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Characterization of genetic diversity on tropical Trichoderma germplasm by sequencing of rRNA internal transcribed spacers.

ABSTRACT: OBJECTIVE:Trichoderma species are found in soil and in association with plants. They can act directly or indirectly in the biological control of plant diseases and in the promotion of plant growth, being among the most used fungi in the formulation of bioproducts applied to agricultural systems. The main objective of this study was to characterize at a first-tier level a collection of 67 Trichoderma isolates from various tropical sources, based solely on sequencing of the internal transcribed spacer (ITS) region of the rRNA genes. Our goal was to provide a preliminary idea of the baseline diversity in this collection, to combine this information later with an array of other isolate-specific physiological data. This study provides a required knowledge at molecular level for assessment of this germplasm potential as a source of biotechnological products for beneficial effects in plants. RESULTS:Sequencing of the ITS region showed that the 67 Trichoderma isolates belonged in 11 species: T. asperellum, T. atroviride, T. brevicompactum, T. harzianum, T. koningiopsis, T. longibrachiatum, T. pleuroticola, T. reesei, T. spirale, T. stromaticum and T. virens. A total of 40.3% of the isolates were very closely related to each other and similar to T. harzianum. The baseline genetic diversity found indicates that the collection has different genotypes, which can be exploited further as a source of bioproducts, aiming at providing beneficial effects to plants of interest to cope with biotic and abiotic stresses.

SUBMITTER: Feitosa YB

PROVIDER: S-EPMC6798453 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Genetic diversity of Pneumocystis carinii f. sp. hominis based on variations in nucleotide sequences of internal transcribed spacers of rRNA genes.

Project description:A variety of genes have been used to type Pneumocystis carinii. In the present study, nucleotide sequence variations in the ITS1 and ITS2 internal transcribed spacer (ITS) regions of the rRNA genes were used to type Pneumocystis carinii f. sp. hominis DNA obtained from the lungs of 60 human immunodeficiency virus-infected individuals. These regions were amplified by PCR, cloned, and sequenced. Multibase polymorphisms were identified among samples. Several new genotypes are reported on the basis of the nucleotide sequence variations at previously unreported positions of both the ITS1 and the ITS2 regions. Twelve new ITS1 sequences were observed, in addition to the nine sequence types reported previously. The most common was type E, which was observed in 60.5% of the samples. The sequence variations in the ITS1 region were mainly located at positions 5, 12, 23, 24, 45, 53, and 54. Sixteen new ITS2 types were also identified, in addition to the 13 types reported previously. The most common was type g (26.6%). The sequences of the ITS2 regions in most specimens were different from the previously published sequence at bases 120 and 166 through 183. The most common variations observed were deletions at positions 177 through 183. The presence of more than one sequence type in some patients (60%) suggested the occurrence of coinfection with multiple P. carinii strains. The genetic polymorphism observed demonstrates the degree of diversity of Pneumocystis strains that infect humans. Furthermore, the high degree of polymorphism suggests that these genes are evolving faster than other genes. Consequently, the sequence information derived is useful for purposes such as examination of the potential of person-to-person transmission and recurrent infections but perhaps not for other genotyping applications that rely on more stable genetic loci.

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Characterization of Microcystis (Cyanobacteria) Genotypes Based on the Internal Transcribed Spacer Region of rRNA by Next-Generation Sequencing.

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Genotypes at the internal transcribed spacers of the nuclear rRNA operon of Pneumocystis jiroveci in nonimmunosuppressed infants without severe pneumonia.

