Caracterización de Fusarium spp. asociado con la pudrición basal de la cebolla de rama / Characterization of Fusarium spp. associated with the basal rot of the green onion

RESUMEN El departamento de Nariño ocupa el cuarto lugar como productor de cebolla de rama, a nivel nacional. En los últimos años, su producción y área de cultivo se han reducido por múltiples limitantes, destacándose, como la más importante, la susceptibilidad al ataque de hongos causantes de pudriciones radicales, los cuales, perjudican el sistema productivo y la rentabilidad. El objetivo del estudio fue caracterizar morfológica y molecularmente las poblaciones de Fusarium, asociados a la enfermedad de pudrición basal de cebolla de rama. Para ello, en los municipios de Pasto, Potosi y Buesaco, se colectaron plantas con síntomas de pudrición basal, acompañada de necrosis de raíces y ablandamiento de tejido. En el laboratorio de Sanidad Vegetal de la Universidad de Nariño, se sembraron tejidos en medio PDA y, a continuación, se purificaron los aislamientos para su posterior caracterización morfológica y molecular. El estudio morfológico, se realizó usando claves taxonómicas para el género Fusarium y la caracterización molecular con cebadores específicos para el género Fusarium y mediante secuenciación. Finalmente, se realizó un análisis filogenético de la variabilidad intraespecífica. Los resultados de la caracterización morfológica y molecular corroboran la presencia de dos especies dentro del género asociadas a esta patología, F. oxysporum f sp. cepae y F. solani. Los análisis filogenéticos muestran alta variabilidad intraespecífica entre los aislamientos de F. oxysporum y F. solani, formando dos complejos Fusarium oxysporum (FOSC) y Fusarium solani (FSSC), manifestando que estas especies no parten de un ancestro común.

ABSTRACT The department of Nariño occupies the fourth place as producer of green onion nationwide. In recent years, its production and cultivation area has been reduced by multiple limitations, highlighting as the most important, the susceptibility to the attack of fungi causing radical problems, which harm the productive system and profitability. This study was carried out in order to morphologically and molecularly characterizes Fusarium populations associated with green onion basal rot disease. For this, in Pasto, Potosi and Buesaco municipalities, plants were collected with basal rot symptoms, tissue deterioration and root necrosis. In the Plant Health laboratory of the University of Nariño, tissues were planted in PDA medium and subsequently the isolates were purified for further morphological and molecular characterization. The morphological study was carried out using taxonomic keys for the genus Fusarium and molecular characterization with specific primers for the genus Fusarium, and by sequencing. Finally, a phylogenetic analysis of the intraspecific variability was carried out. Morphological and molecular characterization results corroborate the presence of two species within the genus associated with this pathology, F. oxusporum f sp. cepae and F. solani. Phylogenetic analyzes show high intraspecific variability between the isolates of F. oxysporum and F. solani, forming two Fusarium oxysporum (FOSC) complexes and Fusarium solani (FSSC), evidencing that these species do not start from a common ancestor.

Padronização de multiplex PCR para detecção de dermatófitos em pelos e crostas de cães e gatos / Standardization of a PCR multiplex for the detection of dermatophytes in dogs and cats fur and crusts

Objetivou-se padronizar uma reação do tipo multiplex PCR (mPCR) para detectar Microsporum canis, Microsporum gypseum e o complexo Trichophyton mentagrophytes em amostras de pelos e/ou crostas de cães e gatos. 250 amostras de pelos e/ou crostas de cães e gatos foram analisadas por meio de exame direto e cultura, o DNA das mesmas foi extraído para mPCR. Primers foram desenhados e como controle positivo da reação utilizou-se o DNA extraído de colônias de M. canis (URM 6273), M. gypseum (URM 6921) e T. mentagrophytes (URM 6211), provenientes da Coleção de Culturas (Micoteca URM), Departamento de Micologia, Centro de Ciências Biológicas da Universidade Federal de Pernambuco (CCB/UFPE). Como controles negativos de reação, utilizou-se água destilada esterilizada e DNA extraído de Alternaria sp. para verificar a especificidade dos primers. Do total de amostras analisadas, 15 (6%) foram identificadas, em cultura, como dermatófitos, e destas, 10 foram M. canis, três M. gypseum e dois T. mentagrophytes (complexo). Destas 15 amostras positivas, 11 (73,3%) foram detectadas por meio da mPCR. Além destas, seis outras, negativas em cultura, foram identificadas como M. gypseum. Verificou-se uma boa concordância entre os resultados da cultura e mPCR (Kappa: 0,66). O protocolo padronizado neste estudo pode ser utilizado como um método de triagem, por apresentar uma sensibilidade maior que a da cultura, usado paralelamente aos exames de rotina, permitindo um diagnóstico em menor tempo.(AU)

