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1.
Rev Inst Med Trop Sao Paulo ; 61: e37, 2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-31411267

RESUMO

Candida glabrata complex includes three species identified through molecular biology methods: C. glabrata sensu stricto , C. nivariensis and C. bracarensis . In Mexico, the phenotypic methods are still used in the diagnosis; therefore, the presence of C. nivariensis and C. bracarensis among clinical isolates is still unknown. The aim of this study was to evaluate the utility of a multiplex PCR for the identification of the C. glabrata species complex. DNA samples from 92 clinical isolates that were previously identified through phenotypic characteristics as C. glabrata were amplified by four oligonucleotides (UNI-5.8S, GLA-f, BRA-f, and NIV-f) that generate amplicons of 397, 293 and 223-bp corresponding to C. glabrata sensu stricto , C. nivariensis , and C. bracarensis , respectively. The amplicon sequences were used to perform a phylogenetic analysis through the Maximum Likelihood method (MEGA6), including strains and reference sequences of species belonging to C. glabrata complex. In addition, recombination and linkage disequilibrium were estimated (DnaSP version 5.0) for C. glabrata sensu stricto isolate s . Eighty-eight isolates generated a 397-bp fragment and only in one isolate a 223-bp amplicon was observed. In the phylogenetic tree, the sequences of 397-bp were grouped with C. glabrata reference sequences , and the sequence of 223-bp was grouped with C. bracarensis reference sequences, corroborating the PCR identification. The number of recombination events for the isolates of C. glabrata sensu stricto was zero, suggesting a clonal population structure. Three isolates that did not amplify any of the expected fragments were identified as Saccharomyces cerevisiae through the sequencing of the D1/D2 domain region within the 28S rDNA gene. The multiplex PCR is a fast, cost-effective and reliable tool that can be used in clinical laboratories to identify C. glabrata complex species.


Assuntos
Candida glabrata/genética , Candidíase/microbiologia , DNA Fúngico/genética , Técnicas de Tipagem Micológica/métodos , Candida glabrata/isolamento & purificação , DNA Ribossômico/genética , DNA Espaçador Ribossômico/genética , Feminino , Humanos , Masculino , México , Reação em Cadeia da Polimerase Multiplex , Filogenia , Análise de Sequência de DNA
2.
World J Microbiol Biotechnol ; 35(9): 138, 2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31451937

RESUMO

Monascus azaphilone pigments, including red, orange, and yellow, are world-famous food colorants. However, the pigments produced by different Monascus species vary in yields and compositions. The underlying mechanism is unclear. In this study, four wild-type Monascus strains, namely M. anka M7, M. purpureus M9, M. ruber C100, and M. aurantiacus M15, were selected as research objects according to the diversification of their pigments fermented in the same mediums and conditions. Twenty-three 3 kbp segments (300 bp overlap with adjacent segments) of the pigment gene cluster were amplified, sequenced, and assembled into the DNA sequences of the clusters. The DNA sequences of pigment biosynthetic gene clusters of the four strains showed 99.94% similarity according to the results of multiple alignment. The expression levels of 17 pigment biosynthetic genes of four strains were determined by using real-time quantitative PCR. The transcriptional regulation contributed more than the DNA sequence variation in Monascus pigments metabolism. Our result gives insight into the study of Monascus pigment biosynthesis.


Assuntos
Monascus/genética , Monascus/metabolismo , Pigmentos Biológicos/biossíntese , Transcrição Genética , Sequência de Aminoácidos , Sequência de Bases , Cor , DNA Fúngico/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Variação Genética , Monascus/química , Monascus/classificação , Família Multigênica , Filogenia , Pigmentos Biológicos/química
3.
Int J Syst Evol Microbiol ; 69(10): 3161-3169, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31390326

RESUMO

During a survey of fungal diversity in a deserted rocky area in Yunnan, PR China, a new species, Memnoniella sinensis, was identified. This new species is characterized by having phialidic conidiogenous cells with conspicuous collarettes, and aseptate, verrucose, ellipsoidal to sometimes ovoid, olivaceous brown to dark brown conidia. Morphologically, M. sinensis is similar to M. dichroa, but can be easily distinguished due to its hyaline conidiophores and smaller conidia. Phylogenetic analysis based on DNA sequences at five loci showed that our strain grouped together with M. dichroa and M. oenanthes. Here, the new species is described and illustrated, and a key to the species of the genus Memnoniella is provided.