Project description:The frequency of Pneumocystis jiroveci (human-derived Pneumocystis) in immunocompetent infants developing acute respiratory syndromes has recently been evaluated and has been shown to be close to 25%. Until now, there have been no data on the genomic characteristics of the fungus in these patients, while molecular typing of P. jiroveci organisms was mostly performed with samples from immunosuppressed patients with pneumocystosis (Pneumocystis carinii pneumonia [PCP]). The present report describes the genotypes of P. jiroveci organisms in 26 nonimmunosuppressed infants developing a mild Pneumocystis infection contemporaneously with an episode of bronchioloalveolitis. The typing was based on sequence analysis of internal transcribed spacers (ITSs) 1 and 2 of the rRNA operon, followed by the use of two typing scores. By use of the first score, 11 P. jiroveci ITS types were identified: 10 were previously reported in immunosuppressed patients with PCP, while 1 was newly described. By use of the second score, 13 types were identified, of which 2 were newly described. The most frequent type was identified as type B(1)a(3) (first score), which corresponds to type Eg (second score). Mixed infections were diagnosed in three infants. The occurrence of such diversity of P. jiroveci ITS types, an identical main type, and mixed infections has previously been reported in immunosuppressed patients with PCP. Thus, the P. jiroveci ITS genotypes detected in immunocompetent infants and immunosuppressed patients developing different forms of Pneumocystis infection share characteristics, suggesting that both groups of individuals make up a common human reservoir for the fungus. Finally, the frequency of P. jiroveci in nonimmunosuppressed infants with acute respiratory syndromes and the genotyping results provide evidence that this infant population is an important reservoir for the fungus.

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Project description:The internal transcribed spacers of the nuclear ribosomal RNA gene cluster, termed ITS1 and ITS2, are the most frequently used nuclear markers for phylogenetic analyses across many eukaryotic groups including most plant families. The reasons for the popularity of these markers include: 1.) Ease of amplification due to high copy number of the gene clusters, 2.) Available cost-effective methods and highly conserved primers, 3.) Rapidly evolving markers (i.e. variable between closely related species), and 4.) The assumption (and/or treatment) that these sequences are non-functional, neutrally evolving phylogenetic markers. Here, our analyses of ITS1 and ITS2 for 50 species suggest that both sequences are instead under selective constraints to preserve proper secondary structure, likely to maintain complete self-splicing functions, and thus are not neutrally-evolving phylogenetic markers. Our results indicate the majority of sequence sites are co-evolving with other positions to form proper secondary structure, which has implications for phylogenetic inference. We also found that the lowest energy state and total number of possible alternate secondary structures are highly significantly different between ITS regions and random sequences with an identical overall length and Guanine-Cytosine (GC) content. Lastly, we review recent evidence highlighting some additional problematic issues with using these regions as the sole markers for phylogenetic studies, and thus strongly recommend additional markers and cost-effective approaches for future studies to estimate phylogenetic relationships.

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Oligonucleotide primers designed to differentiate pathogenic pseudomonads on the basis of the sequencing of genes coding for 16S-23S rRNA internal transcribed spacers.

Project description:Universal primers targeting conserved sequences flanking the 3' end of the 16S and the 5' end of the 23S rRNA genes (rDNAs) were used to amplify the 16S-23S rDNA internal transcribed spacers (ITS) from eight species of pseudomonads which have been associated with human infections. Amplicons from reference strains of Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas gladioli, Pseudomonas mallei, Pseudomonas mendocina, Pseudomonas pickettii, Pseudomonas pseudomallei, and Xanthomonas maltophilia were cloned from each species, and sequence analysis revealed a total of 19 distinct ITS regions, each defining a unique sequevar with ITS sizes ranging from 394 (P. cepacia) to 641 (P. pseudomallei) bp. Five distinct ITS sequevars in P. cepacia, four in P. mendocina, three in P. aeruginosa, two each in P. gladioli and P. pseudomallei, and one each in P. mallei, P. pickettii, and X. maltophilia were identified. With the exception of one P. cepacia ITS, all ITS regions contained potential tRNA sequences for isoleucine and/or alanine. On the basis of these ITS sequence data, species-specific oligonucleotide primers were designed to differentiate P. aeruginosa, P. cepacia, and P. pickettii. The specificities of these primers were investigated by testing 220 clinical isolates, including 101 strains of P. aeruginosa, 103 strains of P. cepacia, and 16 strains of P. pickettii, in addition to 24 American Type Culture Collection (ATCC) Pseudomonas strains. The results showed that single primer pairs directed at particular ITSs were capable of specifically identifying the ATCC reference strains and all of the clinical isolates of P. aeruginosa and P. pickettii, but this was not the case with several ITS-based primer pairs tested for P. cepacia. This pathogen, on the other hand, could be specifically identified by primer pairs directed against the 23S rDNA.

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Molecular characterization of hard tick Haemaphysalis longicornis from China by sequences of the internal transcribed spacers of ribosomal DNA.

