Morphological and molecular identification and PCR amplification to determine the toxigenic potential of Fusarium spp. isolated from maize ears in southern Poland

Aflatoxigenic fungi are species of fungi that produce aflatoxins in food commodities. This study was aimed at screening different food samples in our local market for aflatoxigenic fungi using the aflatoxin regulatory gene (aflR gene). Six food samples (wheat, cowpea, rice, maize, melon and groundnut), were sourced from three different markets in Lagos metropolis (Mushin, Oyingbo and Mile 12). Fungi were isolated from these food samples and identified morphologically and microscopically. The genomic DNA was obtained using DNA isolation kits. The aflR gene was amplified from genomic DNA, nested, subjected to agarose gel electrophoresis and gel imaging. The Internal Transcribed Spacer (ITS) was also amplified from the genomic DNA for molecular identification of the organisms. The results showed that Aspergillus flavus were isolated from all the food samples from the three markets, while Aspergillus niger was present in rice, melon and wheat from Mile 12 market, maize and groundnut from Mushin market, rice and cowpea from Oyingbo market. A. flavus and A.niger were isolated from all the food samples when similar food samples from different market were mixed together. Only A. flavus amplicon from the nested polymerase chain reaction (PCR) showed approximately 400bp DNA fragment on the gel. This study has shown that PCR amplification of aflR gene has high specificity for detection of aflatoxigenic fungi in food samples thus, may be employed in screening food samples for contamination by aflatoxigenic fungi.

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PCR-based identification of Mycobacterium murphy causing Canine Leproid Granuloma Syndrome in Niterói, southeast Brazil - case report

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/1678-4162-10079 ◽

2018 ◽

Vol 70 (6) ◽

pp. 1699-1702

Author(s):

M.A.A. Pereira ◽

V. Nowosh ◽

P.N. Suffys ◽

G.B. Queiroz ◽

K.M.O. Silva ◽

...

Keyword(s):

Molecular Identification ◽

Rio De Janeiro ◽

Skin Diseases ◽

Pcr Amplification ◽

Infectious Agent ◽

Skin Lesions ◽

Molecular Techniques ◽

Hsp65 Gene ◽

Janeiro State

ABSTRACT Canine Leproid Granuloma Syndrome (CLGS), also known as canine leprosy, is a cutaneous nodular infectious disease caused by Mycobacterium sp.. Despite being reported worldwide, it is still quite unknown and underdiagnosed. Diagnosis may be achieved by cytopathology or histopathology of skin lesions, but identification of the infectious agent is complex, since bacterial in vitro growth is not possible, relying upon molecular techniques such as PCR to confirm Mycobacterium DNA in the sample. We report a CLGS case in Niteroi, Rio de Janeiro state, Brazil, diagnosed by cytopathology and submitted to molecular identification of the agent. PCR amplification of hsp65 gene was performed and revealed 100% genetic homology to M. murphy strain. This is the first CLGS report with molecular identification in Rio de Janeiro state, and this finding should raise awareness about CLGS as a differential diagnosis among granulomatous skin diseases in this region.

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Morphology, Pathogenicity and Molecular Identification of Fusarium spp. Associated with Anise Seeds in Serbia

Notulae Botanicae Horti Agrobotanici Cluj-Napoca ◽

10.15835/nbha44210488 ◽

2016 ◽

Vol 44 (2) ◽

pp. 411-417 ◽

Cited By ~ 1

Author(s):

Snežana PAVLOVIC ◽

Danijela RISTIC ◽

Ivan VUCUROVIC ◽

Miloš STEVANOVIC ◽

Saša STOJANOVIC ◽

...

