Lack of correlation between micro fungi species and chemical control method of Atta treated with toxic baits

ABSTRACT: Atta sexdens rubropilosa (leaf-cutter ants) has a symbiotic association with a fungus and has a negative interaction with other fungi due to parasitism of the fungus cultivated by ants; also, there are several other fungi with no exact known role occurring in their cultivated fungus garden. In the present study, we use the ITS region (internal transcribed spacer) to identify fungi in colonies treated with toxic baits. Experiments using two toxic baits were carried out: 0.75g of sulfluramid [0.3%] and 0.75g fipronil [0.003%]. Samples of fungi were collected and cultured in Czapek medium for seven days to allow fungal growth and subsequent identification. Total DNA was isolated from 100-150 mg of mycelium using the CTAB method and using PCR, with the universal primers (ITS4 and ITS5), to amplify the ITS region. Sequencing was performed using the Sanger method. Sequences were subjected to BLAST, allowing the identification of nine different species of the orders Agaricales, Eurotiales, Hypocreales, Pleosporales, Saccharomycetales and Tremellales showing a variation in identity of 96-100%. Using “The Automatic Barcode Gap Discovery” analysis, nine groups were identified, corresponding to species described in NCBI. The K2P distances were used to generate a tree using Neighbour-joining, demonstrating that the species were grouped according to phylogenetic groups. We concluded that leaf-cutter ant colonies exhibited a wide variety of fungi and this study suggested that there is no correlation between the species of fungi isolated with the control method used on the ant nest.

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Higher-level relationships among the eucalypts are resolved by ITS-sequence data

Australian Systematic Botany ◽

10.1071/sb00039 ◽

2002 ◽

Vol 15 (1) ◽

pp. 49 ◽

Cited By ~ 82

Author(s):

Dorothy A. Steane ◽

Dean Nicolle ◽

Gay E. McKinnon ◽

René E. Vaillancourt ◽

Brad M. Potts

Keyword(s):

Internal Transcribed Spacer ◽

Sequence Data ◽

Its Region ◽

Molecular Data ◽

Nuclear Ribosomal Dna ◽

Its Sequence ◽

Phylogenetic Groups ◽

Single Section ◽

Western Australian ◽

Genus Eucalyptus

This expanded survey of ITS sequences represents the largest analysis of molecular data ever attempted on Eucalyptus. Sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA were included in an analysis of 90 species of Eucalyptus s.s. and 28 species representing eight other genera (Allosyncarpia, Angophora, Arillastrum, Corymbia, Eucalyptopsis, Stockwellia, Lophostemon and Metrosideros). The results of the study indicate that Angophora and Corymbia form a well-supported clade that is highly differentiated from Eucalyptus s.s. Corymbia species are divided between two clades, one of which may be the sister to Angophora. Allosyncarpia, Arillastrum, Eucalyptopsis and ‘Stockwellia’ are also highly differentiated from Eucalyptus s.s. If the genus Eucalyptus is to be expanded to include Angophora and Corymbia(sensu Brooker 2000), ITS data suggest that Allosyncarpia, Eucalyptopsis, ‘Stockwellia’ and potentially Arillastrum should also be included in Eucalyptus s.l. The ITS data suggest that subg. Symphyomyrtus is paraphyletic and that subg. Minutifructus should be included within it. Within subg.Symphyomyrtus, only sect. Maidenaria appears to be monophyletic. Sections Adnataria and Dumaria are probably monophyletic; sections Exsertaria and Latoangulatae are very close and probably should be combined in a single section. Section Bisectae is polyphyletic and is divided into two distinct lineages. The phylogenetic groups depicted by ITS data are consistent with the frequency of natural inter-specific hybridisations as well as data from controlled crosses within subgenus Symphyomyrtus. The ITS data illustrate that subg. Idiogenes and western Australian monocalypts are early evolutionary lines relative to E. diversifolia, E. rubiginosa (monotypic subg. Primitiva) and the eastern monocalypts and that subg. Primitiva should be sunk into subg. Eucalyptus. Subgenus Eudesmia may be monophyletic, grouping with subgenera Idiogenes and Eucalyptus. Further work is required to confirm the phylogenetic positions of the monotypic subgenera Alveolata, Cruciformes, Acerosae and Cuboidea.