Assuntos
Hypocreales/classificação , Filogenia , China , DNA Fúngico/genética , Hypocreales/isolamento & purificação , Técnicas de Tipagem Micológica , Análise de Sequência de DNA , Esporos Fúngicos
4.
Plant Dis ; 103(10): 2624-2633, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31397632

RESUMO

Brown spot disease caused by Colletotrichum species was found on leaves of mulberry (Morus alba L.) in Dujiangyan, Sichuan Province, China. Fungal isolates from leaf lesions were identified as six Colletotrichum species based on morphological characteristics and DNA analysis of the combined sequences ITS, GAPDH, ACT, CHS-1, TUB2, and GS. These included Colletotrichum fioriniae, C. fructicola, C. cliviae, C. karstii, C. kahawae subsp. ciggaro, and C. brevisporum. Results showed that the most important causal agent of mulberry anthracnose was C. fioriniae, causing typical brown necrotic spots or streaks, followed by C. brevisporum, C. karstii, and C. kahawae subsp. ciggaro, whereas the two other species (C. fructicola and C. cliviae) showed no pathogenicity to mulberry. This study is the first report of these species associated with mulberry in China.


Assuntos
Colletotrichum , Morus , Filogenia , Virulência , China , Colletotrichum/patogenicidade , DNA Fúngico/genética , Morus/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Especificidade da Espécie
5.
Int J Syst Evol Microbiol ; 69(9): 2828-2833, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31274406

RESUMO

Strain SYSU-17, representing a novel acid-tolerant yeast species which can grow at pH 2.0 weakly, was isolated from acid mine drainage collected in a tailing impoundment of the Fankou Lead/Zinc Mine, Guangdong Province, PR China. Phylogenetic analysis of strain SYSU-17 based on the internal transcribed spacer (ITS) region and the D1/D2 domains of the large subunit ribosomal RNA (LSU rRNA) gene suggested that strain SYSU-17 was a novel species belonging to the genus Spencerozyma (class Microbotryomycetes, subphylum Pucciniomycotina). It differed from the type strain of the closest related species, Spencerozyma crocea CBS 2029T, by 0.7 % sequence divergence (three gaps and one nucleotide substitution out of 594 bp) in the D1/D2 domains of the LSU rRNA gene and 7.6 % sequence divergence (32 gaps and 22 nucleotide substitutions out of 714 bp) in the ITS region. In contrast to the physiological properties of S. crocea, the novel yeast species was unable to assimilate galactose, d-ribose, xylitol, succinate, d-xylose, ethanol, nitrate and nitrite. The name Spencerozyma acididurans sp. nov. is proposed and SYSU-17 is designated as the holotype.


Assuntos
Basidiomycota/classificação , Mineração , Filogenia , Microbiologia da Água , Ácidos , Basidiomycota/isolamento & purificação , China , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Técnicas de Tipagem Micológica , Análise de Sequência de DNA
6.
Int J Syst Evol Microbiol ; 69(10): 3087-3092, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31329532

RESUMO

Four strains, NYNU 15610, NYNU 15612, NYNU 15613 and NYNU 15615, of a novel ascomycetous yeast were isolated from the gut of Allomyrina dichotoma (Coleoptera: Scarabeidae) collected from two different localities in Henan Province, Central PR China. The four strains shared identical sequences in both of the D1/D2 domains of the large subunit rRNA gene and the internal transcribed spacer regions. Sequence analyses revealed that this novel species represents a member of the genus Metschnikowia. It differed from its closest known species Metschnikowia zobellii, Metschnikowiaaustralis and Metschnikowia bicuspidata, by 8.4-9.2 % sequence divergence (33-40 nt substitutions and 7-12 gaps over 509 bases) in the D1/D2 sequences. The formation of ascospores was not observed on various sporulation media. In contrast to M. zobellii, M. australis and M. bicuspidata, the novel yeast species was unable to assimilate succinate, ethanol, ethylamine, cadaverine and 10 % NaCl plus 5 % glucose, but was able to grow in vitamin-free medium. The name Metschnikowia baotianmanensis f.a., sp. nov. is proposed to accommodate these strains, with NYNU 15613 as the holotype.


