Determination of yeast diversity in fermented Sumbawa mare’s milk using internal transcribed spacers (ITS) fragment analysis

Aims@#Molecular identification of yeast has been conducted on various fermentation products. However, the identification of yeast in fermented Sumbawa mare’s milk based on the genotyping method has not been carried out. This study was aimed to determine the diversity profile of yeasts in fermented Sumbawa mare’s milk using phenetic characters and PCR-RFLP analysis technique based on the ITS region.@*Methodology and results@#Yeast isolates were phenotypically characterized and visualized in a dendrogram using CLAD97 software. Then, the yeast DNA was extracted using heat treatment and amplified using ITS1 and ITS4 primers. The amplicons were analyzed by RFLP using HindIII and HaeIII enzymes. The phylogenetic tree was constructed using MEGA 7.0. Based on the result of grouping by phenetic analysis and PCR-RFLP, the 12 isolates were divided into four groups with different members. The results of the phenetic analysis were divided into group I (all isolates of Dompu), group II (isolate B3, B4, S3), group III (isolate B5) and group IV (isolate S1). The types of yeast that were identified molecularly and represented each group of PCR-RFLP results included in group I were Kluyveromyces marxianus D1A and K. marxianus D1B, group II: K. marxianus D7, group III: Kazachstania humilis D4, while milk from Bima and Sumbawa has one yeast species as a member of group IV, namely Pichia kudriavzevii B3. Kluyveromyces marxianus was the yeast frequently found in Sumbawa fermented mare’s milk.@*Conclusion, significance and impact of study@#Various yeast species as a consortium of the milk samples can contribute to the increasing quality of fermented Sumbawa mare’s milk.

Clinical effectiveness of live preparation of lactobacillus in treatment of bacterial vaginosis in pregnancy: A meta-analysis

Objective: To evaluate the clinical effectiveness of live preparation of lactobacillus in treatment of bacterial vaginosis in pregnancy. Methods: Randomized controlled trials of live preparation of lactobacillus in the treatment of bacterial vaginosis in pregnancy were collect by searching PubMed, Web of Science, OVID, CNKI, WanFang, VIP, CBM and Elsevier databases. Quality of the included trials were evaluated by two researchers independently, and data were extracted according to Cochrane systematic evaluation. RevMan 5.3 software was used for meta-analysis. Results: Twenty-one randomized controlled trials involving 2 930 patients were included, which showed that there was significant difference in the clinical effectiveness between vaginal medication of live preparation of lactobacillus and vaginal medication of metronidazole [total effective rate (RR=1.05, 95% CI: 1.02-1.07, P=0.000 4]; significant differences were found in premature delivery rate (RR=0.49, 95% CI: 0.32-0.73, P=0.000 4), premature rupture of membrane rate (RR=0.54, 95% CI: 0.38-0.77, P=0.000 7), infant of low-birth weight rate (RR=0.45, 95% CI: 0.22-0.94, P=0.03), puerperal infection rate (RR=0.60, 95% CI: 0.39-0.94, P=0.03) between the two groups. Conclusions: Vaginal medication of live preparation of lactobacillus was more clinically effective than vaginal medication of metronidazole for bacterial vaginosis in pregnancy. Live preparation of lactobacillus is associated with a lower premature delivery rate, a lower premature rupture of membrane rate, a lower low-birth weight rate and a lower puerperal infection rate.

Prevalence and genotyping of Enterocytoon bieneusi in farmed black goats from Hainan Province, the southernmost part of China

Objective: To investigate the infection rate and genotype distribution of Enterocytoon (E.) bieneusi in farmed black goats from the Hainan Province, China. Methods: A total of 341 fresh fecal samples were collected from black goats farmed in five different locations of the Hainan Province, China. E. bieneusi was examined and genotyped through PCR and sequencing analysis of the internal transcribed spacer (ITS) region of this pathogen. Results: The average prevalence of E. bieneusi in black goats from from the five locations was 24.0% (82/341) ranging from 6.3% (4/63) to 37.2% (32/86) (χ2 =17.252, P<0.01). The detected 82 E. bieneusi isolates belonged to eight ITS genotypes including six known genotypes (AHG1, CHG2, CHG3, CHG5, CM21 and D) and two novel genotypes (HNG-Iand HNG-II). Amongst the genotypes, CHG5 was the the most prevalent with a prevalence of 57.3% (47/82), followed by CHG3 (28.0%, 23/82), CHG2 (4.9%, 4/82), CM21 (3.7%, 3/82), D (2.4%, 2/82), AHG1 (1.2%, 1/82), HNG-I(1.2%, 1/82) and HNG-II(1.2%, 1/82). In those genotypes, only genotype D was found in humans previously. Conclusions: This represents the first report identifying E. bieneusi in black goats from Hainan Province of China. The results indicate that E. bieneusi has a high prevalence and a wide distribution in those animals from Hainan Province, but the risk of zoonotic transmission of E. bieneusi from them to human is low.