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New Isolated Metschnikowia pulcherrima Strains from Apples for Postharvest Biocontrol of Penicillium expansum and Patulin Accumulation

Toxins ◽

10.3390/toxins13060397 ◽

2021 ◽

Vol 13 (6) ◽

pp. 397

Author(s):

Laura Settier-Ramírez ◽

Gracia López-Carballo ◽

Pilar Hernández-Muñoz ◽

Angélique Fontana ◽

Caroline Strub ◽

...

Keyword(s):

Liquid Medium ◽

Fungal Growth ◽

Its Region ◽

Antagonistic Activity ◽

Penicillium Expansum ◽

Metschnikowia Pulcherrima ◽

Potential Activity ◽

Different Strains ◽

Main Producer

Wild yeasts isolated from the surface of apples were screened for antagonistic activity against Penicillium expansum, the main producer of the mycotoxin patulin. Three antagonistic yeasts (Y33, Y29 and Y24) from a total of 90 were found to inhibit P. expansum growth. Identification by ITS region sequence and characterization showed that three selected isolates of yeast should be different strains of Metschnikowia pulcherrima. Several concentrations of the selected yeasts were used to study their in vitro antifungal effectivity against P. expansum on Petri dishes (plates with 63.6 cm2 surface) whereas their potential activity on patulin reduction was studied in liquid medium. Finally, the BCA that had the best in vitro antifungal capacity against P. and the best patulin degradation capacity was selected to be assessed directly on apples. All the selected strains demonstrated antifungal activity in vitro but the most efficient was the strain Y29. Isolated strains were able to reduce patulin content in liquid medium, Y29 being the only strain that completely reduced patulin levels within 120 h. The application of Y29 as biocontrol agent on the surface of apples inoculated with P. expansum, inhibited fungal growth and patulin production during storage. Therefore, the results shown that this yeast strain could be used for the reduction of P. expansum and its mycotoxin in apples or apple-based products by adapting the procedure application.

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Comparison of the ITS Sequences of 5 Common Potentilla Species in Jilin Province of China

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.554-556.1690 ◽

2012 ◽

Vol 554-556 ◽

pp. 1690-1693 ◽

Cited By ~ 2

Author(s):

Shao Xuan Zhang ◽

Xin Rui Liu ◽

Bo Chuan Wang ◽

Yun Hui Ling ◽

De Jun Sun ◽

...

Keyword(s):

Internal Transcribed Spacer ◽

Sequence Data ◽

Its Region ◽

Scientific Data ◽

Jilin Province ◽

Its Sequences ◽

Universal Primers ◽

Its Sequence ◽

Its Sequence Analysis ◽

Pcr Products

To find the differences in the internal transcribed spacer(ITS) sequences and provide scientific data for the authentication of Potentilla chinensis and its related species, we extracted the genome DNA from the leaves of 5 common Potetilla species in Jilin Province, amplified the ITS region using ITS universal primers of angiosperm, and sequenced the purified PCR products directly. Polymorphism of ITS sequences was found within P. chinensis and the sequence data suggested that our samples of this species might be related to hybridization. Other 4 species showed intraspecies-stability in ITS sequence. The ITS sequences of these 5 Potentilla species are significantly different. So ITS sequence analysis and other methods derived from it can be used in authentication of Potentilla.

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Genetic and Metabolic Intraspecific Biodiversity ofGanoderma lucidum

BioMed Research International ◽

10.1155/2015/726149 ◽

2015 ◽

Vol 2015 ◽

pp. 1-13 ◽

Cited By ~ 4

Author(s):

Anna Pawlik ◽

Grzegorz Janusz ◽

Iwona Dębska ◽

Marek Siwulski ◽

Magdalena Frąc ◽

...