The aim of this study was to standardize a multiplex PCR (mPCR) reaction to detect Microsporum canis, Microsporum gypseum and the Trichophyton mentagrophytes complex in dog and cat fur and/or crusts. 250 fur and/or crusts samples from dogs and cats were analyzed by direct examination and culture, DNA from them was extracted for mPCR. Primers were designed and the DNA extracted from colonies of M. canis (URM 6273), M. gypseum (URM 6921) and T. mentagrophytes (URM 6211) from the Collection of Cultures - URM Micoteca - Department of Mycology, Biological Sciences Center of the Federal University of Pernambuco (CCB / UFPE). As negative controls, sterile distilled water and DNA extracted from Alternaria sp., were used to verify the specificity of the primers. Of the total samples analyzed, 15 (6%) were identified in culture as dermatophytes, and of these, 10 were M. canis, three M. gypseum and two T. mentagrophytes (complex). Of these 15 positive samples, 11 (73.3%) were detected by mPCR. Besides these, six others, negative in culture, were identified as M. gypseum. There was good agreement between culture results and mPCR (Kappa: 0.66). The protocol standardized in this study can be used as a screening method, because it has a sensitivity greater than that of the culture, used in parallel to the routine exams, allowing a diagnosis in a shorter time.(AU)

PCR Identification and Phylogenetic Analysis of Trichomonas gallinae from Domestic Pigeons in Guangzhou, China

Avian trichomoniasis caused by Trichomonas gallinae is a serious protozoan disease worldwide. The domestic pigeon (Columba livia domestica) is the main host for T. gallinae and plays an important role in the spread of the disease. Based on the internal transcribed spacers of nuclear ribosomal DNA of this parasite, a pair of primers (TgF2/TgR2) was designed and used to develop a PCR assay for the diagnosis of T. gallinae infection in domestic pigeons. This approach allowed the identification of T. gallinae, and no amplicons were produced when using DNA from other common avian pathogens. The minimum amount of DNA detectable by the specific PCR assay developed in this study was 15 pg. Clinical samples from Guangzhou, China, were examined using this PCR assay and a standard microscopy method, and their molecular characteristics were determined by phylogenetic analysis. All of the T. gallinae-positive samples detected by microscopic examination were also detected as positive by the PCR assay. Most of the samples identified as negative by microscopic examination were detected as T. gallinae positive by the PCR assay and were confirmed by sequencing. The positive samples of T. gallinae collected from Guangzhou, China, were identified as T. gallinae genotype B by sequencing and phylogenetic analyses, providing relevant data for studying the ecology and population genetic structures of trichomonads and for the prevention and control of the diseases they cause.

Internal transcribed spacer guided multiplex PCR for species identification of Convolvulus prostratus and Evolvulus alsinoides

Shankhpushpi is a reputed drug from an Indian system of medicine for treating mental disorders and enhancing memory. Two herbs, namely Convolvulus prostratus Forssk. and Evolvulus alsinoides (L.) L., are commonly known as Shankhpushpi. Ambiguous vernacular identity can affect the scientific validity of the Shankpushpi-based herbal drug therapy. In the present investigation, a novel and sensitive multiplex PCR method based on polymorphism in the internal transcribed spacer (ITS) region was developed to establish the molecular identity of C. prostratus and E. alsinoides. DNA was isolated and the ITS region was amplified, sequenced and assembled. Sequences were aligned to identify variable nucleotides in order to develop plant-specific primers. Primers were validated in singleplex reactions and eventually a multiplex assay was developed. This assay was tested for sensitivity and validated by amplifying DNA isolated from the simulated blended powdered plant material. Primers developed for C. prostratus resulted into a 200 bp amplicon and 596 bp for E. alsinoides. The assay was found to be sensitive enough for amplification of low quantities of DNA. The method can detect 10% of the mixing of plants with each other in blended material. This PCR assay can be used for rapid botanical identification of Shankhpushpi plant materials and will improve evidence-based herbal drug therapy.

Laboratory Experience in Phenotypic and Molecular Identification of Blastomyces dermatitidis First Isolated in Korea / 대한임상미생물학회지

Blastomycosis, endemic in North America, has been hardly reported in Korea. We describe laboratory experience in phenotypic and molecular identification of Blastomyces dermatitidis first isolated in Korea. The patient was a 45-year-old male with pulmonary blastomycosis mimicking pulmonary tuberculosis. Diagnosis was based on culture and dimorphism combined with DNA target sequencing of internal transcribed spacers (ITS) and D1/D2 regions.

Identification of Candida Species by Multiplex Polymerase Chain Reaction / 대한임상미생물학회지

BACKGROUND: Polymerase chain reacation (PCR)-based methods have been described for rapid detection and identification of Candida spp. Multiplex PCR assay was developed using internal transcribed spacers and topoisomerase II gene for the accurate identification of Candida species. METHODS: We designed Dual Specificity Oligo (DSO) primers for multiplex PCR. Multiplex PCR was followed by agarose gel electrophoresis to test 8 type strains (C. albicans, C. parapsilosis, C. glabrata, C. tropicalis, C. krusei, C. guilliermondii, C. lusitaniae, C. dubliniensis) and 96 clinical isolates (C. albicans 51 isolates, C. parapsilosis 10 isolates, C. glabrata 10 isolates, C. tropicalis 9 isolates, C. krusei 6 isolates, C. guilliermondii 5 isolates, C. lusitaniae 5 isolates) of Candida spp. RESULTS: With multiplex PCR using DSO primers, the eight Candida type strains each could be easily differentiated and all 96 clinical isolates were identified as the same species as were identified by the conventional method. CONCLUSION: Multiplex PCR followed by electrophoresis can be useful for the simple and rapid identification of Candida species in routine laboratories.

