ITS Amplification Research Articles

Fungi colonizing wood sticks of Chinese fir incubated in subtropical urban soil growing with Ficus microcarpa trees

Fungi colonized on wood sticks of the Chinese fir Cunninghamia lanceolata after incubation in soil around the Ficus microcarpa trees were isolated. In total, 1189 wood sticks from 28 F. microcarpa trees were sampled and analyzed in this study. A combined approach of culture morphological observation and PCR amplification of ITS of purified fungal isolates in revealing restriction fragment length polymorphism patterns was applied to group the purified fungal isolates for further species identification. Fungal species were identified based on phylogenetic analysis of ITS and β-tubulin gene-amplified sequences. Overall, 25 species belonging to 17 genera from four classes were identified, and all of them were Ascomycota. Among them, Penicillium spp., Fusarium spp., and Phoma insulana were the most frequently isolated. In this study, Dichotomonyces cejpii, Fusicolla acetilerea, and Purpureocillium lilacinum are recorded in Hong Kong for the first time. Some species of the fungal cultures obtained in this study may have potential application as biocontrol agents to control tree disease.

Ecoepidemiología de la Leishmaniasis cutánea no ulcerada en Honduras

La leishmaniasis es una enfermedad infecciosa causada por un protozoo del género Leishmania. En Honduras es una importante causa de morbilidad. El propósito del estudio fue caracterizar la eco epidemiología de la leishmaniasis cutánea no ulcerada (LCNU) en Honduras. Se seleccionaron 4 municipios endémicos a LCNU (Amapala, San Francisco de Coray, Reitoca y Alubarén). Para realizar la captura de Lutzomyia se usaron trampas miniatura tipo CDC, los individuos capturados fueron identificados según los criterios de Young & Duncan. Se realizaron búsquedas de pacientes en conjunto con personal de la Secretaría de Salud de Honduras para realizar la caracterización de las especies de Leishmania usando PCR. Se determinó la ingesta alimenticia de las hembras ingurgitadas utilizando la amplificación de ITS de los principales animales domésticos y humanos. Se colectaron 156 muestras de pacientes con sospecha clínica de LCNU 130 (83.33 %), resultaron positivas a Leishmania spp., siendo Leishmania infantum/chagasi la única especie circulante en la zona. Por medio de la utilización de criterios de Young & Duncan, se logró identificar un total de 5,951 Lutzomyia, de la cuales 3,543 (60 %) son machos y 2,404 (40 %) son hembras con o sin ingesta de sangre. De esta población de insectos capturados, se identificaron diez especies de Lutzomyia, siendo Lu. longipalpis, Lu. evansi y Lu. cruciata las de mayor predominio. Los cuatro municipios presentaron una baja diversidad de especies de Lutzomyia (DMg =1.04). Se analizaron 450 hembras ingurgitadas de las especies: Lu. longipalpis, Lu. cruciata, Lu. evansi, Lu. chiapanensis y Lu. sanguinaria. Las principales fuentes alimenticias encontradas fueron: Gallus gallus, Sus scrofa, Canis familiaris. Se encontró una tasa de infección del 0.27 %, encontrándose a Lu. longipalpis infectada con Leishmania infantum/ chagasi. Con este estudio se logró caracterizar la eco epidemiología de la LCNU en Honduras, confirmando a L. chagasi/infantum como el principal agente etiológico y a Lu. longiplapis su principal vector. DOI: http://dx.doi.org/10.5377/rct.v0i14.1799 Revista Ciencia y Tecnología No. 14, junio 2014: 115-128

Phenotypic and Genotypic Identification of Ticks Sampled from Wildlife Species in Selected Conservation Sites of Kenya