Keyword(s):

Essential Oils ◽

Molecular Identification ◽

Fusarium Species ◽

Fusarium Spp ◽

First Report ◽

Pimpinella Anisum ◽

Health Inspection ◽

The Family ◽

The World

Anise (Pimpinella anisum L.) is an important medicinal spice plant that belongs to the family Apiaceae. Anise seeds are rich in essential oils and this is a reason why anise production in Serbia has increased over the last decade. During a routine health inspection on anise seeds collected from three localities in the province of Vojvodina (MoÅ¡orin, Veliki Radinci and OstojiÄ‡evo) during 2012 and 2013, it was found out that Fusarium spp. were a commonly observed fungi. The presence of Fusarium fungion the seed samples ranged from 3.75-13.75%. The aim of this study was to isolate and identify the strains of Fusarium species present on anise seed samples as it is necessary that commercially used anise seeds are completely free of Fusarium. Based on morphological, microscopic characteristics and a molecular identification by sequencing of TEF gene, the presence of the following species was confirmed on the anise seeds: F. tricinctum, F. proliferatum, F. equiseti, F. oxysporum, F. sporotrichoides, F. incarnatum and F. verticillioides. According to our knowledge and research, this is the first report of F. tricinctum and F. sporotrichoides as pathogens on anise seeds in the world. All seven isolates of Fusarium species are pathogenic to the anise seedlings, while the most virulent species were F. oxysporum, F. tricinctum and F. incarnatum.

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An improved method for the molecular identification of single dinoflagellate cysts

PeerJ ◽

10.7717/peerj.3224 ◽

2017 ◽

Vol 5 ◽

pp. e3224 ◽

Cited By ~ 4

Author(s):

Yangchun Gao ◽

Hongda Fang ◽

Yanhong Dong ◽

Haitao Li ◽

Chuanliang Pu ◽

...

Keyword(s):

Success Rate ◽

Ultrasonic Wave ◽

Molecular Identification ◽

Algal Blooms ◽

Single Cells ◽

Pcr Amplification ◽

Deep Understanding ◽

Dinoflagellate Cysts ◽

Accurate Identification ◽

Cleaning Method

BackgroundDinoflagellate cysts (i.e., dinocysts) are biologically and ecologically important as they can help dinoflagellate species survive harsh environments, facilitate their dispersal and serve as seeds for harmful algal blooms. In addition, dinocysts derived from some species can produce more toxins than vegetative forms, largely affecting species through their food webs and even human health. Consequently, accurate identification of dinocysts represents the first crucial step in many ecological studies. As dinocysts have limited or even no available taxonomic keys, molecular methods have become the first priority for dinocyst identification. However, molecular identification of dinocysts, particularly when using single cells, poses technical challenges. The most serious is the low success rate of PCR, especially for heterotrophic species.MethodsIn this study, we aim to improve the success rate of single dinocyst identification for the chosen dinocyst species (Gonyaulax spinifera,Polykrikos kofoidii,Lingulodinium polyedrum,Pyrophacus steinii, Protoperidinium leonisandProtoperidinium oblongum) distributed in the South China Sea. We worked on two major technical issues: cleaning possible PCR inhibitors attached on the cyst surface and designing new dinoflagellate-specific PCR primers to improve the success of PCR amplification.ResultsFor the cleaning of single dinocysts separated from marine sediments, we used ultrasonic wave-based cleaning and optimized cleaning parameters. Our results showed that the optimized ultrasonic wave-based cleaning method largely improved the identification success rate and accuracy of both molecular and morphological identifications. For the molecular identification with the newly designed dinoflagellate-specific primers (18S634F-18S634R), the success ratio was as high as 86.7% for single dinocysts across multiple taxa when using the optimized ultrasonic wave-based cleaning method, and much higher than that (16.7%) based on traditional micropipette-based cleaning.DiscussionThe technically simple but robust method improved on in this study is expected to serve as a powerful tool in deep understanding of population dynamics of dinocysts and the causes and consequences of potential negative effects caused by dinocysts.

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Morphological and Molecular Characterization of Trichoderma spp. from Rhizosphere Soil and Their Antagonistic Activity against Fusarium spp.

International Journal of Plant & Soil Science ◽

10.9734/ijpss/2021/v33i1930605 ◽

2021 ◽

pp. 100-112

Author(s):

Jaygendra Kumar ◽

Mukesh Kumar ◽

Akash Tomar ◽

. Vaishali ◽

Pushpendra Kumar ◽

...

