M13 Primer Research Articles

Isolation, identification, antibiotic resistance profile and molecular analysis of Ornithobacterium rhinotracheal isolates from turkeys.

Ornithobacterium rhinotracheal (ORT) infects numerous birds, particularly chickens and turkeys. ORT is an emerging bacterial pathogen of global concern in the poultry industry. As ORT is rapidly spreading throughout commercial poultry, it requires intensive studies of its epidemiology, diagnostic procedures, molecular typing, virulence genes and antimicrobial resistance. The present study was conducted in isolation and identification of ORT from slaughtered turkeys. Cleft palate swabs of 200 were collected from slaughtered turkeys and cultured on blood agar. ORT was characterized using biochemical tests and PCR targeting the ORT 16S rRNA gene. Virulence genes of isolates were determined targeting adenylate kinase (adk), copA and virulence-associated protein D (vapD) genes. Additionally, diversity of ORT isolates was performed by enterobacterial repetitive intergenic consensus (ERIC) and RAPD PCR. Disk diffusion was used to determine the antibiotic sensitivity of the isolates. ORT was identified in 23 (11.5%) samples using both the biochemical tests and PCR. The result of detecting virulence genes showed that all the isolates (23: 100%) had the adk gene, whereas two (8.7%) isolates had the copA gene, and seven (30.43%) isolates had the vapD gene. Molecular typing of isolates revealed 21 different patterns by RAPD PCR assay using M13 primer and 20 distinct patterns by ERIC PCR test. Both ERIC and RAPD PCR were distinctive methods for investigating the genetic diversity of ORT isolates. The antibiotic resistance test showed that 18 (78.26%) isolates were resistant to gentamicin, amikacin, cefazolin, streptomycin and penicillin. All isolates (100%) were resistant to cloxacillin and fosfomycin. This study showed the prevalence of ORT in turkey and high resistance of this bacterium to many common veterinary antibiotics. Moreover, both ERIC and RAPD PCR are distinctive methods for investigating the genetic diversity of ORT isolates. These data may help monitor antibiotic resistance and typing of ORT in epidemiological studies and serve as the foundation for designing region-specific vaccines for future use.

Developing a novel selection method for alcoholic fermentation starters by exploring wine yeast microbiota from Greece.

The selection of native yeast for alcoholic fermentation in wine focuses on ensuring the success of the process and promoting the quality of the final product. The purpose of this study was firstly to create a large collection of new yeast isolates and categorize them based on their oenological potential. Additionally, the geographical distribution of the most dominant species, Saccharomyces cerevisiae, was further explored. Towards this direction, fourteen spontaneously fermented wines from different regions of Greece were collected for yeast typing. The yeast isolates were subjected in molecular analyses and identification at species level. RAPD (Random Amplified Polymorphic DNA) genomic fingerprinting with the oligo-nucleotide primer M13 was used, combined with Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique. All yeast isolates were scrutinized for their sensitivity to killer toxin, production of non-desirable metabolites such as acetic acid and H2S, β-glucosidase production and resistance to the antimicrobial agent; SO2. In parallel, S. cerevisiae isolates were typed at strain level by interdelta - PCR genomic fingerprinting. S. cerevisiae strains were examined for their fermentative capacity in laboratory scale fermentation on pasteurized grape must. Glucose and fructose consumption was monitored daily and at the final point a free sorting task was conducted to categorize the samples according to their organoleptic profile. According to our results, among the 190 isolates, S. cerevisiae was the most dominant species while some less common non-Saccharomyces species such as Trigonopsis californica, Priceomyces carsonii, Zygosaccharomyces bailii, Brettanomyces bruxellensis and Pichia manshurica were identified in minor abundancies. According to phenotypic typing, most isolates were neutral to killer toxin test and exhibited low acetic acid production. Hierarchical Cluster Analysis revealed the presence of four yeast groups based on phenotypic fingerprinting. Strain level typing reported 20 different S. cerevisiae strains from which 65% indicated fermentative capacity and led to dry wines. Sensory evaluation results clearly discriminated the produced wines and consequently, the proposed yeast categorization was confirmed. A novel approach that employs biostatistical tools for a rapid screening and classification of indigenous wine yeasts with oenological potential, allowing a more efficient preliminary selection or rejection of isolates is proposed.