Hard ticks are blood feeding ectoparasites that infest humans and animals and are vectors of pathogenic microorganism that cause severe infectious diseases. Morphological identification has been the main approach of identifying ticks but the technique is considered inaccurate and difficult. Molecular techniques have recently been considered to be appropriate approaches for accurate and rapid identification and Internal Transcribed Spacer 2 (ITS 2) has been shown to differentiate genus of hard ticks. Currently, genetic identification of ticks using ITS 2 has not been carried out in Kenya. In this study, 80 tick samples were collected from Lake Nakuru and Tsavo National Parks and were identified morphologically using appropriate identification keys. DNA was extracted from the appendages using DNA extraction kit followed by partial amplification of ITS 2 gene. The PCR products were then analyzed by gel electrophoresis and positive PCR products were sequenced. Of the tick samples four genera were identified morphologically; Amblyomma, Hyalomma, Rhipicephalus and Dermacentor. Of the tick samples identified and compared with the sequences in the GenBank, six and seven samples showed 98-100% homology with A. variegatum and R. pulchellus respectively and they clustered in their respective monophyletic group in the phylogeny tree with a bootstrap of 99%. Two samples showed 92% homology with H. dromedarii and the study sequences clustered with the reference sequence with a bootstrap of 99% while six samples showed 95% homology with H. marginatum rufipes, however, only four of these samples clustered together with the reference sequence in the phylogeny with a bootstrap of 95%. One sample showed 91% homology with A. humerali and did not cluster together in the phylogeny tree. Congruency between both techniques was high with a correlation coefficient of 0.941. This is the first report of phenotypic and genotypic traits of tick species in Kenya and the findings will add value to the existing knowledge in identification of ticks.

Microbial community diversity associated with Sarcotragus sp. and Petrosia ficiformis from the Aegean Sea based on 16S rDNA-DGGE fingerprinting

Marine sponges are prolific sources for new bioactive compounds and they may contain microorganisms that can comprise up to 40% of the total sponge biomass. Therefore, it is important to understand the function and the specific interactions of sponge-associated microorganisms. In this study, we assessed the composition of predominant bacterial and fungal communities using DGGE fingerprinting of Sarcotragus sp. and Petrosia ficiformis collected from the Aegean Sea. Total community DNA extracted with the indirect DNA isolation method by using collagenase followed by 16S rDNA and ITS amplification of 16S rDNA and ITS amplicons were subjected to DGGE profiling. A total of 8 and 21 individual DGGE bands from 16S rDNA-V3 and ITS1 fragments were sequenced, respectively, and matched to corresponding bacteria and fungi in the GenBank database. Methylobacterium sp. and Chalastospora gossypii from Sarcotragus sp. and Gibberella intermedia and Fusarium subglutinans from Petrosia ficiformis are reported for the first time from marine sponges. Associated microorganisms of Sarcotragus sp. and Petrosia ficiformis are considered as host-specific, as tested sponges from the same geographical location showed different dominant bacterial and fungal diversities.

<i>Tulasnella irregularis</i> (basidiomycota: tulasnellaceae) from roots of <i>Encyclia tampensis</i> in south Florida, and confirmation of its mycorrhizal significance through symbiotic seed germination

Las relaciones micorríticas obligadas de las orquídeas epífitas – un componente clave en el modelo integrado de conservación – siguen desconocidas. Los estudios existentes han revelado que estas plantas, así como sus equivalentes terrestres, se asocian normalmente con basidiomicetos ubícuitos (e.g. Tulasnellaceae); sin embargo pocos estudios han verificado su papel fisiológico. Dos cepas del hongo micorrítico (UAMH 11541, UAMH 11543) fueron aisladas de las raíces de una especie de orquídea epifítica del sur de Florida, Encyclia tampensis – una obtenida de una plántula joven y la otra obtenida de una planta madura. Las semillas de cuatro taxones epífitos fueron inoculadas por separado con los dos hongos aislados in vitro: E. tampensis, Epidendrum amphistomum, Epidendrum nocturnum y Prosthechea cochleata. Más de un tercio de las semillas de E. tampensis y E. nocturnum (ambos inoculados) desarrollaron hojas cuando fueron mantenidos en total obscuridad por más de cien días. No se detectaron diferencias significativas entre las dos cepas del hongo con respecto a la germinación y la interacción entre el hongo y las semillas procedentes de diferentes fuentes (ANOVA, α= 0.05). Usando amplificación y secuenciación ITS, ambas cepas fueron identificadas como teleomorfos, Tulasnella irregularis (Basidiomycota: Tulasnellaceae) y ambas resultaron genéticamente idénticas con un elevado grado (98%) de seguridad. De esta manera, tanto la germinación simbiótica así como los resultados de la secuenciación ITS concuerdan en identificar con certeza ambas cepas como el mismo hongo. Este artículo trata de elucidar las interacciones entre orquídeas epífitas y hongos micorríticos y también subraya la necesidad de identificar, comprobar (a través de germinación simbiótica), y preservar los hongos micorríticos necesarios para fines de conservación.

Molecular approach to characterize ectomycorrhizae fungi from Mediterranean pine stands in Portugal.

