Detection Of Arbuscular Mycorrhizal Fungi Research Articles

Identification and molecular detection of Arbuscular Mycorrhizal Fungi colonized naturally in citrus roots in nurseries

Abstract. Sariasih Y, Subandiyah S, Widyaningsih S, Khurshid T, Mo J. 2023. Identification and molecular detection of Arbuscular Mycorrhizal Fungi colonized naturally in citrus roots in nurseries. Biodiversitas 24: 6283-6290. Citrus plants heavily depend on arbuscular mycorrhizal (AMF), a diverse collection of fungi. The objective of this study was to investigate the root colonization, spore density, spore morphology, and molecular identification of AMF colonizing four citrus root species: Japansche Citroen (Citrus limonia Osbeck.), Cleopatra mandarin (Citrus reshni), Salam (Fortunella japonica cv. "Salam"), and Rough Lemon (Citrus jambhiri). AMF was identified by observing spore morphology and molecular PCR methods using the group-specific primers of Glomeromycota, AML1/AML2. Based on the morphological observation, C. limonia was found with the highest root colonization (50%), followed by F. japonica cv Salam (44%), C. reshni (38%), and C. jambhiri (36%). The highest number of AMF spores was found on C. jambhiri rhizosphere soil, which had 1,379 spores/100 g of soil with the lowest percentage of root colonization. Three mycorrhizal genera were identified, with Funneliformis (syn. Glomus) being the most dominant genus, followed by Gigaspora and Acaulospora. The sequence of PCR products confirmed the highest similarity range from 83.7-99.0% to that of Funneliformis, followed by Acaulospora at a similarity range from 83.3-99.0% and found on all of the citrus species. The similarity sequence of the PCR products to that of Gigaspora ranged between 83.3-95.0%, which was only found on C. reshni and C. jambhiri.

Molecular dialogue between arbuscular mycorrhizal fungi and the nonhost plant Arabidopsis thaliana switches from initial detection to antagonism.

Approximately 29% of all vascular plant species are unable to establish an arbuscular mycorrhizal (AM) symbiosis. Despite this, AM fungi (Rhizophagus spp.) are enriched in the root microbiome of the nonhost Arabidopsis thaliana, and Arabidopsis roots become colonized when AM networks nurtured by host plants are available. Here, we investigated the nonhost-AM fungus interaction by analyzing transcriptional changes in Rhizophagus, Arabidopsis and the host plant Medicago truncatula while growing in the same mycorrhizal network. In early interaction stages, Rhizophagus activated the Arabidopsis strigolactone biosynthesis genes CCD7 and CCD8, suggesting that detection of AM fungi is not completely impaired. However, in colonized Arabidopsis roots, fungal nutrient transporter genes GintPT, GintAMT2, GintMST2 and GintMST4, essential for AM symbiosis, were not activated. RNA-seq transcriptome analysis pointed to activation of costly defenses in colonized Arabidopsis roots. Moreover, Rhizophagus colonization caused a 50% reduction in shoot biomass, but also led to enhanced systemic immunity against Botrytis cinerea. This suggests that early signaling between AM fungi and Arabidopsis is not completely impaired and that incompatibility appears at later interaction stages. Moreover, Rhizophagus-mediated defenses coincide with reduced Arabidopsis growth, but also with systemic disease resistance, highlighting the multifunctional role of AM fungi in host and nonhost interactions.

Detection of arbuscular mycorrhizal fungi associated with pecan (Caryaillinoinensis) trees by molecular and morphological approaches.