Keyword(s):

Ganoderma Lucidum ◽

Mycelial Growth ◽

Carbon Sources ◽

Fungal Growth ◽

Its Region ◽

Mitochondrial Activity ◽

Genomic Relationship ◽

Profile Similarity ◽

Geographic Regions ◽

Its Region Sequencing

FourteenGanoderma lucidumstrains from different geographic regions were identified using ITS region sequencing. Based on the sequences obtained, the genomic relationship between the analyzed strains was determined. AllG. lucidumstrains were also genetically characterized using the AFLP technique.G. lucidumstrains included in the analysis displayed an AFLP profile similarity level in the range from 9.6 to 33.9%. Biolog FF MicroPlates were applied to obtain data on utilization of 95 carbon sources and mitochondrial activity. The analysis allowed comparison of functional diversity of the fungal strains. The substrate utilization profiles for the isolates tested revealed a broad variability within the analyzedG. lucidumspecies and proved to be a good profiling technology for studying the diversity in fungi. Significant differences have been demonstrated in substrate richness values. Interestingly, the analysis of growth and biomass production also differentiated the strains based on the growth rate on the agar and sawdust substrate. In general, the mycelial growth on the sawdust substrate was more balanced and the fastest fungal growth was observed for GRE3 and FCL192.

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Illumina-based identification of arbuscular mycorrhizal fungi from Karachay-Cherkessia soils for development of bio-fertilizers

E3S Web of Conferences ◽

10.1051/e3sconf/202128503001 ◽

2021 ◽

Vol 285 ◽

pp. 03001

Author(s):

Andrey Yurkov ◽

Alexey Kryukov ◽

Yulia Mikhaylova ◽

Peter Zhurbenko

Keyword(s):

Mycorrhizal Fungi ◽

Biological Diversity ◽

Arbuscular Mycorrhizal ◽

Its Region ◽

Am Fungi ◽

Illumina Miseq ◽

Universal Primers ◽

Its2 Region ◽

North Caucasus ◽

The Future

The aim of the study was to investigate the species diversity of AM fungi in different parts of the North Caucasus, biodiversity hotspot, the center of the world’s biological diversity. Samples were taken from 5 locations (stationary trial plots, STPs) in different ecosystems and at various altitudes. Identification was performed using sequencing for ITS1 and ITS2 regions, amplified with universal primers, Illumina MiSeq was employed. 19 genera of AM fungi were found on all STPs. The work did not reveal a correlation between the altitude and the species composition of AM fungi. At the same time, it should be assumed that a correlation could be found between the biodiversity of AM fungi and the type of ecosystem, which should be done in the future. The study shows it is necessary to use an analysis for both ITS regions, since the data obtained for each ITS region differ and complement each other. Analysis for the ITS2 region revealed 1.3 times more virtual taxa than for the ITS1, while the number of OTUs identified per species was similar for both regions. The highest biodiversity of AM fungi was found in STP #3 (with meadow flora). Only 4 species (Rhizophagus irregularis, R. intraradices, Paraglomus laccatum, and Claroideoglomus claroideum) were found on all five analyzed STPs. We found unexpectedly that with such a high biodiversity among the identified fungi, no different species were found in the Paraglomus genus, all the sequences of Paraglomus belonged to Paraglomus laccatum, whereas at least 9 species are distinguished in the genus by morphology. Further research will allow us to identify new strains of AM fungi, the efficiency of which may be higher than already studied ones. In the future this will make it possible to create more effective microbial biofertilizers for agriculture.

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First Report of Powdery Mildew Caused by Golovinomyces biocellatus on Peppermint in California

Plant Disease ◽

10.1094/pdis-94-2-0276c ◽

2010 ◽

Vol 94 (2) ◽

pp. 276-276 ◽

Cited By ~ 2

Author(s):

D. B. Marcum ◽

K. Perez ◽

R. M. Davis

Keyword(s):