Cloning, sequencing, and characterizing of soil antibiotic active-producing Streptomyces species-specific DNA markers

Association of the antibiotic activity of the soil Streptomyces isolates to their genetic profiles analyzed through RAPD-PCR fingerprints prompted us here in this study to use the most common bands as specific markers to identify homologous proteins within these isolates by cloning, sequencing, and characterizing these markers. Six out of twelve DNA bands ranged between 600 and 1350 bp previously obtained by RAPD-PCR analysis were purified out of the RAPD gels, and then cloned into pGEM-T Easy vector system. Success of the cloning process was confirmed by digesting purified plasmids with EcoRI. The clones namely No. 54, 55, 20, 56, 57, and 58 were sequenced using the DNA BigDye Terminator Sequencing System utilizing the M13 primer. Results indicated that the size of the inserted sequences is 599, 566, 522, 870, 857, and 254 bp, in clones No. 54. 55, 20, 56, 57, and 58, respectively. Homologous proteins of the six cloned sequences generated by DNA blast software indicated that the highest score of protein homology was scored for clone No. 54 with 87 % homology to putative secreted pectate lyase [Streptomyces coelicolor A3(2)]. The other clones showed less homology with 77 % homology for the clones No. 55 and 56, 73 % homology for the clone No. 20, and 55 % homology for the clones No. 57 and 58. The association of homologous proteins to the reported RAPD pattern is confirmed here for the first time, and the resulting DNA cloned fragments deserve further molecular analysis.

Genetic Structure and Molecular Identities of 46 Apple Landraces (Malus Mill.) in China

In this study, we used genotyping to determine the genetic structure and molecular identities of apple landraces from six species of Malus Mill. in China, based on the fingerprints revealed by microsatellite markers with tailed primer M13. A total of 46 apple landraces of Malus Mill. selected from the National Apple and Pear Germplasm Repository in Xingcheng, China were genotyped with 14 SSR markers. The primers differentiated all the accessions. At least three SSR primers, CH04h02, CH01f07a and CH04g07, with a higher heterozygosity and Shannon’s information index than other combinations can distinguish all the accessions. All the alleles for these three primers were arranged in descending order, and they were assigned values beginning with 01. Character strings were constituted by combining all the codes of the three primers for every accession. By such means, separate and special molecular identities were obtained for every apple landrace, which could be expressed in the form of a bar code. Using such bar codes, trees can be labeled and scanned, which aids in the identification and tracking of genebank collections. The eight-step method for establishing the molecular identities of apple landraces reported here may serve as a reference when determining the molecular identities establishment of other apple germplasms of Malus Mill. This method might also be used for the establishment of a molecular database to aid the preservation of Malus Mill., which is in imminent danger in China. It may also be used to improve the gene bank management of Malus Mill.

Selection of Autochthonous Yeasts Isolated from the Intestinal Tracts of Cobia Fish (Rachycentron canadum) with Probiotic Potential

Some yeast strains have been proposed as probiotics to improve the health of cultured fish. Cobia is a tropical benthopelagic fish species with potential for marine aquaculture; however, one of the main limitations to its large-scale production is the high mortality of fish larvae. In this study, we evaluated the probiotic potential of autochthonous yeasts from the intestines of cobia. Thirty-nine yeast isolates were recovered from the intestinal mucosa of 37 adult healthy cobia by culture methods. Yeasts were identified by sequencing of the ITS and D1/D2 regions of the 28S rRNA gene and typed by RAPD-PCR using the M13 primer. Yeast strains with unique RAPD patterns were characterized in terms of their cell biomass production ability; anti-Vibrio, enzymatic, and hemolytic activity; biofilm production; hydrophobicity; autoaggregation; polyamine production; safety; and protection of cobia larvae against saline stress. Candida haemuloni C27 and Debaryomyces hansenii C10 and C28 were selected as potential probiotics. They did not affect the survival of larvae and showed biomass production >1 g L-1, hydrophobicity >41.47%, hemolytic activity γ, and activity in more than 8 hydrolytic enzymes. The results suggest that the selected yeast strains could be considered as potential probiotic candidates and should be evaluated in cobia larvae.