Identification of molecular markers to study the Garcinia spp. diversity.

The genus Garcinia shows a considerable variation in its morphological characters such as leaf, flower and fruit with taxonomic ambiguity. It is a potential under-exploited multipurpose crop that gained considerable attention for the presence of (-) hydroxycitric acid, an anti-obesity compound, in its fruit rind and leaves. Here, we evaluated the genetic relationship through molecular markers among the selected 9 species commonly available in the Western Ghats and the Northeastern Himalayan foot hills of India. The nucleotide sequence data obtained from two prominent monomorphic bands generated in ISSR profiling of the species was utilized for the study. The selected bands were found to be of ITS region (700 bp) and partial region of KNOX-1 gene (600 bp). The evolutionary cluster was formed using MEGA5 software. The study indicated 2 major clusters, influenced by floral morphology of the species and availability of (-) hydroxycitric acid in their fruit rinds. In the subclusters, one species from the Western Ghats were paired with another from Northeastern Himalayas with relatively similar morphological traits.

Identification of Pseudostellaria heterophylla from different idioplasms by analysis of rDNA ITS sequences / 中草药

Objective: To provide the DNA molecular marker for the identification of Pseudostellaria heterophylla from the different idioplasms by analysis of rDNA ITS sequences. Methods: PCR amplification, cloning, and sequencing were carried out using specified primer, and the rDNA ITS base sequences were compared. Results: The ITS mutation extension was 623-624 pb among nine idioplasms of P. heterophylla. Thereinto, the ITS-1 was 224 pb and its G + C content was 52.91%-54.26%, the 5.8S rDNA was 155 bp and its G + C content was 54.49%-55.13%, the ITS-2 was 244-245 bp and its G + C content was 55.55%-56.41%. There were 17 mutation sites (2.72%) in the whole ITS sequences. There were 7, 7, and 3 mutation sites in ITS1, ITS2, and 5.8S, respectively. The different idioplasms had a number of specific single nucleotide mutation sites. Their homologies with each other were upwards 99.9% and their sequence genetic distances were 0.003-0.013. These results showed that the mutation in species from different producing areas and idioplasms was within no more than one species. Conclusion: The mutation of ITS sequences could be used to authenticate P. heterophylla from different idioplasms.

Molecular characterization of 'Bhut Jolokia' the hottest chilli.

The northeast region of India, considered as ‘hot spot’ of biodiversity, having unique ecological environment with hot and high-humidity conditions, has given rise to the world’s hottest chilli, ‘Bhut Jolokia’, which is at least two times hotter than Red Savina Habanero in terms of Scoville heat units (SHU). This study was undertaken to determine the distinctiveness of ‘Bhut Jolokia’ from Capsicum frutescens or Capsicum chinense through sequencing of the ribosomal RNA (rRNA) gene-internal transcribed (ITS) region along with its phylogenetic analysis. Although a compensatory base change (CBC) in the ITS2 region was not observed between the closely related species of C. frutescens and C. chinense when compared with Bhut Jolokia; phylogenetic analysis using ITS1, 5.8S and ITS2 sequences indicated a distinct clade for all the accessions of ‘Bhut Joloikia’, while C. frutescens and C. chinense occupied discrete lineages. Further, a unique 13-base deletion was observed in all the representative accessions of ‘Bhut Jolokia’, making it distinct from all other members within the genus and beyond. The degree of genetic variations along with its extreme pungency might be related to ambient environmental factors of northeastern India.

Characterization of Species of Cladobotryum which Cause Cobweb Disease in Edible Mushrooms Grown in Korea

Four Cladobotryum isolates were collected from four different commercially grown mushroom types infected with cobweb disease in Cheongdo-gun and Chilgok-gun of Gyeongbuk Province, Korea in 2010. The isolates were identified as C. mycophilum from Agaricus bisporus and Pleurotus eryngii, C. varium from Flammulina velutipes and Hypsizygus marmoreus. The cultural characteristics of the four isolates were investigated using potato dextrose agar (PDA) media under nine different temperatures ranging from 5~32degrees C. Rapid growth of the isolates to colony diameters of 47~82 mm was observed at conditions of 18~22degrees C. No growth was observed at 32degrees C. C. mycophilum produced a yellowish red pigment while C. varium produced a cream colored pigment after cultivation for 25 days on PDA. Phylogenetic analysis of the internal transcribed spacer region and partial 28S rDNA from the four isolates confirmed they were C. mycophilum and C. varium. Cross pathogenicity tests revealed that the two isolates of C. mycophilum were highly pathogenic toward three mushroom types, but not toward H. marmoreus. The two isolates of C. varium were less pathogenic than those of C. mycophilum, but were pathogenic toward all mushroom types evaluated.