Stone pine (Pinus pinea L.), like other conifers, forms ectomycorrhizas (ECM), which have beneficial impact on plant growth in natural environments and forest ecosystems. An in vitro co-culture of stone pine microshoots with pure mycelia of isolated ECM sporocarps was used to overcome the root growth cessation not only in vitro but also to improve root development during acclimation phase. Pisolithus arhizus (Scop.) Rauschert and Lactarius deliciosus (L. ex Fr.) S.F. Gray fungi, were collected, pure cultured and used in in vitro co-culture with stone pine microshoots. Samples of P. arhizus and L. deliciosus for the in vitro co-cultures were collected from the pine stands southwest Portugal. The in situ characterization was based on their morphotypes. To confirm the identity of the collected material, ITS amplification was applied using the pure cultures derived from the sporocarps. Additionally, a molecular profile using PCR based genomic fingerprinting comparison was executed with other genera of Basidiomycetes and Ascomycetes. Our results showed the effectiveness of the techniques used to amplify DNA polymorphic sequences, which enhances the characterization of the genetic profile of ECM fungi and also provides an option to verify the fungus identity at any stage of plant mycorrhization.

Isolation and Identification of Potential DNA Helicase from Local Isolate of Candida Albicans

Candida albicans is a most common fungal pathogen that causes oral and genital infections in human. This pathogen can causes candidiasis, especially in person with immunocompromised. Local isolated C. albicans has been successfully identified based on its phenotype and genotype characteristics. ITS amplification was performed using ITS1 and ITS4 primers. C. albicans is also exhibits as mesophilic fungus. Due to this reason, we believed that this microorganism has the potential to produce a mesophilic form of DNA helicase. In this study, a potential DNA repair helicase gene has been successfully amplified and identified from the local isolated C. albicans. DNA helicase was known as a motor protein for unwinding of duplex DNA into single stranded DNA using energy derived from ATP hydrolysis. However, a further study is needed to verify and validate the DNA helicase unwinding activity in future.

Molecular Phylogeny of Ophiognominialeptostyla Isolates Collected from Iran Based on ITS nrDNA Sequences

Anthracnose is the most important fungal disease on Persian walnut in Iran. The current study was performed on 16 Ophiognomonia leptostyla isolates collected from northwest region of Iran. ITS amplification was performed using ITS1 and ITS4 primers and the sequences were analyzed. Phylogenetic trees made based on ITS sequences showed that all Iranian isolates are in the same branch in a clade with Ophiognomonia leptostyla from CBS isolated from Juglans regia. The Iranian isolates are divided into two closely related sub-clades with low bootstrap support, the smaller sub-clade comprised of only three isolates were collected from wild walnut trees located in woodland areas. Keywords-Gnomonia leptostyla; walnut leaf blotch; phylogentic tree; ITS sequencing I. I NTRODUCTION

Validation of Melampsora larici-populina reference genes for in planta RT-quantitative PCR expression profiling during time-course infection of poplar leaves

The foliar rust caused by Melampsora larici-populina ( Mlp) is the main disease affecting poplar plantations in Europe. The biotrophic status of this fungus is a major limitation to address in planta transcripts profiling. Thus, identification of reference rust genes steadily expressed during plant tissue colonization is a crucial point. A quantitative PCR approach to assess fungal ITS amplification profile and Reverse Transcription quantitative-PCR was set to compare candidate reference genes amplification profiles in poplar infected tissues. We selected two M. larici-populina genes encoding an alpha-tubulin and the elongation factor-1-alpha that showed the highest expression stability across biological samples and for which transcript levels were correlated with fungal ITS amplification during time-course infection of poplar leaves. We report the use of these reference genes to assess in planta expression profiles of two genes involved in thiamine biosynthesis ( THI1 and THI2) for which specific haustorium expression was previously described in the bean rust fungus Uromyces fabae. Mlp- THI1 and Mlp-THI2 showed similar expression profiles. Trancripts were barely detectable in urediniospores as well as during the early stages of infection compared to those reported in the bean rust, whereas a strong induction was observed after haustorial formation after 24 hpi. These data are in frame with the results obtained in U. fabae and consistent with a metabolic reorientation that likely occurs after the fungus derived nutrients from its host in the haustorial structure essential for fungal biotrophy.