Powdery Mildew ◽

Fungal Growth ◽

Universal Primers ◽

Mentha Piperita ◽

Exact Match ◽

First Report ◽

Plastic Bag ◽

The One ◽

Shasta County ◽

Disease Free

In August of 2009, powdery mildew was observed on peppermint (Mentha piperita L.) in several commercial fields in the Fall River Valley of eastern Shasta County, California. Plant growth was apparently reduced by the disease, but its impact on yield was unknown. White fungal growth was restricted to the adaxial surfaces, where colonies were thin and effused. Heavily infected leaves developed a reddish tint as growth prematurely ceased. Doliform conidia ([26.6-] 29.2 [-31.7] × [13.2-] 15.6 [-16.8] μm) were produced in chains of approximately six conidia. Foot cells were cylindrical ([41.3-] 55.2 [-75.0] × [11.2-] 12.0 [-12.8] μm). Immature chasmothecia were yellowish brown and approximately 100.0 μm in diameter with flexuous, mycelium-like appendages up to 200 μm long. All these features were consistent with those of Golovinomyces biocellatus. Asci were not observed. To confirm the identity of the fungus, nuclear rDNA internal transcribed spacer (ITS) regions were amplified by PCR with universal primers ITS4 and ITS5. The sequence (537 bp) was an exact match for several submissions of G. biocellatus in GenBank (e.g., Accession No. EU035602, a sequence of the fungus from mint in Australia [1]). Pathogenicity was confirmed by brushing spores from naturally infected leaves onto three rooted cuttings of M. piperita ‘Black Mitchum’. After the plants were covered with a plastic bag for 36 h to maintain high humidity, they were kept on a greenhouse bench at 23 to 28°C. Three noninoculated plants, which served as controls, were placed in another greenhouse in similar conditions. The experiment was repeated once. All inoculated plants developed signs of powdery mildew within 7 days of inoculation whereas noninoculated plants remained disease free. The fungus on inoculated leaves was morphologically indistinguishable from the one used to inoculate the plants. To our knowledge, this is the first report of G. biocellatus on peppermint in California. References: (1) J. R. Liberato and J. H. Cunnington. Australas, Plant Dis. Notes 2:38, 2007.

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Panfungal PCR and Multiplex Liquid Hybridization for Detection of Fungi in Tissue Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.38.11.4186-4192.2000 ◽

2000 ◽

Vol 38 (11) ◽

pp. 4186-4192 ◽

Cited By ~ 85

Author(s):

Panu H. Hendolin ◽

Lars Paulin ◽

Pirkko Koukila-Kähkölä ◽

Veli-Jukka Anttila ◽

Henrik Malmberg ◽

...

Keyword(s):

Fungal Pathogens ◽

Candida Parapsilosis ◽

Fungal Growth ◽

Its Region ◽

Deep Tissue ◽

Detection Level ◽

5.8S Rrna ◽

Panfungal Pcr ◽

Tissue Specimens ◽

Dna 2

A procedure based on panfungal PCR and multiplex liquid hybridization was developed for the detection of fungi in tissue specimens. The PCR amplified the fungal internal transcribed spacer (ITS) region (ITS1-5.8S rRNA-ITS2). After capture with specific probes, eight common fungal pathogens (Aspergillus flavus,Aspergillus fumigatus, Candida albicans,Candida krusei, Candida glabrata, Candida parapsilosis, Candida tropicalis, andCryptococcus neoformans) were identified according to the size of the amplification product on an automated sequencer. The nonhybridized products were identified by sequencing. The performance of the procedure was examined with 12 deep-tissue specimens and 8 polypous tissue biopsies from the paranasal sinuses. A detection level of 0.1 to 1 pg of purified DNA (2 to 20 CFU) was achieved. Of the 20 specimens, PCR was positive for 19 (95%), of which 10 (53%) were hybridization positive. In comparison, 12 (60%) of the specimens were positive by direct microscopy, but only 7 (35%) of the specimens showed fungal growth. Sequencing of the nonhybridized amplification products identified an infecting agent in six specimens, and three specimens yielded only sequences of unknown fungal origin. The procedure provides a rapid (within 2 days) detection of common fungal pathogens in tissue specimens, and it is highly versatile for the identification of other fungal pathogens.