Molecular characterization of Ornithobacterium rhinotracheale strains isolated from chickens in the Northwestern Iran.

Ornithobacterium rhinotracheale is the etiological agent of chickens and turkeys' respiratory diseases, reduction of eggs, growth retardation, and death. Present research aimed tocond uct the isolation, recognition, and molecular investigations of the bacterium in commercial broiler chicken flocks in East Azerbaijan province, Northwest of Iran, by the partial sequencing of 16S rRNA gene, ERIC‑PCR, and RAPD‑PCR with the OPG11 and M13 primers. We obtained 330 specimens from tracheal swabs of 33 slaughtered broiler flocks, of which we found 14 isolates (4.24%) of five flocks (15.15%) to be O. rhinotracheale. Typing by RAPD assay with the OPG11 primer, and ERIC‑PCR, classified the isolates in two types of 1 and 2 molecular patterns, most of which belonged to type 1. However, the M13 primer‑based RAPD technique was inappropriate for distinguishing and categorizing the isolates andgenerating all of them in the same pattern. In a phylogenetic analysis of O. rhinotracheale based on 16S rRNA sequences, the strains generated three clusters (I‑III), in which all of the studied isolates fell in one cluster (cluster I). Based on the results obtained from the RAPD and ERIC‑PCR assays, the genetic patterns of broiler‑chicken‑isolated O. rhinotracheale strains in Northwestern Iran had no significant differences.

Survival During Simulated Digestion, Bacteriocin Production, and Bile Salt Hydrolase Activities of Lactic Acid Bacteria in Poi, a Fermented Hawaiian Food

ObjectivesPoi, a paste from pounded, cooked taro corms, is culturally significant in Hawaii. This study aimed to investigate the probiotic properties of lactic acid bacteria (LAB) in poi.MethodsNaturally fermented poi was subjected to simulated oral, gastric, and intestinal digestions in vitro. The LAB count of poi after each digestion step was determined. After in vitro digestion, ten LAB colonies were randomly isolated. Poi from three different batches were tested independently. Thirty LAB isolates were differentiated using RAPD-PCR with M13, P4, and P7 primers. Strains with unique band patterns were identified by 16s rRNA sequencing. Overnight cultures of representative LAB isolates were collected for cell-free supernatant (CFS) and neutralized to pH 6.5. Antimicrobial properties of CFS were determined against Listeria monocytogenes using agar well diffusion method. Strains that produced CFS forming inhibition zones were identified as bacteriocin positive. Bile salt hydrolase (BSH) activities were assessed by spotting the LAB cultures on MRS agars containing 0.5% sodium salts of taurodeoxycholic acid (TDCA) or glycodeoxycholic acid (GDCA) and 0.037% CaCl2. Strains that formed white precipitation zones were identified as BSH positive, which is associated with bile acid resistance and cholesterol reduction ability.ResultsThe LAB counts of poi before digestion, and after simulated oral, gastric, and intestinal digestions were 10.67, 9.59, 6.55, and 7.15 log CFU, respectively. After digestion, 14 unique LAB strains (A1-A14) were identified from poi, among which nine strains (A1, A2, A4, A5, A7, A8, A10, A11, and A14) belonged to Leuconostoc lactis, two strains (A3 and A6) belonged to Lactococcus lactis, two strains (A12 and A13) belonged to Weissella confuse, and one strain (A9) belonged to Leuconostoc pseudomesenteroides. Strain A13 showed bacteriocin-producing activity against L. monocytogenes. Strains A2, A3, A4, A6, A7, A8, A9, A12, and A13 showed TDCA hydrolase activities. Strains A3 and A6 showed GDCA hydrolase activities.ConclusionsApproximately 0.03% of LAB in poi survived human digestion conditions. Some surviving LAB strains showed probiotic properties including bacteriocin-producing and bile salt hydrolase activities, which warrant further analysis.Funding SourcesUSDA-Hatch, USDA-ARS.