In vitro cultivation and fruit body formation of the black bolete, Phlebopus portentosus, a popular edible ectomycorrhizal fungus in Thailand

The tropical black bolete Phlebopus portentosus is highly favored in the cuisine of northern Thailand. It is suspected to form ectomycorrhizae with many host trees. Mycelium of P. portentosus isolated from a basidiome in Chiang Rai Province in 2003 grew well on modified Gamborg, modified Melin–Norkans, and Murashige and Skoog media at 30°C and at pH 4. In vitro fructification of P. portentosus on sorghum grain medium without a host plant is presented for the first time. Basidiomes emerged 3 months after inoculation on the medium, and the produced basidiospores germinated on agar, indicating the completion of its life cycle in vitro without a host. Five putative host plants (Castanopsis tribuloides, Dipterocarpus alatus, Dimocarpus longan, Pinus kesiya, and Syzygium cumini) were inoculated with mycelium on sorghum grain medium in a greenhouse to confirm its ectomycorrhizal status. Ectomycorrhizal roots were observed only on Pinus kesiya, suggesting that P. portentosus may be facultatively ectomycorrhizal. Identification of the synthesized ectomycorrhizae was confirmed by PCR amplification of ITS with a designed specific primer (HAR2A).

In vitro cultivation and fruit body formation of the black bolete, <i>Phlebopus portentosus</i>, a popular edible ectomycorrhizal fungus in Thailand

The tropical black bolete Phlebopus portentosus is highly favored in the cuisine of northern Thailand. It is suspected to form ectomycorrhizae with many host trees. Mycelium of P. portentosus isolated from a basidiome in Chiang Rai Province in 2003 grew well on modified Gamborg, modified Melin–Norkans, and Murashige and Skoog media at 30°C and at pH 4. In vitro fructification of P. portentosus on sorghum grain medium without a host plant is presented for the first time. Basidiomes emerged 3 months after inoculation on the medium, and the produced basidiospores germinated on agar, indicating the completion of its life cycle in vitro without a host. Five putative host plants (Castanopsis tribuloides, Dipterocarpus alatus, Dimocarpus longan, Pinus kesiya, and Syzygium cumini) were inoculated with mycelium on sorghum grain medium in a greenhouse to confirm its ectomycorrhizal status. Ectomycorrhizal roots were observed only on Pinus kesiya, suggesting that P. portentosus may be facultatively ectomycorrhizal. Identification of the synthesized ectomycorrhizae was confirmed by PCR amplification of ITS with a designed specific primer (HAR2A).

Molecular identification, typing and traceability of cyanobacteria from freshwater reservoirs

In order to assess the potential of several molecular targets for the identification, typing and traceability of cyanobacteria in freshwater reservoirs, molecular techniques were applied to 118 cyanobacterial isolates mostly sourced from Portuguese freshwater reservoirs and representative of three orders of cyanobacteria: Chroococcales (54), Oscillatoriales (15) and Nostocales (49). The isolates were previously identified by morphological methods and subsequently characterized by composite hierarchical cluster analysis of STRR and LTRR (short and long tandemly repeated repetitive sequences) PCR fingerprinting profiles. Representative isolates were selected from each cluster and their molecular identification, at the species level, was obtained or confirmed by phylogenetic positioning using 16S rRNA gene and rpoC1 phylogenies. A highly congruent association was observed between STTR- and LTRR-based clusters and taxonomic affiliation, revealing the usefulness of such PCR fingerprinting profiles for the identification of cyanobacteria. Composite analysis of hierarchical clustering of M13 and ERIC PCR fingerprints also appeared suitable for strain typing and traceability within a reservoir, indicating its potential for use in cyanobacterial monitoring, as a quality management control. Based on Simpson (D) and Shannon-Wiener (J') indices a high diversity was observed within all species, with Planktothrix agardhii showing the lowest diversity values (D=0.83; J'=0.88) and Aphanizomenon flos-aquae the highest ones (D=J'=0.99). A diagnostic key based on 16S-ARDRA, ITS amplification and ITS-ARDRA for identification purposes is also presented.

Ectomycorrhizal Inocybe species associate with the mycoheterotrophic orchid Epipogium aphyllum but not its asexual propagules.