Arbuscular mycorrhizal (AM) fungal community associated with pecan (Caryaillinoinensis) roots and rhizospheric soils was assessed by spore isolation and morphological characterisation and by pyrosequencing of AM molecular markers. The AM fungal community associated with pecan growing in the field, was always more diverse than that associated with pecan growing in containers. This was not observed when AM richness was studied, suggesting that soil disturbance by a reduction in host plant richness leads to a less equitable distribution of AM fungal species, in contrast to natural soils. The chosen primers (AMV4.5F/AMDGR) for pyrosequencing showed high AM fungal specificity. Based on 97% sequence similarity, 49 operational taxonomic units (MOTUs) were obtained and, amongst these, 41 MOTUs corresponded to the Glomeromycotaphylum. The number of obtained AM sequences ranged from 2164, associated with field samples, to 5572 obtained from pecan trap pot culture samples, defining 30 and 29 MOTUs, respectively. Richness estimated by conventional species identification was 6 and 9 AM fungal species in soil and pot samples, respectively. Claroideoglomuslamellosum, Funneliformismosseae and Entrophosporainfrequens were the only taxa detected using both techniques. Predominant sequences in the pecan rhizosphere samples, such as Rhizoglomusirregulare and other less abundant (Dominikiairanica, Dominikiaindica, Sclerocystissinuosa, Paraglomuslaccatum), were detected only by pyrosequencing. Detection of AM fungal species based on spore morphology, in combination with molecular approaches, provides a more comprehensive estimate of fungal community composition.

<b>Assessment of DNA extraction methods for detection of arbuscular mycorrhizal fungi in plant roots by nested-PCR

DNA extraction methods were evaluated for the yield and purity of DNA recovered from mycorrhized roots and whether the recovered DNA is suitable for amplification of arbuscular mycorrhizal (AM) fungal SSU rDNA. The DNeasy Plant Mini Kit and three extraction buffers were used alone or in combination with either polyvinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP) and/or activated charcoal (AC). Among the extraction methods tested, those based on the CTAB buffers yielded more DNA than those based on the TE buffer and the DNeasy Plant Mini Kit. Moreover, the use of AC alone or in combination with PVPP reduced DNA yield, while it significantly improved the purity of recovered DNA, whatever the extraction buffer. On the other hand, the success of nested-PCR amplification was negatively correlated with the amount of template DNA and positively correlated with the purity of recovered DNA. Three methods based on the TE buffer, two on the CTAB-βM buffer and one on the DNeasy Plant Mini Kit produced high-quality DNA in terms of purity and PCR performance. However, the TE buffer-based methods are less time consuming than the CTAB-βM buffer-based methods, and cheaper than the method based on the DNeasy Plant Mini Kit.

Root-Associated Fungi of Rosa rugosa Grown on the Frontal Dunes of the Baltic Sea Coast in Lithuania

The aim of the present study was to assess fungal communities associated with fine living roots of Rosa rugosa Thunb grown on the frontal dunes of Curonian Spit at the Baltic Sea coast in Lithuania. The roots of R. rugosa were sampled at five sites (Nida, Preila, Pervalka, Juodkrante and Smiltyne) situated at a distance ca. 5-15 km from each other. Direct amplification, cloning and sequencing of fungal ITS rRNA from the fine roots resulted in 134 high-quality sequences, representing 31 fungal taxa among which saprotrophs and endophytes Mycena sp. (14.2 %), Tumularia sp. (14.2 %), Penicillium spinulosum (11.9 %) and Cadophora malorum (9.0 %) were most common. Arbuscular mycorrhizal fungi including Entrophospora baltica (0.7 %) and Rhizophagus irregularis (0.7 %) and potentially root pathogenic fungi--Ceratobasidium sp. (4.5 %), Fusarium oxysporum (3.0 %), Fusarium culmorum (0.7 %) and Ilyonectria crassa (0.7 %)--were also detected at low proportions. In conclusion, the results demonstrated that the fine roots of R. rugosa are inhabited by various groups of fungi. Although saprotrophs and endophytes were dominant, the detection of arbuscular mycorrhizal fungi indicated that these may be important for mineral nutrition of R. rugosa established on dry and poor fertility coastal dunes.

