ATCC 10231) and Cryptococcus neoformans. GC‐MS analysis of both PAs was carried out to obtain their chemical composition. Regression analysis was performed, and models were adjusted, with diameter of inhibition halos and PA concentration (100, 50 and 20%) as parameters. Identity of regression models and equality of parameters in polynomial orthogonal equations were verified. Inhibition halos were observed in the range 15–25 mm of diameter.

Conclusions

All micro‐organisms were inhibited by both types of PA even in the lowest concentration of 20%.

Significance and Impact of the Study

The feasibility of the usage of PAs produced with wood species planted in large scale in Brazil was evident and the real potential as a basis to produce natural antibacterial and antifungal agents, with real possibility to be used in veterinary and zootechnical applications.     

Reversing expansion of Calamagrostis epigejos in a grassland biodiversity hotspot: Hemiparasitic Rhinanthus major does a better job than increased mowing intensity

Questions

Can hemiparasitic Rhinanthus major originating from a local population suppress the competitive clonal grass Calamagrostis epigejos and reverse its expansion in species‐rich semi‐natural grasslands? Does sowing seeds of R. major facilitate restoration of target meadow vegetation? Is R. major more beneficial for biodiversity restoration/conservation than increased mowing intensity, a conventional measure to suppress C. epigejos?

Location

?ertoryje National Nature Reserve, Bílé Karpaty (White Carpathians) Protected Landscape Area, Czech Republic.

Methods

We conducted a before‐after‐control‐impact experiment in meadow patches heavily infested by C. epigejos: eight blocks, each containing four plots with four treatment combinations: (1) traditional management, i.e. mowing once in summer, (2) mowing in summer and autumn (3) mowing in summer and seed sowing of R. major, (4) mowing in summer and autumn and seed sowing of R. major. Above‐ground biomass of C. epigejos and vegetation composition of each of the plots were monitored every year from 2013 to 2016. To assess the effects of treatments, we analysed biomass production of C. epigejos, herb layer cover and vegetation composition.

Results

Both sowing R. major and an additional autumn meadow cut significantly suppressed C. epigejos. Their effects were additive and of comparable size. Both treatments also had significant but markedly different effects on community composition. Rhinanthus major facilitated directional community composition change towards the regional Brachypodio‐Molinetum meadows. In contrast, increased mowing intensity significantly decreased frequency of threatened species, which however may have also been influenced by R. major.

Conclusions

Sowing of autochthonous R. major seeds was demonstrated as an efficient tool to suppress C. epigejos and facilitate community restoration. It can be combined with an additional meadow cut to further accelerate decline of the grass. The additional cut should however be used as a short‐term practice (1–2 years) only to minimize potential negative effects of its long‐term application on some threatened plant species. The effects of R. major are comparable to those of Rhinanthus alectorolophus reported previously. As a species occurring naturally in species‐rich dry grasslands, R. major has a broader and longer‐term application potential than R. alectorolophus in ecological restoration and conservation of these communities.