Epipogium aphyllum is a Eurasian achlorophyllous, mycoheterotrophic forest orchid. Due to its rarity, it is often protected, and its biology is poorly known. The identity and pattern of colonization of fungal associates providing carbon to this orchid have not been studied previously. Using samples from 34 individuals from 18 populations in Japan, Russia and France, the following were investigated: (a) colonization patterns of fungal associates of E. aphyllum by microscopy; (b) their identity by PCR amplification of nuclear ribosomal ITS carried out on rhizome fragments and hyphal pelotons. Microscopic investigations revealed that thick rhizomes were densely colonized by fungi bearing clamp-connections and dolipores, i.e. basidiomycetes. Molecular analysis identified Inocybe species as exclusive symbionts of 75 % of the plants investigated and, more rarely, other basidiomycetes (Hebeloma, Xerocomus, Lactarius, Thelephora species). Additionally, ascomycetes, probably endophytes or parasites, were sometimes present. Although E. aphyllum associates with diverse species from Inocybe subgenera Mallocybe and Inocybe sensu stricto, no evidence for cryptic speciation in E. aphyllum was found. Since basidiomycetes colonizing the orchid are ectomycorrhizal, surrounding trees are probably the ultimate carbon source. Accordingly, in one population, ectomycorrhizae sampled around an individual orchid revealed the same fungus on 11.2 % of tree roots investigated. Conversely, long, thin stolons bearing bulbils indicated active asexual multiplication, but these propagules were not colonized by fungi. These findings are discussed in the framework of ecology and evolution of mycoheterotrophy.

Molecular characterisation and development of rapid molecular methods to identify species of Gracilariaceae from the Atlantic coast of Morocco

In Gracilariaceae, species identification is traditionally based on gross morphology; therefore the taxonomic status of terete individuals remains frequently problematic due to the lack of diagnostic characters to identify specimens. Different morphospecies have been recorded along the Atlantic coast of Morocco; however, no clear diagnostic characters were available to discriminate between terete species. Rapid molecular techniques have been developed recently to resolve many taxonomic problems and to re-assess the global diversity and biogeography in algae. In this study, molecular markers were used as DNA barcoding to characterise species. The sequence of the Rubisco spacer allowed identification of six species of Gracilariaceae: Gracilaria gracilis, Gracilaria dura, Gracilaria conferta, Gracilaria vermiculophylla, Gracilaria multipartita and Gracilariopsis longissima. In order to identify species with certainty, two simple and rapid methods based on the amplification of rDNA ITS and PCR-RFLP of the large subunit of the Rubisco were developed.

Cryptic long internal repeat sequences in the ribosomal DNA ITS1 gene of the dinoflagellate Cochlodinium polykrikoides (dinophyceae): a 101 nucleotide six-repeat track with a palindrome-like structure

Extremely long PCR fragments were generated by PCR amplification of ITS and 5.8S rDNA from Cochlodinium polykrikoides against other dinoflagellates. These patterns were consistent among geographically different isolates of C. polykrikoies. DNA sequencing reactions revealed that the PCR products were 1,166 bp in length and consisted of 813 bp of ITS1, 160 bp of 5.8S rDNA and 193 bp of ITS2. Thus, the long length was caused mainly by the long ITS1 sequence. Cryptically, the ITS1 contained a tract of 101 bp that occurs six times in tandem. The six repeated elements had identical nucleotide sequences. ITS1, therefore, separated three distinct regions: the 5' end (122 bp), the six parallel repeats (606 bp), and the 3' region (85 bp). Interestingly, both the single and six-repeat sequences should be palindrome-like sequences. In inferred secondary structures, both repeat sequences formed a long helical structure. This is the first reported discovery of comparatively long internal repeats in the ITS1 of dinoflagellates.

Coprodiagnosis of Hammondia heydorni in Dogs by PCR Based Amplification of ITS 1 rRNA: Differentiation from Morphologically Indistinguishable Oocysts of Neospora caninum

Hammondia heydorni is thought to be a non-pathogenic coccidian parasite of dogs that is closely related to Neospora caninum, an important parasite of cattle and dogs. Oocysts of these two species are morphologically indistinguishable from each other. A population of 2240 dogs in the Czech Republic was screened for the presence of H. heydorni/N. caninum oocysts and five (0.22%), represented by five of 3135 faecal samples (0.16%), were positive. The internal transcribed spacer 1 region of the rRNA gene (ITS1) from two isolates were cloned and the DNA sequences were identical with those of the ITS1 of H. heydorni. Based on the rRNA sequences available for H. heydorni and related coccidia, the primer pair JS4-JS5 was designed to amplify the 3′ end of the small subunit (SSU) rRNA gene and ITS1 of H. heydorni. When tested on DNA extracted from a variety of parasites, the primers amplified a specific 267bp fragment in our isolates only. The presence of DNA equivalent to 10 oocysts was sufficient for the amplification of the ITS1. We present a PCR-based diagnostic method as the only fast and reliable method for the diagnosis of H. heydorni in dogs.