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Quantitative Microplate-Based Growth Assay for Determination of Antifungal Susceptibility of Histoplasma capsulatum Yeasts

Journal of Clinical Microbiology ◽

10.1128/jcm.00795-15 ◽

2015 ◽

Vol 53 (10) ◽

pp. 3286-3295 ◽

Cited By ~ 22

Author(s):

Kristie D. Goughenour ◽

Joan-Miquel Balada-Llasat ◽

Chad A. Rappleye

Keyword(s):

Fungal Pathogens ◽

Histoplasma Capsulatum ◽

Dynamic Range ◽

Antifungal Susceptibility ◽

Fungal Growth ◽

Invasive Fungal Disease ◽

Phylogenetic Groups ◽

Morphological Form ◽

Content Type

Standardized methodologies for determining the antifungal susceptibility of fungal pathogens is central to the clinical management of invasive fungal disease. Yeast-form fungi can be tested using broth macrodilution and microdilution assays. Reference procedures exist forCandidaspecies andCryptococcusyeasts; however, no standardized methods have been developed for testing the antifungal susceptibility of yeast forms of the dimorphic systemic fungal pathogens. For the dimorphic fungal pathogenHistoplasma capsulatum, susceptibility to echinocandins differs for the yeast and the filamentous forms, which highlights the need to employHistoplasmayeasts, not hyphae, in antifungal susceptibility tests. To address this, we developed and optimized methodology for the 96-well microtiter plate-based measurement ofHistoplasmayeast growthin vitro. Using optical density, the assay is quantitative for fungal growth with a dynamic range greater than 30-fold. Concentration and assay reaction time parameters were also optimized for colorimetric (MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction) and fluorescent (resazurin reduction) indicators of fungal vitality. We employed this microtiter-based assay to determine the antifungal susceptibility patterns of multiple clinical isolates ofHistoplasmarepresenting different phylogenetic groups. This methodology fulfills a critical need for the ability to monitor the effectiveness of antifungals onHistoplasmayeasts, the morphological form present in mammalian hosts and, thus, the form most relevant to disease.

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First Report of Citrus Root Rot Caused by Phytophthora palmivora in Egypt

Plant Disease ◽

10.1094/pdis-02-13-0206-pdn ◽

2014 ◽

Vol 98 (1) ◽

pp. 155-155 ◽

Cited By ~ 1

Author(s):

Y. Ahmed ◽

A. M. D'Onghia ◽

A. Ippolito ◽

T. Yaseen

Keyword(s):

Mating Type ◽

Root Rot ◽

Its Region ◽

Selective Medium ◽

Phytophthora Palmivora ◽

Universal Primers ◽

Potential Threat ◽

First Report ◽

Citrus Root ◽

International Mycological Institute

During spring-summer 2009, a survey was conducted to determine the species of Phytophthora present in citrus nurseries in Egypt. A total of 300 samples of soil and fibrous roots were collected from the rhizosphere of symptomatic Volkameriana lemon (Citrus volkameriana Tan. & Pasq.) plants growing in Delta (Benha-Qalyubia) and a desert (Cairo/Alexandria desert road) citrus nurseries. Plants showed various symptoms. Canopies of affected plants showed few and yellowish leaves, a general stunted growth, no new vegetation, and sometimes sudden desiccation; the root system showed few dark brown feeder roots, no new yellow-white apexes, and a fibrous appearance of the rootlets due to disintegration of the cortical bark but not of the xylem. Collected rootlets and soil were plated in Petri dishes containing a selective medium for the oomycete Phytophthora (2) and incubated for 3 to 6 days at 19 ± 1°C as described by Ippolito et al. (1). Pure cultures were obtained by single-hypha transfers and the isolates were identified as Phytophthora palmivora (Butler) Butler on the basis of morphological and cultural characteristics (3). Isolates formed stoloniferous colonies on potato dextrose agar (PDA) and grew between 10 and 30°C, with the optimum at 25°C. On V8 juice agar, they showed a highly fluffy pattern and produced terminal and intercalary globose chlamydospores. Sporangia were papillate, elliptical (45 to 51 × 29 to 35 μm; length/breadth ratio of 1.3:1.8), and were caducous with short pedicel. All isolates were A2 mating type, forming spherical oogonia and amphigynous antheridia in dual cultures with reference P. palmivora isolate of A1 mating type. Identification of the isolates was further confirmed by amplification and sequencing of the internal transcribed spacer (ITS) region using the universal primers ITS4 and ITS6. BLASTn analysis of ITS sequences (GenBank Accession No. HE583183) showed 99% homology with P. palmivora isolates available in GenBank. Pathogenicity tests for P. palmivora were conducted by inoculating three groups of ten 6-month-old Volkameriana lemon plants, transplanted into 1.4 liter pots with growing medium artificially inoculated at the rate of 1% (v/v) of P. palmivora inoculum produced according to Yaseen (4). Ten uninoculated plants served as a control. Two months after the inoculation, plants were analyzed for canopy symptoms and the presence of pathogen in feeder roots. More than 50% of inoculated plants showed foliage symptoms and extensive decay of feeder roots. Colonies of Phytophthora were recovered from necrotic rootlets and identified as P. palmivora, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of P. palmivora as a pathogen to citrus plants in the Egyptian nurseries. P. palmivora should be considered a potential threat to the Egyptian citrus industry since it may negatively influence the nurseries and orchards production in the future. References: (1) A. Ippolito, V. De Cicco, and M. Salerno. Rivista di Patologia Vegetale 2:57, 1992. (2) H. Masago, M. Yoshikawa, M. Fukada, and N. Nakanishi. Phytopathology 67:425, 1977. (3) D. J. Stamps. Revised tabular key to the species of Phytophthora. CAB International Mycological Institute, Kew, Surrey, 1990. (4) T. Yaseen. Molecular diagnosis and biological control of Phytophthora-citrus root rot. PhD thesis. University of Bari, Italy, 2004.