Strain Typing of Trichosporon asahii Clinical Isolates by Random Amplification of Polymorphic DNA (RAPD) Analysis.

Objective The aim of this study was to identify and isolate Trichosporon asahii ( T. asahii ) from clinical samples and to assess the genetic relatedness of the most frequently isolated strains of T. asahii using random amplification of polymorphic DNA (RAPD) primers GAC-1 and M13. Methods All the clinical samples that grew Trichosporon species, identified and confirmed by polymerase chain reaction (PCR) using Trichosporon genus-specific primers, were considered for the study. Confirmation of the species T. asahii was carried out by T. asahii- specific PCR. Fingerprinting of the most frequently isolated T. asahii isolates was carried out by RAPD using random primers GAC-1 and M13. Results Among the 72 clinical isolates of Trichosporon sp. confirmed by Trichosporon -specific PCR, 65 were found to be T. asahii as identified by T. asahii- specific PCR. Fingerprinting of the 65 isolates confirmed as T. asahii using GAC-1 RAPD primer yielded 11 different patterns, whereas that of M13 primer produced only 5 patterns. The pattern I was found to be the most predominant type (29.2%) followed by pattern III (16.9%) by GAC-1 primer. Conclusions This study being the first of its kind in India on strain typing of T. asahii isolates by adopting RAPD analysis throws light on genetic diversity among the T. asahii isolates from clinical samples. Fingerprinting by RAPD primer GAC-1 identified more heterogeneity among the T. asahii isolates than M13.

Microbiological characteristics and identification of yeast microbiota of traditional mouldy civil cheese

Investigation of the traditional cheeses and bringing them to the industry contributes to the development of dairy technology in terms of quality and diversity. For this purpose, traditional Turkish mouldy Civil cheese, which has a rich microbial diversity and is lovingly consumed, was studied and its yeast flora revealed. Samples were evaluated in terms of aerobic plate count, psychrotrophic bacteria, proteolytic bacteria, yeast, mould, coliform, enterococci, presumptive lactobacilli, presumptive lactococci, Staphylococcus aureus and Enterobacteriaceae counts. Seventy isolates were characterised by RAPD-PCR with M13 primer. Six diverse species were detected: Yarrowia lipolytica, Kluyveromyces marxianus, Pichia membranifaciens, Debaryomyces hansenii, Pichia fermentans and Candida zeylanoides. As a result, yeast diversity in mouldy Civil cheese may have an important role on development of characteristic blue cheese flavour and selected yeast strains could be used as adjunct culture to improve the quality of the final product.

Diversity and technological characterization of Pediococcus pentosaceus strains isolated from Nigerian traditional fermented foods

The diversity and technological properties of Pediococcus pentosaceus strains, isolated from some traditional fermented foods in Nigeria, were studied with the aim of selecting suitable strains as a starter and/or adjunct cultures. Eighteen lactic acid bacteria isolates from ogi, gari and fufu were presumptively identified as Pediococcus species using the conventional methods, these isolates were screened for technological and safety properties such as exopolysaccharide production, acidifying activity, antimicrobial activity, and biogenic amine production. The LAB isolates were further differentiated by using internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP), randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and pulsed-field gel electrophoresis (PFGE) molecular typing techniques. The Pediococcus sp. displayed antimicrobial activities against Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecium. They all had high acidifying activities and were able to reduce the pH to lower than 5.20 after 24 h. Pediococcus pentosaceus OF31 showed interesting potential technological characteristics due to its antimicrobial activity and exopolysaccharide production. The combination of RAPD-PCR (using primers OPA20 and M13) and PFGE-SfiI genotypic fingerprinting technique allows successful intraspecific differentiation. The present study confirmed the presence of exopolysaccharide-producing strain of P. pentosaceus in indigenous fermented foods which could be used as autochthonous starter culture to impart more functional attributes to fermented foods.