Assuntos
Besouros/microbiologia , Metschnikowia/classificação , Filogenia , Animais , China , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Metschnikowia/isolamento & purificação , Técnicas de Tipagem Micológica , Análise de Sequência de DNA , Esporos Fúngicos
7.
Int J Syst Evol Microbiol ; 69(9): 2823-2827, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31259676

RESUMO

Strains of yeast were isolated under a nitrogen-depleted culture condition from decaying tree bark (strain N-12.1) and from mangrove forest water (strain 1-7W.1) sampled at different locations within a mangrove forest site in Ranong province, Thailand. They were found to be genetically and phenotypically different from any currently recognised yeast species. Phylogenetic analysis of nucleotide sequence of three genes, the internal transcribed spacer region 1 and 2 plus 5.8S ribosomal RNA gene (ITS), D1/D2 domain of the large subunit ribosomal RNA gene (LSU D1/D2) and the small subunit of the ribosomal RNA gene (SSU), revealed that these two strains were related to but distinguished from Heterocephalacriaarrabidensis. Several distinct physiological characteristics of these two strains were detected, namely inability to assimilate glycerol, dl-lactic acid, succinic acid, citric acid, d-gluconic acid, and ability to grow well at 25 °C, which were different from those of H. arrabidensis. Accordingly, the name Heterocephalacria mucosa sp. nov., is proposed to accommodate this novel species. The MycoBank number is MB 828624.


Assuntos
Basidiomycota/classificação , Filogenia , Casca de Planta/microbiologia , Basidiomycota/isolamento & purificação , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Técnicas de Tipagem Micológica , RNA Ribossômico 5,8S , Análise de Sequência de DNA , Tailândia , Áreas Alagadas
8.
Int J Syst Evol Microbiol ; 69(9): 2899-2906, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31274407

RESUMO

A facultative halo-tolerant Aspergillus strain was isolated from olive brine waste, the effluent from the debittering process of table olives. Phenotypic and molecular characteristics showed clearly that the isolate represents a novel species. Based on the source of isolation, the new species has been named Aspergillus olivimuriae. It was found tolerant to high concentrations of NaCl (15 %) or sucrose (60 %) and it exhibits substantial growth under these conditions. Although the new species grew profusely at 37 °C, no growth was observed at 40 °C, conidia en masse were avellaneous on all media. The description of the new species Aspergillus olivimuriae brings the total species of Aspergillus sect. Flavipedes to 15. The type strain of A. olivimuriae sp. nov. is NRRL 66783 (CCF 6208), its whole genome has been deposited as PRJNA498048.


Assuntos
Aspergillus/classificação , Microbiologia de Alimentos , Olea/microbiologia , Filogenia , Sais , Aspergillus/isolamento & purificação , DNA Fúngico/genética , Técnicas de Tipagem Micológica , Pigmentação , Análise de Sequência de DNA , Esporos Fúngicos
9.
Zhongguo Zhong Yao Za Zhi ; 44(10): 2046-2050, 2019 May.
Artigo em Chinês | MEDLINE | ID: mdl-31355559

RESUMO

The endophytic fungi from root,main stem,branch and leaf of Scrophularia ningpoensis were isolated from Zhejiang,whether these strains could yield harpagide or harpagoside were tested by HPLC and LC-MS. According to the morphological characteristic and the similarity of the nucleotide sequence of internal transcribed spacer( ITS) between r DNAs,the strains producing harpagide or harpagoside were identified. The results showed that 210 strains were isolated from the samples,which were classified into 9 orders,13 families and 17 genera by morphological study. Harpagide was detected in endogenous fungi ZJ17 and harpagoside was detected in endogenous fungi ZJ25 by HPLC coupled with LC-MS. ZJ17 was identified as Alternaria alternate and ZJ25 was identified as A.gaisen by its morphology and authenticated by ITS( ITS4 and ITS5 regions and the intervening 5. 8 S rDNA region).