Detection of Arbuscular Mycorrhizal Fungi in the Roots of Strawberry Plants Fertilized with Organic Bioproducts

Summary In organic farming, mineral fertilizers are replaced by various preparations to stimulate plant growth and development. Introduction of new biopreparations into horticultural production requires an assessment of their effects on the growth and yielding of plants. Among the important indicators of the impact on plants of beneficial microorganisms contained in bioproducts is determination of their effectiveness in stimulating the growth and yielding of plants. Moreover, confirmation of the presence of arbuscular mycorrhizal (AM) fungi in the roots and plant growth promoting rhizobacteria (PGPR) in the rhizosphere is also necessary. In addition to conventional methods, molecular biology techniques are increasingly used to allow detection and identification of AM fungi in plant roots. The aim of this study was identification and initial taxonomic classification of AM fungi in the roots of ‘Elkat’ strawberry plants fertilized with various biopreparations using the technique of nested PCR. Tests were performed on DNA obtained from the roots of ‘Elkat’ strawberry plants: not fertilized, treated with 10 different biopreparations, or fertilized with NPK. Amplification of the large subunit of ribosomal gene (LSU rDNA) was carried out using universal primers, and then, in the nested PCR reaction, primers specific for the fungi of the genera Glomus, Acaulospora, and Scutellospora were used. Colonization of strawberry roots by arbuscular mycorrhizal fungi was determined on the basis of the presence of DNA fragments of a size corresponding to the types of the fungi tested for. As a result of the analyses, the most reaction products characterizing AM fungi were found in the roots of plants treated with the preparation Florovit Eko. The least fragments characteristic of AM fungi were detected in the roots of plants fertilized with NPK, which confirms the negative impact of mineral fertilizers on the occurrence of mycorrhizal fungi in the roots of strawberry plants. The roots of plants fertilized with Tytanit differed from the control plants by the presence of one of the clusters of fungi of the genus Glomus and by the absence of a cluster of fungi of the genus Scutellospora. In the roots of plants treated with other biopreparations there were reaction products indicating the presence of fungi of the genera Glomus, Scutellospora and Acaulospora, like in the roots of the control plants. The results will be used to assess the suitability of microbiologically enriched biopreparations in horticultural production.

Detection of arbuscular mycorrhizal fungi within colonised roots of the Gramineae family members by nested-polymerase chain reaction (PCR)

In this study, it is aimed to asses the association of arbuscular mycorrhizal (AM) fungi within colonised rhizosphere of Gramineae family members through a survey by using nested- polymerase chain reaction method in Van province (Turkey). From 24 agro-ecological fields, a total of 82 samples belonging to Gramineae family were tested by molecular methods. The presence of Glomus intraradices and Glomus mosseae was ascertained in 10 plants belonging to eight different species by using fungus specific primers. Root colonisation ranged from 6 to 37% within rhizosphere of Gramineae family members and the average root colonisation by AM fungi was 22%.

Uvitex2B: a rapid and efficient stain for detection of arbuscular mycorrhizal fungi within plant roots

The study of arbuscular mycorrhiza often requires the staining of fungal structures using specific dyes. Fluorescent dyes such as acid fuchsin and wheat germ agglutinin conjugates give excellent results, but these compounds are either hazardous or very expensive. Here, we show that a safer and inexpensive dye, Uvitex2B, can be efficiently used to stain intraradical fungal structures formed by the arbuscular mycorrhizal fungus Glomus intraradices in three plant species: carrot, Casuarina equisetifolia, and Medicago truncatula. The intensity and stability of Uvitex2B allow the acquisition of high-quality images using not only confocal laser scanning microscopy but also epifluorescence microscopy coupled with image deconvolution. Furthermore, we demonstrate that Uvitex2B and β-glucuronidase staining are compatible and can thus be used to reveal arbuscular mycorrhizal structures in the context of promoter activation analysis.

Detection of Arbuscular Mycorrhizal Fungi in the Roots of Strawberry with Nested PCR Technique