Phaeoacremonium tuscanicum, a new fungal pathogen associated with oak decline in Zagros forests, Iran

An extensive survey on phylogeny and pathology of fungi associated with the oak decline in Zagros forests located in Western Azarbaijan, Kurdistan, Kermanshah, Lorestan, and Ilam provinces, a large number of collected fungal isolates seventeen isolates morphologically resembled the members of Phaeoacremonium. Similar DNA fingerprinting patterns were generated for all isolates using M13 primer. Thus, one representative isolate (IRAN 4348C) was analyzed based on tub2 sequence data using maximum parsimony and neighbor-joining algorithms and identified as Phaeoacremonium tuscanicum. Pathogenicity was confirmed following Koch’s postulates on two-year-old Quercus brantii seedlings under greenhouse conditions. To our knowledge, it is the first time Ph. tuscanicum is reported from oak trees and confirmed as a pathogenic fungal species on Q. brantii in the world.

Поліморфізм штамів Acidithiobacillus ferrooxidans, ізольованих із відходів вугільної та енергетичної промисловості України

The results of Acidithiobacillus ferrooxidans strains genetic variability studies that were first isolated from waste by coal and energy industries of Ukraine are reported in this article. These strains, according to the results of previous studies, are fully consistent with the biological properties of A. ferrooxidans bacteria given in Bergey’s Manual of Determinative Bacteriology and other original works [8, 10, 12, 18] also strains studied, regardless of habitat, were resistant to temperature and pH, had a mixed type of food, similar energy sources used by strains, etc. [1, 10, 11]. Using PCR was confirmed the affiliation of isolated from different of origin dumps of acidophilic chemolithotrophic strains with A. ferrooxidans. Genetic polymorphism of the strains was studied by RAPD-PCR using universal primer M13. It was shown that strains of A. ferrooxidans that were isolated from various dumps have a wide genetic diversity. By PCR, their affiliation with the species A. ferrooxidans was confirmed. Comparison analysis of the obtained RAPD profiles showed the variability of the strains which coincides with their main phenotypic properties, as described earlier [11]. The most heterogeneous profiles were characteristic of A. ferrooxidans DTV 1, A. ferrooxidans Lad 5 and A. ferrooxidans Lad 27. The obtained RAPD profiles served as the basis for the generation of the dendrogram constructed using the Neighbor-Joining method and calcula­ting the similarity matrix, based on the coefficient of similarity of Nei & Li. Based on the obtained dendrogram, the formation of two clusters that combine similar strains is shown. The obtained indicators of the probability of formation of nodes of the constructed dendrogram range from 65.0 to 76.0 %%, which confirms the high degree of similarity between strains grouped by clusters. It is also shown that the first of the cluster includes strains that were isolated from coal and waste from its enrichment, and the second cluster includes strains that were isolated from coal waste. The obtained data confirm that the genetic variability of the strains depends on the primary location of isolation of the strains, and the coefficients of similarity between them varies from 0.182 to 0.80.

Screening and identification of bacteriocin-like inhibitory substances producing lactic acid bacteria from fermented products

Abstract A total of 544 isolates of lactic acid bacteria isolated from fermented products: 451 isolates from fermented products from Thailand and 93 isolated from French fermented products were screened for bacteriocin production. 10 isolates showed antimicrobial activities. Identification of the selected strains by random amplification of polymorphic DNA-polymerase change reaction using the primers E1, E2 and M13 showed three different RAPD patterns. One of the isolates from each product was chosen and good antimicrobial activity were displayed by 3 strains including MP6/2, PKS2-1 and OV1-1 producing in MRS broth bacteriocin-like substance (BLIS) inhibiting Candida albicans ATCC 90028, Carnobacterium maltraromaticum NCDO 2760, Lactobacillus sakei subsp. sakei JCM 1157, Listeria innocula CIP 80.11, Listeria ivanovii SLCC 2379, Pseudomonasc aeruginosa ATCC 27853, Staphylococcus aureus CIP 76.25 and Streptococcus mutans used as indicator strains. However, inhibitory activities of MP6/2 and PKS2-1 were related to the production of either organic acid, while OV2-1 was not affected by heating. The inhibitory activities of these three isolates were sensitive to proteolytic enzymes. 16S rDNA gene sequencing allowed to identify the MP6/2 as Pediococcus pentosaceus while PKS2-1 and OV1-1 were identified as Enterococcus faecium. It was concluded that the obtained isolates may be used as bio-preservative cultures to produce fermented foods.

