Main Biotypes Research Articles

Анализ генетического разнообразия сортов масличного льна селекции ВНИИМК с применением молекулярно-генетических маркеров

Oil flax (Linum usitatissimum L.) is one of the most important technical crops. The success of the breeding work is reached mostly by the genetic diversity of used initial germplasm. The molecular-genetic markers determine this diversity. Five cultivars of oil flax: Danik, Snegurok, Nilin, Y 117, and August, bred at the V.S. Pustovoit All-Russian Research Institute of Oil Crops were used as research material. To estimate genetic structure of cultivars we used 40 plants of each cultivar. Ten microsatellite loci markers were used for analysis. The amplification was conducted in a termocycler MiniAmp (Thermo Scientific, USA). The electrophoretic segregation of amplicons was carried out in 8% polyacrilamide gel in the native conditions. The primary indicators of genetic varia-tion (a number of alleles per a locus (Na), an effective number of alleles (Ne), observed (Ho) and wanted (He) heterozigosity, an informative Shennon’s index (I), fixation index (F), percentage of polymorphic loci (%P), genetic distances (D) were determined, analysis of molecular variation and principle coordinate analysis were done. The indicators of intraspecific genetic diversity decreased in the direction of cultivar range Nilin, Danik, August, Y 117, and Snegurok. Snegurok is a cultivar of linear type with some number of non-typical plants. The cultivar Y 117 is divided into two main biotype. Nilin, Danik, and August do not possess clear-marked structure. Most of the total variation is conditioned by differences between cultivars. The genetic distances between cultivars are quite big and equal from 0.752 to 0.986. All cultivars are distant from each other by two main coordinates. The conducted research showed small intravarietal and signif-icant intervarietal genetic diversity of five oil flax cultivars bred at the V.S. Pustovoit All-Russian Re-search Institute of Oil Crops and their potential in as initial material for breeding of new cultivars.

Структура сорта масличного льна Ы 117 на основе полиморфных микросателлитных локусов

Oil flax is a high-demand highly profitable crop. The modern standards of breeding and seed growing expect to the genetic passportization of the breeding achievement. Microsatellite sequences of DNA are an effective instrument to conduct the passportization of the genetic material. Oil flax cultivars self-pollinators are known to be able to possess the intravarietal heterogeneity. That’s why using a system of microsatellite DNA-markers, we estimated the genetic structure of the cultivar Y 117 of oil flax bred at the V.S. Pustovoit All-Russian Research Institute of Oil Crops. The estimation of the genetic structure was conducted in a series of 40 plants. We used 16 microsatellite markers in our work. The analysis of PCR products showed the series of plants is homogenous by 13 loci. Loci Lu3, Lu21, and Lu27b had two alleles. The analysis by three polymorphic loci of allele state of the separate plants of the cultivar reviled four groups of plants having a different type of allele combination (biotypes): A (Lu3175Lu21´109 Lu27b135), B (Lu3166Lu21´117Lu27b142), C (Lu3166Lu21´109Lu27b135), and D (Lu3175,166Lu21´109 Lu27b135) with a frequency of 57.5%, 37.5, 2.5, and 2.5%, respectively. Our results certifies that the cultivar Y 117 can be considered as a complex of biotypes. The main biotypes A and B totally reach 95% of the series. The biotypes D and C (totally 5% of the series) possibly are a result of pollination between plants of biotypes A and B. In a view of origin of this cultivar from some unique by the resistance to flax sickness plants, the amount of reviled biotypes is little. However taking in to attention an admissible level of allogamy within a cultivar, with time a shift in a combination of biotypes and some increase in their amount is possible. The combination of biotypes can be used as a parameter to control the stability of a cultivar, which is one of the definitive criteria of a cultivar according to the requirements of the International Union for the Protection of New Varieties of Plants (UPOV).

Nitrogen management at sowing and topdressing with the time of supply in the main biotype of oats grown in southern Brazil

Adjusting the nitrogen dose at sowing and topdressing with the time of application can improve nutrient management and increase oat productivity. The objective of the study is the most efficient and sustainable management of nitrogen use in the adjusted combination of nutrient dose at sowing and for topdressing with the moment of application on the productivity of biomass and oat grains, considering the main systems of cereal succession in southern Brazil.. The experiment was conducted in the years 2015, 2016 and 2017, in Augusto Pestana, RS, Brazil. The design was a randomized block with four replications, in a 4 x 4 factorial model, consisted of four doses of nitrogen at sowing time (0, 10, 30 and 60 kg ha-1), and topdressing dose by the total supplied of 70 and 100 kg ha-1 in succession system soybeans / oats and corn / oats, respectively, in the expectation of grain yield of 4000 kg ha-1, with the supply for topdressing considering four application times (0, 10, 30 and 60 days after emergence). The most efficient and sustainable management of nitrogen use for biomass and oat grains productivity occurs with the total supply of the covered nutrient around 30 days after emergence, regardless of the condition of the agricultural year and succession system.