Phylogenetic Analysis of Caterpillar Fungi by Comparing ITS 1-5.8S-ITS 2 Ribosomal DNA Sequences

This study was carried out to identify the phylogenetic relationships among several caterpillar fungi by comparing the sequences of internal transcribed spacer regions (ITS1 and ITS2) and 5.8S ribosomal DNA (rDNA) repeat unit. The sequences of ITS1, ITS2, and the 5.8S rDNA from 10 strains of Cordyceps species, 12 strains of Paecilomyces, 3 strains of Beauveria, 2 strains of Metarhizium and 1 strains of Hirsutella were amplified, determined and compared with the previously known Cordyceps species. The sequences of 5.8S rDNA were more conserved in length and variation than those of ITS regions. Although the variable ITS sequences were often ambiguously aligned, the conserved sites could be found. In the phylogenetic tree, the species generally divided into three clusters, supported by their morphology and/or host ranges. The 5.8S rDNA and ITS1 sequences among 10 species of Cordyceps militaris were identical and only one base pair in ITS2 sequence was different. Cordyceps sinensis and Cordyceps ophioglossoides were also clearly different, although they belonged to the same cluster. The GenBank database search of species revealed sister taxa of an entomogenous fungus. Metarhizium was used as an outgroup in all taxa.

ITS sequence analysis on medicinal plants of Euphorbia L. in Anhui and Jiangsu Provinces / 中草药

Objective To study the correlation and variation between plants of internal transcribed spacers (ITS) sequence of the six medicinal plants of Euphorbia L. in Anhui and Jiangsu Provinces, in (order) to provide their DNA molecular marker to identify and explore the phylogenetic relationship of the plants of [WTBX]Euphorbia L. Methods To determine rDNA ITS sequence for the plants of Euphorbia L. by PCR technology. Results The sequences of ITS1 in the six species ranged from 255 to 262 bp in length and those of ITS2 from 214 to 236 bp. The dendrogram was obtained with Mega2 analysis. The analysis result was consistent with those from morphology. Conclusion The method can be used to identify the plants of [WTBX](Euphorbia) L. among different species and to differentiate their fakes.

Ribosomal DNA-ITS sequence analysis and molecular identification of Morinda officinalis and its counterfeit species / 中草药

Objective To compare the rDNA-ITS differentiation and its regulation between Morinda (officinalis) and its counterfeit species, and provide DNA molecular markers for the fingerprint identification of them. Methods The rDNA-ITS regions of M. officinalis and its counterfeit species were amplified and sequenced, then analyzed by means of CLUSTRAL X and MEGA softwares. Results The internal transcribed spacers (ITS) including ITS1, 5.8S, ITS2, and partial 18S and 26S were determined. In DNA DIST analysis, the range of diversity among M. officinalis and M. shughuaeusis, M. umbellata was (2.9%-)5.8% and 2.9%-4.2% based on ITS1 and ITS2; the range of diversity between M. officinalis and Damnacanthus indicus was 21.2% and 18.9% based on ITS1 and ITS2. Phylogenetic tree based on ITS and 5.8S sequence data indicated the M. umbellata and M. shuanghuaensis were closely related then with M. officinalis, while D. indicus was monophyletic group. Conclusion The rDNA-ITS sequence is a better molecular marker for idertification of M. officinalis and its counterfeit species.

Comparison on internal transcribed spacers (ITS) sequences of Tibetan medicine Saussurea medusa and its easily confusable species / 中草药

Object In order to identify the medicine at the molecular level, the internal transcribed spacers (ITS) of Saussurea medusa Maxim and its easily confusable species were sequenced Methods The double stranded DNA was amplified using PCR systems 9 600 kits and sequenced on an ABI 377 automated sequencer from both directions Results The ITS sequences of S medusa of different populations showed no variation, but there existed distinct variation between S medusa and its confusable species Conclusion ITS sequences can be used for the molecular authentication between S medusa and its confusable species

The Identification of Two Isolates from Cadaver of Brontispa longissima (Gestro) and Their Phylogeny / 微生物学通报

The strains Dz01 and Ma4 were isolated from cadavers of Brontispa longissima(Gestro),and were confirmed to be pathogenicity to Brontispa longissima(Gestro).After microscopical observation of the morphological characters of mycelium,phialide and conidia from two isolates,they were found to be identical to Metarhizium anisopliae var anisopliae,so they were identificated as M.anisopliae var anisopliae.The Maximum Parsimony tree constructed based on the sequences of ITS1-5.8S-ITS2 regions in ribosomal DNA from two isolates and 31 other isolates which represent different species or varity species of genus Metarhizium obtained from GenBank database showed that two isolates clustered together in the clade which was composed of the isolates classified as Metarhizium anisopliae var anisopliae.This provided the molecular data for the result of morphological identification of Dz01 and Ma4 isolates.