Assuntos
Fungos/classificação , Glicosídeos/biossíntese , Glicosídeos Iridoides/metabolismo , Piranos/metabolismo , Scrophularia/microbiologia , China , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Endófitos/classificação , Endófitos/metabolismo , Fungos/metabolismo
10.
Hautarzt ; 70(8): 618-626, 2019 Aug.
Artigo em Alemão | MEDLINE | ID: mdl-31263912

RESUMO

In this study, a novel real-time polymerase chain reaction (PCR) assay (DermaGenius®2.0, PathoNostics BV, Maastricht, The Netherlands) and a recently developed microarray test (EUROArray Dermatomycosis, Euroimmun, Lübeck, Germany) were evaluated regarding their diagnostic specificity to identify dermatophyte DNA. The tests were compared to conventional methods and sequencing. The microarray Dermatomycosis test allows the detection of 50 dermatophytes and definitive identification of 23 dermatophyte species, 6 yeasts and moulds combined in one test. In comparison, real-time PCR is able to identify 11 dermatophytes and one yeast at the species level. Using the EUROArray, 22 out of 24 dermatophyte species were correctly identified. Using real-time PCR, 9 out of the 11 different dermatophytes included in the test kit were correctly identified. Both molecular tests for detection and differentiation of dermatophytes are useful tools for daily clinical practice. The real-time PCR test does not detect as many species, and specificity is slightly lower. However, real-time PCR is a very fast and easy to perform test, especially since no post-PCR step is necessary. Real-time PCR detects the most frequent dermatophytes like T. rubrum, T. interdigitale, and M. canis without any problems. The EUROArray is more elaborate to perform in the lab, due to the hybridization step. However, the EUROArray shows higher specificity and can detect a much broader range of causative agents, including rare species, in dermatomycology.


Assuntos
DNA Fúngico/classificação , DNA Fúngico/genética , Dermatomicoses , Reação em Cadeia da Polimerase em Tempo Real/métodos , Trichophyton/classificação , Trichophyton/genética , DNA Fúngico/isolamento & purificação , Alemanha , Humanos , Microsporum/classificação , Microsporum/genética , Microsporum/isolamento & purificação , Países Baixos , Trichophyton/isolamento & purificação
11.
Int J Syst Evol Microbiol ; 69(10): 3217-3223, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31339482

RESUMO

Phalangispora sinensis, an aquatic hyphomycete collected from south-western PR China, is described as a new species. This new species is characterized by having multicellular branched conidia composed of a curved main axis and one or two laterals, with the laterals arising from the third or fourth cell of the base of the main axis. Combined analyses of the LSU, SSU, RPB2 and TEF1 gene sequence data revealed that Phalangispora and another aquatic hyphomycete genus, Setosynnema, belonged to Wiesneriomycetaceae, Tubeufiales, Dothideomycetes, Ascomycota.


Assuntos
Ascomicetos/classificação , Filogenia , Microbiologia da Água , Ascomicetos/isolamento & purificação , China , DNA Fúngico/genética , Técnicas de Tipagem Micológica , Análise de Sequência de DNA , Esporos Fúngicos
12.
Int J Syst Evol Microbiol ; 69(10): 3262-3267, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31343399

RESUMO

Ethanol production at high temperatures has garnered much interest in recent years and a key factor is the availability of thermotolerant yeasts. During an investigation on the diversity of thermotolerant yeasts from different habitats, a novel yeast species from the spent wash of a distillery unit associated with a sugar factory was isolated. Phylogenetic analysis of D1/D2 large subunit and ITS rRNA genes placed this species in the ascomycetous genus Wickerhamiella. The novel species can be distinguished from the closely related species Wickerhamiella pararugosa using these rRNA gene regions. The cells of the new species are ovoid to ellipsoid with a diameter of 3.5-6.0×2.4-3.10 µm, while W. pararugosa cells are cylindrical with a cell diameter of 1.5-3.0×6-23 µm. This novel species represents, together with Wickerhamiella cacticola, one of the two most thermotolerant yeast species in the genus Wickerhamiella, able to grow at 42 °C. Wickerhamiella shivajii sp. nov. is proposed during this study.