Development of Feline Ileum- and Colon-Derived Organoids and Their Potential Use to Support Feline Coronavirus Infection.

Feline coronaviruses (FCoVs) infect both wild and domestic cat populations world-wide. FCoVs present as two main biotypes: the mild feline enteric coronavirus (FECV) and the fatal feline infectious peritonitis virus (FIPV). FIPV develops through mutations from FECV during a persistence infection. So far, the molecular mechanism of FECV-persistence and contributing factors for FIPV development may not be studied, since field FECV isolates do not grow in available cell culture models. In this work, we aimed at establishing feline ileum and colon organoids that allow the propagation of field FECVs. We have determined the best methods to isolate, culture and passage feline ileum and colon organoids. Importantly, we have demonstrated using GFP-expressing recombinant field FECV that colon organoids are able to support infection of FECV, which were unable to infect traditional feline cell culture models. These organoids in combination with recombinant FECVs can now open the door to unravel the molecular mechanisms by which FECV can persist in the gut for a longer period of time and how transition to FIPV is achieved.

Investigating the virulence genes and antibiotic susceptibility patterns of Vibrio cholerae O1 in environmental and clinical isolates in Accra, Ghana

BackgroundCholera has been endemic in Ghana since its detection in 1970. It has been shown that long-term survival of the bacteria may be attained in aquatic environments. Consequently, cholera outbreaks may be triggered predominantly in densely populated urban areas. We investigated clinical and environmental isolates of Vibrio cholerae O1 in Accra to determine their virulence genes, antibiotic susceptibility patterns and environmental factors maintaining their persistence in the environment.MethodsWater samples from various sources were analyzed for the presence of V. cholerae O1 using culture methods. Forty clinical isolates from a previous cholera outbreak were included in the study for comparison. Antibiotic susceptibility patterns of the bacteria were determined by disc diffusion. Virulence genes were identified by analyzing genes for ctx, tcpA (tcpAEl Tor tcpACl), zot, ompW, rbfO1 and attRS using PCR. Physicochemical characteristics of water were investigated using standard methods. One-way ANOVA and student t - test were employed to analyze the relationship between physicochemical factors and the occurrence of V. cholerae O1.ResultsEleven V. cholerae O1 strains were successfully isolated from streams, storage tanks and wells during the study period. All isolates were resistant to one or more of the eight antibiotics used. Multidrug resistance was observed in over 97% of the isolates. All isolates had genes for at least one virulence factor. Vibrio cholerae toxin gene was detected in 82.4% of the isolates. Approximately 81.8% of the isolates were positive for tcpAEl Tor gene, but also harbored the tcpAcl gene. Isolates were grouped into thirteen genotypes based on the genes analyzed. High temperature, salinity, total dissolved solids and conductivity was found to significantly correlate positively with isolation of V. cholerae O1. V. cholerae serotype Ogawa biotype El tor is the main biotype circulating in Ghana with the emergence of a hybrid strain.ConclusionsMultidrug resistant V. cholerae O1 with different genotypes and pathogenicity are present in water sources and co-exist with non O1/O139 in the study area.

The effect of human interferon alpha on replication of different bovine viral diarrhea virus strains.

Bovine viral diarrhea virus (BVDV) exists in two main biotypes: cytopathic (cp) and noncytopathic (ncp). Although some studies were done on the effect of interferon alpha (IFN-α) on BVDV, the effect of exogenous IFN against BVDV biotypes remains unclear. In the present study, we evaluated the comparative effect of exogenous human IFN-α (HuIFN-α) on different BVDV biotypes and genotypes. The results showed that exogenous HuIFN-α greatly inhibited the growth of different BVDV biotypes and genotypes. However, HuINF-α has a significant inhibitory effect on cp biotype compared to ncp one without significant variation between different genotypes. The effect of HuIFN-α on BVDV reached the maximum level at early stages of infection (0-20 h post infection) and increased in a dose-dependent manner (10-500 U/ml). Quantitative real-time RT-PCR was used to evaluate the effect of exogenous HuIFN-α on RNA synthesis of both BVDV biotypes. HuIFN-α reduced RNA production of cp by 4 logs compared to only 2 logs for ncp strains. Additionally, the antiviral effect of IFN-α against both BVDV biotypes seems to be independent of the RNA-dependent protein kinase (PKR) activation as assayed by direct analysis of in vivo phosphorylation of eIF2-α and by 2-aminopurine (2-AP) treatment. Collectively, these results indicated that the exogenous HuIFN-α treatment has an inhibitory effect not only on cp BVDV biotype but also on the ncp BVDV. The antiviral effect of exogenous HuIFN-α was biotype, time, dose but not genotype dependent. PKR has no role in the inhibitory effect suggesting that other IFN-antiviral pathways were involved. Keywords: BVDV biotypes; HuIFN-α; RNA synthesis; PKR-independent.