Project description:In the present study, the entire first and second internal transcribed spacer (ITS-1 and ITS-2) regions of nuclear ribosomal DNA (rDNA) of Haemaphysalis longicornis from China were amplified by polymerase chain reaction. The 45 representative amplicons were sequenced, and sequence variation in the ITS was examined. The ITS sequences of H. longicornis were 3644 bp in size, including the part of 18S rDNA, 28S rDNA sequences and the complete ITS-1, 5.8S rDNA and ITS-2 sequences. Sequence analysis revealed that the ITS-1, 5.8S rDNA and ITS-2 of this hard tick were 1582, 152, and 1610 bp in size, respectively. The intra-specific sequence variations of ITS-1 and ITS-2 within H. longicornis were 0-2 and 0-2.2%; however, the inter-specific sequence differences among members of the genus Haemaphysalis were significantly higher, being 35.1-55.2 and 37-52% for ITS-1 and ITS-2, respectively. The molecular approach employed in this study provides the foundation for further studies of the genetic variation of H. longicornis from different hosts and geographical origins in China.

| S-EPMC5847221 | biostudies-literature

Large-Scale Characterization of the Soil Microbiome in Ancient Tea Plantations Using High-Throughput 16S rRNA and Internal Transcribed Spacer Amplicon Sequencing.

Project description:There is a special interaction between the environment, soil microorganisms, and tea plants, which constitute the ecosystem of tea plantations. Influenced by environmental factors and human management, the changes in soil microbial community affected the growth, quality, and yield of tea plants. However, little is known about the composition and structure of soil bacterial and fungal communities in 100-year-old tea plantations and the mechanisms by which they are affected. In this regard, we characterized the microbiome of tea plantation soils by considering the bacterial and fungal communities in 448 soil samples from 101 ancient tea plantations in eight counties of Lincang city, which is one of the tea domestication centers in the world. 16S and Internal Transcribed Spacer (ITS) rRNA high-throughput amplicon sequencing techniques were applied in this study. The results showed that the abundance, diversity, and composition of the bacterial and fungal communities have different sensitivity with varying pH, altitude, and latitude. pH and altitude affect soil microbial communities, and bacterial communities are more sensitive than fungi in terms of abundance and diversity to pH. The highest α-diversity of bacterial communities is shown in the pH 4.50-5.00 and 2,200-m group, and fungi peaked in the pH 5.00-5.50 and 900-m group. Because of environmental and geographical factors, all microbes are similarly changing, and further correlations showed that the composition and structure of bacterial communities are more sensitive than fungal communities, which were affected by latitude and altitude. In conclusion, the interference of anthropogenic activities plays a more important role in governing fungal community selection than environmental or geographical factors, whereas for the bacterial community, it is more selective to environment adaptation than to adaptation to human activities.

| S-EPMC8555698 | biostudies-literature

Typing of Pneumocystis carinii strains that infect humans based on nucleotide sequence variations of internal transcribed spacers of rRNA genes.

Project description:Small portions of the 18S and the 26S rRNA genes, the entire 5.8S rRNA gene, and internal transcribed spacers ITS1 and ITS2 (located between the 18S and 5.8S rRNA genes and between the 5.8S and 26S rRNA genes, respectively) of Pneumocystis carinii that infect humans were cloned and sequenced. The nucleotide sequences of the 18S, 5.8S, and 26S rRNA genes determined in the study were approximately 90% homologous to those of P. carinii that infect rats, while the sequences of ITS1 and ITS2 of P. carinii from the two different hosts were only 60% homologous. The 18S, 5.8S, and 26S rRNA gene sequences of P. carinii from 15 patient specimens were determined and were found to be identical to each other, whereas the ITS sequences were found to be variable. With the observed sequence variation, it was possible to classify the ITS1 sequences into two types and the ITS2 sequences into three types. P. carinii strains that had the same type of ITS1 sequence could have a different type of ITS2 sequence. On the basis of the sequence types of the two ITS regions, P. carinii from the 15 patients were classified into four groups. P. carinii from three patient specimens were found to contain two different ITS sequence patterns. More surprisingly, one additional specimen was found to have one ITS sequence typical of P. carinii isolates that infect humans and another typical of P. carinii isolates that infect rats. The studies indicate that it is possible to type P. carinii strains on the basis from one patient, suggesting that coinfection with more than one strain of P. carinii may occur in the same patient.

| S-EPMC264199 | biostudies-other

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