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Diversity of Species and Susceptibility Phenotypes toward Commercially Available Fungicides of Cultivable Fungi Colonizing Bones of Ursus spelaeus on Display in Niedźwiedzia Cave (Kletno, Poland)

Diversity ◽

10.3390/d11120224 ◽

2019 ◽

Vol 11 (12) ◽

pp. 224 ◽

Cited By ~ 1

Author(s):

Mariusz Dyląg ◽

Artur Sawicki ◽

Rafał Ogórek

Keyword(s):

Antifungal Activity ◽

Fungal Growth ◽

Its Region ◽

Negative Influence ◽

Fungal Species ◽

Cave Environment ◽

Spot Tests ◽

Susceptibility Tests ◽

And Function ◽

Phenotypic Tests

Underground ecosystems are one of the most inhospitable places for microorganism development and function. Therefore, any organic matter located in these areas can stimulate fungal growth. The main purpose of this study was to find the best solution to effectively preserve (without relapses) paleolithic bones of cave bear (Ursus spelaeus) exhibited in cave without any negative influence on the cave environment. To achieve this aim, unambiguous identification of fungal species and its susceptibility tests toward fungicidal preparations were performed. Fungi were identified based on phenotypic tests and the internal transcribed spacer (ITS) region analysis. The antifungal activity of three preparations (Pufmax, Boramon and Devor Mousse) was evaluated by microdilution assay (protocol M38-A2) and spot tests assay. Phenotypic and molecular research showed that bones were colonized by 11 fungal species: Absidia glauca, Aspergillus fumigatus, Chrysosporium merdarium, Fusarium cerealis, Mortierella alpina, Mucor aligarensis, M. plumbeus, Penicillium chrysogenum, P. expansum, Sarocladium strictum and Scopulariopsis candida. All of the tested preparations were the most active against C. merdarium. In turn, M. plumbeus, M. aligarensis, M. alpina and A. glauca were the least susceptible. The highest antifungal activity was shown for Pufmax (minimum inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) values were in the range of 0.16–0.63% and 1.25–2.50%, respectively). The lowest fungicidal effect was observed for Boramon (MICs and MFCs in the range of 2.5–10% and 5–20%, respectively). Devor Mousse and Pufmax preparations showed fungicidal activity at the concentrations in the range of 1.25–5%. Susceptibility profiles were also confirmed based on spot tests assay. Our study allows for unambiguously identifying isolated fungi and assessing their susceptibility to commercially available fungicides, to prevent fungal outbreak.

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