Analysis of body differences for the design of children’s clothing

This paper discusses anthropometric data obtained through 3D body scanner technology, aiming the dimensioning of Portuguese children´s measurements for the design of clothing. The study comprises boys and girls from the Northern Region of Portugal aged between two and ten years old. The main objective of this part of the study was to evaluate the possibility of grouping children with six and seven years old in a same garment size, proposing the same body measurements in the final standard measurements table. In addition to the children’s anthropometric measurements, it was checked the body conformation of the children and the possibility of identifying the main biotypes. As a preliminary result, through cluster analysis it was found that it is possible to combine these children in the same group in the proposed standard measurements table.

Genotypic and Phenotypic Assays to Distinguish Vibrio cholerae Biotype.

Vibrio cholerae is a motile gram-negative bacterium found in brackish water and the etiological agent of the fecal-oral disease cholera. Classical and El Tor are two main biotypes that make up the V. cholerae O1 serogroup, which each display unique genotypic and phenotypic characteristics that allow for reliable biotype characterization. While treatment for cholera is much the same despite the causative strain's biotype, such classification can be imperative for laboratory experiments and may have broader impacts in the biomedical field. In the early 2000s, clinical isolates were identified that contained genotypic and phenotypic traits from both biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to be more complicated than previous single-assay identification. Herein, we describe a series of PCR-based genetic screens (tcpA and ctxB) and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and Voges-Proskauer). Together, these assays are used for reliable biotype characterization of V. cholerae clinical (and environmental) isolates.

Diphtheria: An Emerging Disease

Diphtheria is caused by a toxigenic bacterium Corynebacterium diphtheriae which remains as one of the important causes of illness and death among children. Globally, epidemic waves of diphtheria have killed thousands of children in early 1920s, i.e., before the vaccine era. There are four main biotypes of C. diphtheriae available namely C.d. Gravis, C.d. Intermedius, C.d. Mitis, and C.d. Belfanti. Globally, the C.d. Intermedius is most often associated with exotoxin production, although all three strains are capable of producing exotoxin. Diphtheria patients are usually prescribed for diphtheria antitoxin and antibiotics such as erythromycin/penicillin. Keeping all the pathogenic information in context, here in the present review we have accumulated all the recent updates on diphtheria and also presented a timely discussion on diagnosis and vaccination procedure, which might assist future therapeutics.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates.

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000's clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

English

The sowing density adjustment in oat can maximize the productivity expression. The aim of this study is to define the behavior of productivity expression of biomass, grains, straw and harvest index through increasing sowing density in the main biotype cultivated in Southern Brazil. It proposes the possibility of indicating higher sowing density to the productivity maximization of biomass and grains. With the density adjusted to the grain productivity to simulate the reflexes on the biological and straw productivity and harvest index compared to recommended density, considering high and reduced tillering cultivars in different succession systems. The study was carried out in 2013, 2014 and 2015 in randomized block design with four replications in a 4 × 2 factorial scheme, for sowing density (100, 200, 300, 600 and 900 m-2) and oat cultivars (Brisasul and URS Taura), respectively, in the corn/oat and soybean/oat succession system. With the increase in sowing density, the biological and straw productivity evidence a linear behavior and the grain productivity and harvest index of quadratic behavior, regardless of the cultivar, agricultural year and succession system. It is possible to indicate a higher sowing density to the biomass and grain productivity maximization with sowing density close to 500 seeds m-2 in the main succession systems. In high and reduced tillering cultivars, the adjusted density compared to the recommended increased the biological and straw productivity, regardless of agricultural year and succession systems. Key words: Avena sativa, succession system, weather condition, regression.