Assuntos
Filogenia , Saccharomycetales/classificação , Águas Residuárias/microbiologia , DNA Fúngico/genética , Etanol , Índia , Técnicas de Tipagem Micológica , Saccharomycetales/isolamento & purificação , Análise de Sequência de DNA
13.
Semin Ophthalmol ; 34(4): 223-231, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31170015

RESUMO

Purpose: To review the value of next-generation sequencing (NGS) in identifying the pathogens which cause ocular infections, thereby facilitating prompt initiation of treatment with an optimal anti-microbial regimen. Both contemporary and futuristic approaches to identifying pathogens in ocular infections are covered in this brief overview. Methods: Review of the peer reviewed literature on conventional and advanced methods as applied to the diagnosis of infectious diseases of the eye. Conclusion: NGS is a novel technology for identifying the pathogens responsible for ocular infections with the potential to improve the accuracy and speed of diagnosis and hastening the selection of the best therapy.


Assuntos
Infecções Oculares/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos , DNA Bacteriano/genética , DNA Fúngico/genética , DNA Ribossômico/genética , Humanos , Reação em Cadeia da Polimerase
14.
Nat Commun ; 10(1): 2862, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253793

RESUMO

DNA double strand breaks (DSBs) pose a high risk for genome integrity. Cells repair DSBs through homologous recombination (HR) when a sister chromatid is available. HR is upregulated by the cycling dependent kinase (CDK) despite the paradox of telophase, where CDK is high but a sister chromatid is not nearby. Here we study in the budding yeast the response to DSBs in telophase, and find they activate the DNA damage checkpoint (DDC), leading to a telophase-to-G1 delay. Outstandingly, we observe a partial reversion of sister chromatid segregation, which includes approximation of segregated material, de novo formation of anaphase bridges, and coalescence between sister loci. We finally show that DSBs promote a massive change in the dynamics of telophase microtubules (MTs), together with dephosphorylation and relocalization of kinesin-5 Cin8. We propose that chromosome segregation is not irreversible and that DSB repair using the sister chromatid is possible in telophase.


Assuntos
Cromátides/metabolismo , Segregação de Cromossomos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Fúngico/genética , Troca de Cromátide Irmã , Telófase/genética , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Recombinação Genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
Int J Syst Evol Microbiol ; 69(9): 2658-2661, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31162007

RESUMO

Three strains of a novel yeast species were isolated from rotting wood in the Xishuangbanna Tropical Rainforest, Yunnan Province, PR China. Sequence analysis of the D1/D2 domains of the large subunit rRNA gene and the internal transcribed spacer (ITS) regions showed that the novel species represents a member of the genus Saturnispora. It differed from its closest known species, Saturnispora sekii CBS 10931T, by 1.3 % nucleotide substitutions in the D1/D2 domains and by 2.2 % nucleotide substitutions in the ITS regions, respectively. In contrast to Saturnispora sekii, the novel yeast species was unable to assimilate glycerol, dl-lactate, succinate and citrate, and grow at 37 °C. The name Saturnispora galanensis sp. nov. is proposed to accommodate these strains, with NYNU 1797 as the holotype.


Assuntos
Filogenia , Floresta Úmida , Saccharomycetales/classificação , Madeira/microbiologia , China , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Técnicas de Tipagem Micológica , Saccharomycetales/isolamento & purificação , Análise de Sequência de DNA
16.
Int J Syst Evol Microbiol ; 69(9): 2674-2680, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31166159

RESUMO

Three strains, representing a novel anamorphic and d-xylose-fermenting yeast species, were isolated from moss (ST-302T), seawater (ST-1169) and peat (DMKU-XE12) collected from the southern part of Thailand. The three strains had identical sequences of the D1/D2 regions of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) regions. Candida flosculorum CBS 10566T and Candida sharkiensis CBS 11368T were the most closely related species with 7.9 % nucleotide substitutions in the D1/D2 regions of the LSU rRNA gene, and 10.3 and 12.6% nucleotide substitutions in the ITS regions, respectively. Phylogenetic analysis based on the concatenated sequences of the ITS and the D1/D2 regions confirmed that the three strains represented a distinct anamorphic species in the Clavispora clade. Therefore, the three strains were described as a novel species, for which we propose the name Candida xylosifermentans sp. nov.