Identification, genetic characterization and virulence of Serbian Erwinia amylovora isolates

Fire blight disease, caused by plant pathogenic bacteria Erwinia amylovora represents a global threat for pome fruit industry, affecting yield and causing serious economic damage. During six year period (2011–2016), a total of 45 isolates typical for E. amylovora were isolated from four different host plants (apple, pear, quince and medlar) in 21 localities within four Serbian regions (Southeast, Central, West, and North). Isolates were primarily identified using biochemical and serological tests, polymerase chain reaction (PCR) with species-specific primer pair (A/B) and their pathogenicity. Polyclonal antibodies used in DAS ELISA and IF were found to be specific and sensitive enough for rapid E. amylovora detection. In virulence test performed on immature pear fruitlets, higher variability (CV) was found between regions than among hosts from which isolates originated. Slighter virulence of isolates originated from North region of Serbia (Vojvodina) was found in comparison to the isolates from Central Serbia, which were the most variable. Principal components analysis (PCA) segregated quince as a host among the most prevalent isolates in group of highly virulent, but failed to identify difference related to geographic origin of Serbian isolates. The same results enabled multivariate statistical analysis. Different DNA fingerprinting techniques such as repetitive element sequence based PCR (rep-) with BOX, ERIC, REP, GTG5 and SERE primers, then Random Amplified Polymorphic DNA (RAPD) with M13-PCR primer revealed genetic homogeneity among all Serbian E. amylovora isolates. ERIC, SERE and M13 primers generated the most complex and visually perceptible patters. Restriction fragment length polymorphism analysis (RFLP) of gyrA and recA genes, digested with three restriction endonucleases BstUI, BgIII and BsmI and rpoS gene digested with BstUI gave the unuque, characteristic patterns for all Serbian isolates.

Detection of osteopontin gene transcripts in bull spermatozoa

The present experiment was aimed to detect osteopontin gene transcript from bull spermatozoa. Fresh semen samples from 12 Jersey cross breed bulls were collected using artificial vagina. The volume and concentration of the individual samples were recorded. The spermatozoa were separated from bull semen samples by swim up protocol using sperm TALP. The initial concentration of semen sample was checked immediately before proceeding for RNA isolation and normalization of initial concentration was done. So, initial amount of every sample was made equal. Total RNA from the bull spermatozoa were extracted by the RNeasy® Mini Kit, Qiagen as per manufacturer’s protocol. The purity and concentration of the samples were measured at A280/260 by using Nano Drop TM 1000 spectrophotometer. The first strand cDNA was synthesized from 1μg total RNA using M-Mu LV Reverse Transcriptase Revert Aid TM H minus first strand cDNA synthesis kit (Thermo Scientific) as per manufacturer’s protocol. PCR product of about 267 bp fragment length of OPN gene was confirmed by conventional PCR. The bulk PCR product of about 100μl was purified by Gen Elute™ PCR Clean-Up Kit Sigma-Aldrich as per manufacturer’s protocol. The concentration and purity of PCR product were 64ng/μl and 1.6 respectively. The cloning and expression of OPN gene was carried out by using TA cloning kit. The ligated product was then transformed into E. coli DH5α cells. The plate was incubated for overnight at 37 ˚C for selection of the colonies and stock cultures were maintained in LB media at -80˚ C. The clones which showed the amplification of 400 bp DNA fragment were considered as positive clone carrying desired insert. Plasmid isolation was done as per the protocol followed in Hi Yield TM Plasmid Mini Kit (RBC Cat. NO. YPD100) and the eluted DNA were stored at -20 ˚C. The concentration and purity of PCR purified product were 67ng/μl and 1.6 respectively. The eluted plasmid DNA was sequenced commercially (Amnion, Bangalore) using M13 primers. The cloned partial sequence of OPN gene isolated from bovine spermatozoa was submitted at DDBJ (Accession No. AB983656). On blast analysis using NCBI nucleotide blast (Online tool), 99 per cent identity was found with reported consensus sequences except single nucleotide variation in DNA sequence at 234th nucleotide. There was substitution of adenine instead of cytosine.