Multicenter prospective epidemiological studies and case-control study on Haemophilus influenza infection in hospitalized neonates

Objective To explore the status of infection, biotype and resistant background of epidemic strains of Haemophilus influenza(Hi) in neonates, and the clinical features of neonatal pneumonia caused by Hi. Methods The multicenter prospective epidemiological cross-sectional design was used; four hospitals in west Sichuan China were chosen as research field, sputum bacterial culture was done and biological typing, PCR identification and drug sensitivity test of Hi epidemic strains were carried out among 0 to 28 days hospitalized neonates with infectious pneumonia in four hospitals located in west Sichuan China.The cases with discharge diagnosis of neonatal infectious pneumonia with Hi positive separation were assumed as case group, and the same number of cases with Hi negative separation were assumed as control group according to 1∶1 extraction at the same time. Results Totally 757 cases with admitting diagnosis of neonatal infectious pneumonia in four hospitals were investigated in west Sichuan from November 2014 to October 2015, and the rate of sputum culture was 95.51%(723/757). The total pathogenic bacteria positive rate of sputum culture was 15.63%(113/723), and Hi positive rate was 1.94%(14/723), Hi accounting for 12.39%(14/113) of the pathogenic bacteria in respiratory system.All the Hi strains(100%) were non-typeable Haemophilus influenzae(NTHi) indentified by PCR.The main biotypes of 14 Hi strains were typeⅠwith 57.1%(8/14), type Ⅲ with 14.3%(2/14) and type Ⅳ with 28.6%(4/14). The total of 35.7%(5/14) bacterial strains of β-lactamase distributed in four hospitals, 7.1%(1/14) bacterial strains of β-lactamase-nonproducing-ampicillin-resistant, and 35.7%(5/14) bacterial strains of β-lactamase-positive-ampicillin-resistant were found in four hospitals.The rate of resistance and mediation to cefuroxime were 14.2%(2/14) respectively, the resistance rate to cefaclor was 35.7%(5/14), and 21.4%(3/14) to ofloxacin.None of the 14 strains was resistant to amoxicillin clavulanic acid and cefotaxime.The 1∶1 matching analysis had been done for 10 cases with discharge diagnosis of neonatal pneumonia caused by Hi.There were no statistical differences in general conditions, main symptoms, lung signs, X-ray appearance, classification of leukocyte and C-reactive protein levels between case group and control group(P>0.05). Conclusion All the Hi isolated from sputum were NTHi among 0 to 28 days inpatients with neonatal pneumonia and the main biotype were type Ⅰ, type Ⅲ and type Ⅳ in west Sichuan China.There were no significant differences in the clinical manifestations of neonatal pneumonia with NTHi infection and other infectious pneumonia. Key words: Haemophilus influenzae; Infectious pneumonia; Epidemiology; Resistance rate; Neonate

Characterization of eight Russian wheat aphid (Hemiptera: Aphididae) biotypes using two-category resistant-susceptible plant responses.

Eight biotypes of the Russian wheat aphid, Diuraphis noxia (Kurdjumov), have been discovered in the United States since 2003. Biotypes are identified by the distinct feeding damage responses they produce on wheat carrying different Russian wheat aphid resistance genes, namely, from Dn1 to Dn9. Each Russian wheat aphid biotype has been named using plant damage criteria and virulence categories that have varied between studies. The study was initiated to compare the plant damage caused by all the eight known Russian wheat aphid biotypes, and analyze the results to determine how Russian wheat aphid virulence should be classified. Each Russian wheat aphid biotype was evaluated on 16 resistant or susceptible cereal genotypes. Plant damage criteria included leaf roll, leaf chlorosis, and plant height. The distribution of chlorosis ratings followed a bimodal pattern indicating two categories of plant responses, resistant or susceptible. Correlations were significant between chlorosis ratings and leaf roll (r(2) = 0.72) and between chlorosis ratings and plant height (r(2) = 0.48). The response of 16 cereal genotypes to feeding by eight Russian wheat aphid biotypes found RWA1, RWA2, RWA6, and RWA8 to differ in virulence, while Russian wheat aphid biotypes RWA3, RWA4, RWA5, and RWA7 produced similar virulence profiles. These biotypes have accordingly been consolidated to what is hereafter referred to as RWA3/7. Our results indicated that the five main biotypes RWA1, RWA2, RWA3/7, RWA6, and RWA8 can be identified using only four wheat genotypes containing Dn3, Dn4, Dn6, and Dn9.