Assuntos
Candida/classificação , Filogenia , Xilose/metabolismo , Briófitas/microbiologia , Candida/isolamento & purificação , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Fermentação , Técnicas de Tipagem Micológica , Água do Mar/microbiologia , Análise de Sequência de DNA , Solo , Microbiologia do Solo , Tailândia
17.
Int J Syst Evol Microbiol ; 69(9): 2775-2780, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31237537

RESUMO

During studies on the yeast communities associated with rotting wood in the Xishuangbanna Tropical Rainforest in PR China, four novel yeast strains were found. Phylogenetic analysis based on the concatenated sequences of the D1/D2 domains of the large subunit rRNA gene and the ITS regions showed that these strains represented two novel species in the Candida albicans/Lodderomyces clade. The novel species, represented by strains NYNU 17948 and NYNU 17981, formed a clade with Candida maltosa and Candida baotianmanensis, with 1-1.8% sequence divergence in the D1/D2 domains and 8.9-10% sequence divergence in the ITS regions. The other novel species, represented by NYNU 17105 and NYNU 17763, is most closely related to Candida blackwelliae with 0.7 % sequence divergence in the D1/D2 domains and 6.9 % sequence divergence in the ITS regions. The two novel species could be distinguished from their closest described species in terms of physiological traits. The two novel species are described as Candida yunnanensis sp. nov. (holotype NYNU 17948) and Candida parablackwelliae sp. nov. (holotype NYNU 17763).


Assuntos
Candida/classificação , Filogenia , Floresta Úmida , Madeira/microbiologia , Candida/isolamento & purificação , China , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Técnicas de Tipagem Micológica , Fenótipo , Análise de Sequência de DNA
18.
Plant Dis ; 103(8): 1865-1875, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31161921

RESUMO

Black root rot of avocado is a severe disease of nursery trees and young orchard transplants, causing tree death within a year after planting. In Australia, key pathogens include species complexes Calonectria ilicicola and Dactylonectria macrodidyma; however, several other Dactylonectria species also cause the disease. Rapid detection of these pathogens in planta is important to speed up implementation of disease management and reduce loss. The purpose of this study was to develop three loop-mediated isothermal amplification (LAMP) diagnostic assays to rapidly identify species within the C. ilicicola and D. macrodidyma complexes and species in the Dactylonectria genus in avocado roots. Primers were designed from ß-tubulin sequence data of C. ilicicola and from histone H3 of D. macrodidyma and the Dactylonectria genus. The LAMP primers were tested for specificity and sensitivity with 82 fungal isolates, which included the target species complexes C. ilicicola and D. macrodidyma; species within the target Dactylonectria genus viz. D. macrodidyma, D. anthuriicola, D. novozelandica, D. pauciseptata, and D. vitis; and isolates of nontarget species, including Calonectria sp., Cylindrocladiella sp., Gliocladiopsis forsbergii, G. peggii, G. whileyi, Ilyonectria sp., Mariannaea sp., Fusarium sp., and Phytophthora cinnamomi. The species-specific LAMP assays were sensitive and specific at DNA concentrations of 1 pg/µl for C. ilicicola and 0.01 ng/µl for D. macrodidyma, whereas the Dactylonectria genus-wide assay was sensitive to 0.1 ng/µl. Detection of C. ilicicola occurred within 10 to 15 or 15 to 30 min when the template was pure DNA or crude extracts obtained from suspending fungal cultures in sterile water, respectively. Detection of D. macrodidyma was between 12 to 29 min with pure DNA and 16 to 30 min with crude extracts. Dactylonectria spp. were detected within 6 to 25 min with pure DNA and 7 to 23 min with crude extracts. The specificity of the assays was found to be dependent on time and isothermal amplification temperature, with optimal specificity occurring in reactions of <30 min and at temperatures of 67°C for C. ilicicola and D. macrodidyma assays and 69°C for Dactylonectria genus-wide assays. The assays were modified to accommodate a DNA extraction step and use of avocado roots as DNA templates. Detection in avocado roots ranged between 12 to 25 min for C. ilicicola, 12 to 26 min for D. macrodidyma, and 14 to 30 min for species in the Dactylonectria genus. The LAMP assays are applicable across multiple agricultural industries, because C. ilicicola, D. macrodidyma, and Dactylonectria spp. are also important pathogens of various crops and ornamental plants.