Detection of Arbuscular Mycorrhizal Fungi in the Roots of Strawberry with Nested PCR Technique Organic farming of strawberry fruit crops is being increasingly developed in Poland. There is no possibility of using mineral fertilizers and chemical plant protection products in such a system of cultivation. The Rhizosphere Laboratory of the Research Institute of Horticulture in the Skierniewice has been working on the development and practical implementation of bioproducts enriched with beneficial soil microorganisms that have a positive effect on the growth and development of strawberry plants. An analysis of the impact of bioproducts on plants requires an assessment of the colonization of the plant roots by beneficial, arbuscular mycorrhizal fungi (AMF). Because of the difficulty in detecting and identifying AM fungi, the mycelium of which cannot be maintained outside the host plant, use is made of molecular biology techniques based on DNA analyses of these fungi. The aim of this study was to use a nested PCR technique to establish the presence of arbuscular mycorrhizal fungi in the roots of strawberry plants treated with bioproducts. The tests were performed on the DNA obtained from the roots of strawberry plants of the cultivars Elsanta and Honeoye treated with bioproducts of natural origin. In terms of fertilization, the control combinations were made up of plants that were not fertilized (control 0) and plants fertilized with NPK (control NPK). In the molecular tests, the control was the DNA obtained from the leaves of strawberry plants of the two cultivars. Colonization of strawberry roots by mycorrhizal fungi was determined on the basis of the presence of PCR products of a specific size, which were characteristic of the genera of the tested fungi. Primers amplifying the region of the large subunit ribosomal gene (LSU rDNA), which were specific to the genera Glomus, Acaulospora and Scutellospora, were used. As a result of the tests, DNA fragments were obtained that confirmed the presence of mycorrhizal fungi in all the samples of roots with the exception of those that came from the control combination of cultivar Honeoye fertilized with NPK. The roots of the plants were found to contain DNA fragments characteristic of three clusters of the genus Glomus. The presence of fungi Acaulospora was detected in all the samples of cultivar Honeoye roots except the roots of the control plants fertilized with NPK. Reaction products characteristic of the Acaulospora fungi in the roots of cultivar Elsanta were observed in the samples taken from the plants treated with bioproducts, with the exception of the control plants and plants fertilized with manure. Moreover, the test samples of both cultivars were found to contain fungi belonging to two clusters of the genus Scutellospora. The tests made it possible to detect the presence of arbuscular mycorrhizal fungi in the roots of strawberry plants. In addition, the analyses have shown the usefulness of most of the primers used for detecting the presence of mycorrhizal fungi of the genera Glomus, Acaulospora and Scutellospora. The results will be used to assess the effects of bioproducts on the growth and yield of strawberry plants of the cultivars Elsanta and Honeoye, and on the colonization of the roots of these plants by AM fungi.

Taxon-specific PCR primers to detect two inconspicuous arbuscular mycorrhizal fungi from temperate agricultural grassland

Taxon-specific polymerase chain reaction (PCR) primers enable detection of arbuscular mycorrhizal fungi (AMF, Glomeromycota) in plant roots where the fungi lack discriminative morphological and biochemical characters. We designed and validated pairs of new PCR primers targeted to the flanking regions of the variable domain 1 of the nuclear ribosomal large subunit RNA gene to specifically detect Acaulospora paulinae and an undescribed member of the Diversisporaceae. These two fungal taxa, sporulating late in soil-trap cultures and showing small, faintly coloured spores and weakly staining intraradical structures, were frequently found in roots of Trifolium repens from a high-input agricultural grassland. The newly developed PCR primers may thus enable studies on two inconspicuous AMF taxa that appear to have been overlooked in previous molecular AMF community analyses and for which no specific PCR primers have been published.

Detection of arbuscular mycorrhizal fungi (Glomales) in roots by nested PCR and SSCP (Single Stranded Conformation Polymorphism)

PCR amplification of a region of the large subunit ribosomal DNA sequence with Glomus specific primers was used to detect arbuscular mycorrhizal fungi in root tissue of four plant species. The primers were specific to Glomus mosseae, Glomus caledonium, Glomus geosporum, Glomus coronatum, Glomus fragilistratum and Glomus constrictum, and did not recognise sequences from Glomus claroideum. Sequence differences between isolates were detected by Single Stranded Conformation Polymorphisms (SSCPs) in polyacrylamide gels under non-denaturing conditions. Isolates of G. mosseae, G. caledonium and G. coronatum could be separated by their SSCP patterns, while three isolates of G. geosporumshowed no variation. Specific SSCP patterns from isolates of G. mosseae and G. caledonium allowed detection of both fungi in the same root segment. Sequence differences leading to variations in SSCP patterns were confirmed by direct sequencing.