Epidemiology, genotypic diversity, and antimicrobial resistance of Lactococcus garvieae in farmed rainbow trout (Oncorhynchus mykiss)

Bacterial agents must be genotypically analyzed for vaccinations, effective control programs, and antimicrobial resistance genes that could transfer from aquaculture settings to terrestrial ecosystems and humans. Therefore, we investigated the prevalence, genotypic characterization, and antimicrobial resistance of Lactococcus garvieae for two years at aquaculture sites throughout Turkey. A total of 137 L. garvieae isolates were obtained from rainbow trout (Oncorhynchus mykiss) farms in different regions of Turkey, and three reference strains were used. The isolates were identified with pLG and ITS-LG (16S-23S internal transcribed spacer region). All isolates were genotyped with RAPD-PCR using M13 primers. Five different genogroups were determined, and the reference strains were found to differ from all the isolates. Some isolates compared with the GeneBank database and most isolates were within the same European, Asian, Australian, and South African genogroups. Isolates showed differing levels of resistance to most of the commonly used antimicrobials. The ermB, ermA, tetM, and tetS genes were identified and confirmed, whereas the floR, sulI, sulII, sulIII, tetA, tetB, and tetE genes were not detected. Conclusion: Lactococcus garvieae is one of the most important diseases of fish, also isolated cows and humans. The identification of antimicrobial resistance genes in rainbow trout fry (weight 0.5 g) showed that genes for antimicrobial resistance could be spread during any of the fishes’ life stages, thereby facilitating transmission of resistance to humans and other animals. The investigation of antimicrobial resistance genes in phenotypically susceptible isolates revealed that it is insufficient to investigate only phenotypic resistance in antimicrobial resistance studies. Also, for vaccination trials, the determination of prevalence, comparison with global isolates, and selection of non-resistant bacteria are very important.

Genetic Diversity of Fusarium oxysporum f. sp. cubense Causing Panama Wilt of Banana in the Philippines.

Panama wilt, caused by Fusarium oxysporum f. sp. cubense (Foc) is considered one of the most devastating banana diseases in recorded history. The disease threatens the banana industry due to Tropical Race 4 (TR4) infecting the Cavendish cultivar. Forty-two of the 45 representative isolates from Luzon were pathogenic, based on leaf symptom index and vascular discoloration rating. Accurate, fast and reliable identification are pre-requisites for effective management considering there are yet no proven effective chemicals to control the disease, thus the confirmation by a PCR-based diagnostic tool is essential. Using race-specific primers, FocTr4-F/FocTr4-R and Foc-1/Foc-2, the absence of TR4 in Luzon has been confirmed, however, the occurrence of Race 4 has been reported, which should also be taken in consideration as the latter can also cause severe damage under favorable conditions. Furthermore, to examine genetic diversity of Foc in bananas, 55 of the 164 isolates collected from Regions I, II, III, IV and Cordillera Administrative Region (CAR) were analyzed by fingerprinting techniques using M13, ERIC and REP primers. Twenty-two reference isolates from Mindanao were also analyzed using the same primers. Foc isolates were differentiated into two clades at 25% similarity level, classifying all Mindanao isolates to clade A. Consistently high genetic variation was obtained from Luzon isolates using M13, an arbitrarily primed fingerprinting technique and repetitive elements, REP and ERIC-PCR, while low genetic variation was obtained from Mindanao isolates. ERIC-PCR was the most informative and predictive fingerprinting method as the TR4 isolates from Mindanao were grouped together. No grouping of Foc isolates was observed with respect to geographical origin, except isolates from Mindanao. In addition, grouping of Foc4 is also regardless of host variety in all analyses conducted. Overall, high genetic variability was recorded in Foc Philippine population for the three primers used, which might render host resistance vulnerable.