16S/23S rRNA intergenic spacer regions for phylogenetic analysis, identification, and subtyping of Bartonella species.

Species of the genus Bartonella are currently recognized in growing numbers and are involved in an increasing variety of human diseases, mainly trench fever, Carrion's disease, bacillary angiomatosis, endocarditis, cat scratch disease, neuroretinitis, and asymptomatic bacteremia. Such a wide spectrum of infections makes it necessary to develop species and strain identification tools in order to perform phylogenetic and epidemiological studies. The 16S/23S rRNA intergenic spacer region (ITS) was sequenced for four previously untested species, B. vinsonii subsp. arupensis, B. tribocorum, B. alsatica, and B. koehlerae, as well as for 28 human isolates of B. quintana (most of them from French homeless people), six human or cat isolates of B. henselae, five cat isolates of B. clarridgeiae, and four human isolates of B. bacilliformis. Phylogenetic trees inferred from full ITS sequences of the 14 recognized Bartonella species using parsimony and distance methods revealed high statistical support, as bootstrap values were higher than those observed with other tested genes. Five well-supported lineages were identified within the genus and the proposed phylogenetic organization was consistent with that resulting from protein-encoding gene sequence comparisons. The ITS-derived phylogeny appears, therefore, to be a useful tool for investigating the evolutionary relationships of Bartonella species and to identify Bartonella species. Further, partial ITS amplification and sequencing offers a sensitive means of intraspecies differentiation of B. henselae, B. clarridgeiae, and B. bacilliformis isolates, as each strain had a specific sequence. The usefulness of this approach in epidemiological investigations should be highlighted. Among B. quintana strains, however, the genetic heterogeneity was low, as only three ITS genotypes were identified. It was nevertheless sufficient to show that the B. quintana population infecting homeless people in France was not clonal.

TRNA genes were found in Piscirickettsia salmonis 16S–23S rDNA spacer region (ITS)

Piscirickettsia salmonis is the etiological agent of Salmonid Rickettsial Septicemia, a disease affecting salmon aquaculture industry. We analyzed the 16S-23S rDNA spacer region (internal transcribed spacer, ITS) of Chilean P. salmonis isolates LF-89 and EM-90. Two main ITS amplification products were obtained by PCR using L1 and G1 primers, differing from that described where only one ITS region was found. By Southern blot, it was established that these two amplification products contained sequences related to P. salmonis ITS. Sequence analysis confirmed that P. salmonis had two ITS regions: ITS A and ITS B. In both isolates, the smaller (ITS B) corresponded to ITS sequences previously described for each one, and the larger (ITS A) were almost the same as their respective ITS B sequences interrupted by an insert which contained two tRNAs genes: tRNA-Ile and tRNA-Ala.

TRNA genes were found in Piscirickettsia salmonis 16S–23S rDNA spacer region (ITS)

Piscirickettsia salmonis is the etiological agent of Salmonid Rickettsial Septicemia, a disease affecting salmon aquaculture industry. We analyzed the 16S–23S rDNA spacer region (internal transcribed spacer, ITS) of Chilean P. salmonis isolates LF-89 and EM-90. Two main ITS amplification products were obtained by PCR using L1 and G1 primers, differing from that described where only one ITS region was found. By Southern blot, it was established that these two amplification products contained sequences related to P. salmonis ITS. Sequence analysis confirmed that P. salmonis had two ITS regions: ITS A and ITS B. In both isolates, the smaller (ITS B) corresponded to ITS sequences previously described for each one, and the larger (ITS A) were almost the same as their respective ITS B sequences interrupted by an insert which contained two tRNAs genes: tRNA-Ile and tRNA-Ala.

MYCOBIONT-SPECIFIC PCR PRIMERS FOR THE AMPLIFICATION OF NUCLEAR ITS AND LSU rDNA FROM LICHENIZED ASCOMYCETES

MYCOBIONT-SPECIFIC PCR PRIMERS FOR THE AMPLIFICATION OF NUCLEAR ITS AND LSU rDNA FROM LICHENIZED ASCOMYCETES