Assuntos
Agricultura/métodos , Hypocreales , Técnicas de Amplificação de Ácido Nucleico , Persea , Austrália , DNA Fúngico/genética , Hypocreales/genética , Persea/microbiologia , Doenças das Plantas/microbiologia
19.
Plant Dis ; 103(8): 1967-1973, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31188736

RESUMO

Rice blast is one of the most serious diseases for rice, and controlling the filamentous fungus Magnaporthe oryzae that causes rice blast is crucial for global food security. Typically, early infected rice does not show symptoms. Therefore, the early diagnosis of rice blast is particularly important to avoid uncontrollable propagation of rice blast fungus. In the present work, a rapid and efficient loop-mediated isothermal amplification (LAMP) method was developed to detect the pathogen at the early infected stage of rice. The Alb1 superfamily hypothetical protein MGG_04322, a nuclear shuttling factor involved in ribosome and melanin biogenesis, was chosen as the target for designing the LAMP primers. The LAMP assay enabled rapid detection of as little as 10 pg of pure genomic DNA of M. oryzae. In addition, we established the quantitative LAMP (q-LAMP) detection system to quantify the conidia of rice blast fungus. The q-LAMP assay enabled rapid detection (within 35 min) of the fungal spores at a sensitivity of 3.2 spores/ml. In addition, the assay sets up the linearization formula of the standard curve as y = 0.3066 + 15.33x (where x = amplification of time), inferring that spore number = 100.60y. In addition, the q-LAMP assay was successfully used to detect the presence of the virulence strains of M. oryzae (wild type) in comparison with that of the two mutant strains by quantifying the biomass within host tissue. These results provide a useful and convenient tool for detecting M. oryzae that could be applied in the incubation period of rice blast before symptoms appear.


Assuntos
Magnaporthe , Técnicas de Amplificação de Ácido Nucleico , Oryza , Primers do DNA , DNA Fúngico/genética , Limite de Detecção , Magnaporthe/genética , Oryza/microbiologia , Doenças das Plantas/microbiologia
20.
Plant Dis ; 103(8): 1998-2009, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31188737

RESUMO

A highly virulent cotton wilt pathogen, Fusarium oxysporum f. sp. vasinfectum VCG0114 (race 4) was found in West Texas in 2017, after being known in California since 2001. Isolates obtained from wilted plants collected in 2017 from Texas, in 2015 from China, and during 2001 to 2014 from California and isolates from historical collections including the race 4 reference isolate were characterized by soil-infestation pathogenicity assays, DNA sequence analysis, and vegetative compatibility analysis. All obtained F. oxysporum f. sp. vasinfectum isolates belonged to VCG0114. All of these isolates, except one isolate from China, caused disease in a soil-infestation assay without nematodes. Thus, they belong to the nematode-independent pathotype. Texas isolates were significantly more virulent than were isolates from China or California on Gossypium barbadense 'Pima S-7'. Four different genotypes (N, T, MT, and MiT) were identified based on the transposable element Tfo1 insertion into the PHO gene and independent MULE or MITE insertions into the Tfo1 transposon. Some significant differences in virulence were detected among the genotypes in some locations. No differences in pathogenicity were observed between the California and China collection isolates on Pima S-7, and the virulence of the major genotypes was similar on the Gossypium hirsutum cultivar 'Stoneville 474' or the Barbren 713 germplasm line. Simple polymerase chain reaction (PCR) methods were developed to specifically determine and detect the four genotypes within VCG0114. A specific PCR method to detect all VCG0114 isolates was also developed. These methods will facilitate the timely identification of infested fields and seed lots and the elucidation of evolutionary relationships among the isolates. This should help to closely monitor the movement of the pathogen and reduce dissemination of these devastating pathogens.


Assuntos
Fusarium , California , China , DNA Fúngico/genética , Fusarium/classificação , Fusarium/genética , Fusarium/isolamento & purificação , Fusarium/patogenicidade , Texas , Virulência
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