Lactic acid bacteria diversity in corn silage produced in Minas Gerais (Brazil)

PurposeThe diversity of lactic acid bacteria (LAB) in silages produced in warm climate countries is not well known. This study aimed to identify and characterise the metabolic and genotypic aspects of autochthonous LAB isolated from corn silage produced in the state of Minas Gerais, Brazil.MethodsEighty-eight LAB were isolated. To evaluate their performance at the strain level, all isolates were distinguished among strains using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) and repetitive extragenic palindromic PCR (REP-PCR) techniques. The organic acid and ethanol production were determined by high-performance liquid chromatography (HPLC).ResultThe fingerprints obtained by RAPD-PCR with a M13 primer were more discriminatory than those obtained with the REP-PCR technique using a (GACA)4 primer. Moreover, 28 representative isolates were identified as Lactobacillus acidophilus, L. buchneri, L. casei, L. diolivorans, L. hilgardii, L. paracasei, L. parafarraginis, L. plantarum, L. rhamnosus, L. zeae and Pediococcus acidilactici. Different fingerprinting profiles between isolates within the same species were observed. However, some strains isolated from different silages showed the same band profile, thus suggesting the presence of clusters with high similar fingerprints in silages from various regions.ConclusionA variation in LAB diversity was observed in the silages of the evaluated regions, with L. rhamnosus and L. buchneri showing the highest distribution. Differences in organic acid production were observed among the strains belonging to the same species. This research contributes to a better understanding of the LAB community present in corn silage produced in warm climates. These strains will be studied as potential silage starters.

Keragaman Isolat Khamir Osmofilik pada Madu Hutan dari Sulawesi Tengah Ditinjau Menggunakan Teknik RAPD

Honey is a sweet substance with high sugar content produced by bees from flower nectar. Osmophilic yeast is one of organisms that can grow in honey because their ability to adapt in high osmotic pressure environment. Twenty-seven osmophilic yeast isolates were obtained from 11 forest honey samples to this research. This research objective is to determine the diversity of yeast isolates from genotypic characterization. Genotypic characterization was done using DNA fingerprint analysis using the RAPD PCR technique. This research used 2 types of primers, namely CDU primer (5′-GCGATCCCCA-3 ′) and M13 primer (5′-GAGGGTGGCGGTTCT GAGGGTGGCGGTTCT-3 ′). Data in the form of electrophoregram are represented in table n (yeast strain) x t (character units) using binary scoring type. The data were analyzed using a numerical taxonomy method with similarity (OTU) and determined using Jaccard coefficient (SJ) in 70% of IS limit. The results of this research showed that 27 yeast isolates were divided into 2 clusters and 10 outliers with a correlation coefficient (r) of 0.924624

Molecular Characterization of Pseudomonas syringae pv. coriandricola and Biochemical Changes Attributable to the Pathological Response on Its Hosts Carrot, Parsley, and Parsnip.

Bacterial leaf spot caused by the plant pathogenic bacterium Pseudomonas syringae pv. coriandricola (Psc) was observed on carrot, parsnip, and parsley grown on a vegetable farm in the Vojvodina Province of Serbia. Nonfluorescent bacterial colonies were isolated from diseased leaves and characterized using different molecular techniques. Repetitive element PCR fingerprinting with five oligonucleotide primers (BOX, ERIC, GTG5, REP, and SERE) and the randomly amplified polymorphic DNA-PCR with the M13 primer revealed identical fingerprint patterns for all tested strains. Multilocus sequence analysis of four housekeeping genes (gapA, gltA, gyrB, and rpoD) showed a high degree (99.8 to 100%) of homology with sequences of Psc strains deposited in the Plant-Associated Microbes Database and NCBI database. The tested strains caused bacterial leaf spot symptoms on all three host plants. Host-strain specificity was not found in cross-pathogenicity tests, but the plant response (peroxidase induction and chlorophyll bleaching) was more pronounced in carrot and parsley than in parsnip.