Keyword(s):

Plant Pathogens ◽

Rhizosphere Soil ◽

Pcr Amplification ◽

Its Region ◽

Culture Method ◽

Dual Culture ◽

Fusarium Spp ◽

Trichoderma Spp ◽

Trichoderma Species

Trichoderma species are well known for their biocontrol activity which colonize many soil and tuber-borne and foliage plant pathogens. In this study, 12 native isolates of Trichiderma spp were collected from various crop rhizosphere soil samples and characterized them phenotypically based on morphological and cultural features and genotypically based on sequence analysis of internal transcribed spacer (ITS) region-PCR amplification. The results obtained from phenotypic and genotypic observation revealed that isolates were belonged to five different species namely T. asperellum, T. harzianum, T. longibrachiatum, T. koningii and T. koningiopsis. All Trichoderma isolates produced ~600 bp amplicon and phylogenetic analysis revealed that all isolates were grouped with respective species. Further, the antagonistic potential of all the isolates was evaluated against Fusarium spp. following in vitro dual culture method. The results showed that isolates of T. harzianum exhibited maximum growth inhibition activity. The highest rate of inhibition was recorded with T. harzianum isolate TBT6 (87.1%) followed by TBT7 (82.2%), while the least inhibition was observed in T. longibrachiatum isolate TBT10 (59.7%) after 7 days of incubation. The antagonistic T. harzianum isolate TBT6 can be used for development of Trichoderma based bio-formulation and served as bio-control agent against Fusaium spp. under field conditions.

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Improvement of long segment ribosomal PCR amplification for molecular identification of Litylenchus crenatae mccannii associated with beech leaf disease

Journal of Nematology ◽

10.21307/jofnem-2020-016 ◽

2020 ◽

Vol 52 ◽

pp. 1-15

Author(s):

L.K. Carta ◽

S. Li

Keyword(s):

Molecular Identification ◽

Pcr Amplification ◽

Leaf Disease

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Diversity of Nematode Microbial Antagonists from Algeria Shows Occurrence of Nematotoxic Trichoderma spp.

Plants ◽

10.3390/plants9080941 ◽

2020 ◽

Vol 9 (8) ◽

pp. 941

Author(s):

Nawal Benttoumi ◽

Mariantonietta Colagiero ◽

Samira Sellami ◽

Houda Boureghda ◽

Abdelaziz Keddad ◽

...

Keyword(s):

Pcr Amplification ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Fusarium Spp ◽

Trichoderma Spp ◽

Bacillus Spp ◽

Phytoparasitic Nematodes ◽

Meloidogyne Spp ◽

Rrna Gene Sequencing

Fungi and bacteria associated to phytoparasitic nematodes Globodera rostochiensis and Meloidogyne spp. in Algeria were identified and characterized. Trichoderma spp. showed the highest prevalence in the cysts of G. rostochiensis. A number of isolates were identified through PCR amplification and the sequencing of the internal transcribed spacer (ITS)1-2 and Rpb2 gene regions. The most represented species were T. harzianum and T. afroharzianum. The latter and T. hirsutum were reported for the first time in Algeria. Fusarium spp., including F. oxysporum and F. solani, comprised a second group of fungi found in cysts. Taxa associated to females of Meloidogyne spp. included T. harzianum, Fusarium spp. and other hyphomycetes. To assess the efficacy of Trichoderma spp., two assays were carried out in vitro with the culture filtrates of two T. afroharzianum and T. harzianum isolates, to check their toxicity versus the second stage juveniles of M. incognita. After 24–48 h exposure, a mortality significantly higher than the control was observed for both filtrates at 1% dilutions. The TRI genes involved in the production of trichothecenes were also amplified with the PCR from some Trichoderma spp. isolates and sequenced, supporting a putative role in nematode toxicity. Bacteria isolated from the cysts of G. rostochiensis included Brucella, Rhizobium, Stenotrophomonas and Bacillus spp., identified through 16S rRNA gene sequencing. The potential of the microbial isolates identified and their mechanisms of action are discussed, as part of a sustainable nematode management strategy.

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