Identification ofYersinia enterocoliticaat the Species and Subspecies Levels by Fourier Transform Infrared Spectroscopy

Yersinia enterocolitica and other Yersinia species, such as Y. pseudotuberculosis, Y. bercovieri, and Y. intermedia, were differentiated using Fourier transform infrared spectroscopy (FT-IR) combined with artificial neural network analysis. A set of well defined Yersinia strains from Switzerland and Germany was used to create a method for FT-IR-based differentiation of Yersinia isolates at the species level. The isolates of Y. enterocolitica were also differentiated by FT-IR into the main biotypes (biotypes 1A, 2, and 4) and serotypes (serotypes O:3, O:5, O:9, and "non-O:3, O:5, and O:9"). For external validation of the constructed methods, independently obtained isolates of different Yersinia species were used. A total of 79.9% of Y. enterocolitica sensu stricto isolates were identified correctly at the species level. The FT-IR analysis allowed the separation of all Y. bercovieri, Y. intermedia, and Y. rohdei strains from Y. enterocolitica, which could not be differentiated by the API 20E test system. The probability for correct biotype identification of Y. enterocolitica isolates was 98.3% (41 externally validated strains). For correct serotype identification, the probability was 92.5% (42 externally validated strains). In addition, the presence or absence of the ail gene, one of the main pathogenicity markers, was demonstrated using FT-IR. The probability for correct identification of isolates concerning the ail gene was 98.5% (51 externally validated strains). This indicates that it is possible to obtain information about genus, species, and in the case of Y. enterocolitica also subspecies type with a single measurement. Furthermore, this is the first example of the identification of specific pathogenicity using FT-IR.

Antimicrobial Susceptibility, Biochemical and Genetic Profiles of Staphylococcus haemolyticus Strains Isolated from the Bloodstream of Patients Hospitalized in Critical Care Units

Staphylococcus haemolyticus strains (n=20), responsible of blood stream infections, were consecutively isolated from patients hospitalized in two different wards at high risk of infection. Strains displayed high rate of resistance to oxacillin (90%). All strains but two with decreased susceptibility (MIC = 4 μg/mL), were sensitive to vancomycin. Ten strains were resistant to teicoplanin. Among the strains susceptible to glycopeptides, three displayed heteroresistance to vancomycin and seven to teicoplanin, when tested by Etest technique with 2 x McFarland inoculum. Biochemical reactions allowed to assign strains to eight biotypes, with 11 strains clustering under two main biotype A and biotype B. Pulsed-field-gel-electrophoresis (PFGE) identified 11 different PFGE-types. Seven strains grouping under the major PFGE-type 1 and three strains clustering in PFGE-type 2, closely correlated to biotype A and biotype B respectively. Seven teicoplanin-resistant isolates clustered in the PFGE-type 1, two in the PFGE-type 2 and one in PFGE-type 5. Therefore, teicoplanin-resistant strains were biochemically and genetically related and clonally distributed, despite different clones of S. haemolyticus circulated in the units during the study period.

The host range of Polymyxa betae in Britain

The host range of Polymyxa betae on common arable weed species in Britain was determined by growing plants in naturally infested soil and examining their root systems for the presence of resting spores (cystosori). Of the 24 species tested, only Atriplex patula and Chenopodium album of the Chenopodiaceae, and Silene alba of the Caryophyllaceae, were found to be heavily infected. S. alba is a newly recorded host species for Polymyxa. The host specificity of isolates of P. betae from Beta vulgaris, C. album and A. patula was investigated by observing which of 11 test plants could be infected by the isolates obtained from this soil. Three main biotypes of P. betae appeared to be distinguishable: one which was able to infect all chenopodiaceous species; one which had a narrower host range; and one which was able to infect S. alba. The role of weed species in the epidemiology of rhizomania is discussed.

Inheritance of Gliadin Proteins Associated with Quality in Durum Wheat1

The inheritance of gliadin protein differences was investigated because prior electrophoretic studies revealed a close relationship between certain endosperm proteins and gluten strength in durum wheat (Triticum turgidum L. var. durum). Particular attention was focused on gliadin band 45, as it is closely associated with dough strength, and on gliadin band 42, which is associated with poor viscoelastic properties. These inheritance studies were conducted on crosses between the two main biotypes of the Australian cv. Duramba. Results from F1, F2, and BC1F1, populations were consistent with the hypothesis that the syntheses of gliadin bands 45 and 42 are controlled by alleles at a single locus. Dosage effects were exhibited by both alleles; however, band 42 displayed a greater degree of dominance than did band 45. These results should prove useful in breeding durum wheat for improved pasta quality.