Molecular Identification of Two Strains of Phellinus sp. by Internal Transcribed Spacer Sequence Analysis

Two species of cultivated Phellinus sp. were identified as P. baumii by internal transcribed spacer (ITS) sequence analysis. The fruit bodies of the examined strains were similar to those of naturally occurring strains, having a bracket-like form, yellow-to-orange color, and poroid hymenial surfaces. The DNA sequences of ITS region of both strains showed a homology of 99% with ITS1 to ITS2 sequences of P. (Inonotus) baumii strain PB0806.

Identification of Candida species using PCR-RFLP in cancer patients in Iran.

Opportunistic infections caused by Non-Candida albicans. have been increasing. Traditional methods that are used to identify clinical isolates of Candida species are time-consuming and not appropriate for rapid, accurate and reliable identification. Purpose: To identify Candida spp isolated from cancer patients using PCR-restriction enzyme. Materials and ethods: Using universal primers, ITS1 and ITS4, in this study, we could amplify ITS1-5.8S-ITS2 rDNA regions at both 80 clinical isolates and 3 standard strains. The PCR products were digested with two restriction enzymes MspI and BlnI separately. Result: We successfully identified all isolated species using two restriction enzymes (MspI, BlnI). Candida albicans was the most common species (77.5%), followed by C. glabrata (15%), C. tropicalis (5%), C. krusei (2.5%). Although the primers and enzyme had the ability to identify C. parapsilosis, C. guilliermondii, C. dubliniensis, present isolates did not include these among identified ones. Conclusion: RFLP-PCR using ITSI and ITS4 primers and restriction enzyme is a rapid, easy, reliable and also applicable method in clinical laboratory for identification of medically important Candida spp.

Development of Species-specific Primers for Rapid Detection of Phellinus linteus and P. baumii

Genus Phellinus taxonomically belongs to Aphyllophorales and some species of this genus have been used as a medicinal ingredients and Indian folk medicines. Especially, P. linteus and morphological-related species are well-known medicinal fungi that have various biological activities such as humoral and cell-mediated, anti-mutagenic, and anti-cancer activities. However, little is known about the rapid detection for complex Phellinus species. Therefore, this study was carried out to develop specific primers for the rapid detection of P. linteus and other related species. Designing the species-specific primers was done based on internal transcribed spacer sequence data. Each primer set detected specifically P. linteus (PL2/PL5R) and P. baumii (PB1/PB4R). These primer sets could be useful for the rapid detection of specific-species among unidentified Phellinus species. Moreover, restriction fragment length polymorphism analysis of the ITS region with HaeIII was also useful for clarifying the relationship between each 5 Phellinus species.

Identification of Some Phellinus spp

Four strains of Phellinus spp. was identified based on internal transcribed spacer (ITS) region of rDNA sequence analysis and morphological characteristics. Basidiocarps of all strains were effused-reflexed and hymenial surface was poroid. Hyphal system was dimitic and basidiospore was globose to ellipsoid. The amplification of ITS regions produced a DNA fragment of 500 to 780 bp in all strains examined. The determined sequences were analyzed for the reconstruction of phylogenetic tree. From these results, Phellinus sp. KM-1, KM-2, and KM-4 was identified as P. hartigii, P. baumii, and P. linteus, respectively.

PCR Assays for Detection of Pseudomonas tolaasii and Pseudomonas agarici

PCR assays were developed to detect Pseudomonas tolaasii and Pseudomonas agarici using primer sets, PTOF/PTOR and PAGF/R23-1R. The primer set, PTOF and PTOR, was designed from the nucleotide sequence of pPTOF2 that showed specificity for P. tolaasii in dot blot previously. For development of primers specific for P. agarici, ITS I regions of seven P. agarici strains were analyzed. P. agarici strains contained from one up to three putative ITS I regions, which were different in size and nucleotide sequence from each other. From the sequence of the band (PaI-III) common to all P. agarici strains, primer PAGF was designed. PAGF was used for forward primer, and R23-1R as reverse primer to detect P. agarici. Multiplex PCR with two primer sets, PTOF/PTOR and PAGF/R23-lR, successfully produced two fragments (PTSF and PASF) specific for P. tolaasii and P. agarici with the mixture of DNA of P. tolaasii and P. agarici.