An improved method for bright-field imaging of arbuscular mycorrhizal fungi in plant roots

Kobae, Yoshihiro; Ohtomo, Ryo

doi:10.1080/00380768.2015.1106923

Cited by 32 publications

(34 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The roots were stored at −80 • C to estimate colonization by AMF and DNA extraction. The fresh roots were stained with a 3,3 -diaminobenzidine (DAB) solution [41]. The colonization by AMF in the tomato plants was estimated according to the method of Giovannetti and Mosse [42].…”

Section: Root Sampling and Stainingmentioning

confidence: 99%

Impact of Phosphorus Fertilization on Tomato Growth and Arbuscular Mycorrhizal Fungal Communities

et al. 2020

View full text Add to dashboard Cite

Understanding the impact of phosphorus (P) addition on arbuscular mycorrhizal fungi (AMF) is crucial to understanding tomato (Solanum lycopersicum L.) P nutrition. However, it remains unknown how P fertilization is associated with the structure of AMF communities on tomato plants. Thus, we investigated whether levels of P fertilizer interacted with the colonization and structure of AMF in tomato roots in a field trial. In this study, we established three different amounts of P fertilizer treatments (0 kg ha −1 , 50 kg ha −1 , and 100 kg ha −1 ). We investigated AMF root colonization and community structure, as well as plant growth in tomatoes at seven weeks following transplantation. The structure of the AMF communities in the roots of tomato were determined by MiSeq amplicon sequencing. As expected, P fertilizer input enhanced the P uptake and plant biomass. In contrast, the P fertilizer level did not affect the AMF root colonization and diversity or the structure of the AMF communities in the tomato. However, we found a negative correlation between AMF colonization and richness in the roots of the tomato plants. Therefore, we need to investigate whether and how AMF communities and P fertilization develop more effective P management for tomato plants.

show abstract

Section: Root Sampling and Stainingmentioning

confidence: 99%

Impact of Phosphorus Fertilization on Tomato Growth and Arbuscular Mycorrhizal Fungal Communities

et al. 2020

View full text Add to dashboard Cite

show abstract

“…We previously established a WGA-HRP-DAB staining method for the bright-field imaging of AM fungal mycelia in roots of diverse plant species (Kobae and Ohtomo 2016). This staining method is high contrast and low background, and allows the recognition of symbiotic structures of AM fungi under low microscopic magnification.…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, in general, these methods require the preparation of specimens and observation under high magnification (McGonigle et al 1990). We previously reported that the application of WGA-conjugated horseradish peroxidase (HRP) with 3,3ʹ-diaminobenzidine (DAB) (WGA-HRP-DAB staining) successfully excludes the background signals of cortex and steles (Kobae and Ohtomo 2016). This method enables the identification of AM fungal structures in roots with extremely high contrast and simple bright-field imaging that can be performed with bright-field stereomicroscopy at only 15× magnification (Kobae and Ohtomo 2016).…”

Section: Introductionmentioning

confidence: 99%

“…We previously reported that the application of WGA-conjugated horseradish peroxidase (HRP) with 3,3ʹ-diaminobenzidine (DAB) (WGA-HRP-DAB staining) successfully excludes the background signals of cortex and steles (Kobae and Ohtomo 2016). This method enables the identification of AM fungal structures in roots with extremely high contrast and simple bright-field imaging that can be performed with bright-field stereomicroscopy at only 15× magnification (Kobae and Ohtomo 2016). However, rhizosphere materials with brownish color often adhere to roots removed from some soils and are difficult to distinguish from DAB staining under low magnification.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Chemiluminescence-based quantification of the colonization rates of Lotus japonicus roots by arbuscular mycorrhizal fungi

Kobae

Ohtomo

Morimoto

et al. 2017

Soil Science and Plant Nutrition

Self Cite

View full text Add to dashboard Cite

Simple evaluation of the levels of root colonization by arbuscular mycorrhizal (AM) fungi is challenging for conventional histochemical staining because most roots show an endogenous background signal. Herein, we report a chemiluminescence-based novel method for the quantification of the relative colonization rates of AM fungi. Lotus japonicus seedlings were grown in two types of soils with different inoculum potential [i.e., the former vegetation was maize (host) or buckwheat (non-host)]. Indigenous AM fungi colonizing roots were labelled with wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP), which specifically targets the N-acetylglucosamine polymers of fungal cell walls. After the roots were removed from the soil and rinsed with water, all procedures were conducted in 24-well plates. AM fungi were detected by chemiluminescence in the presence of enhanced chemiluminescence (ECL) reagents, and the signal strengths were measured by image analyses. Post-staining of AM fungal structures in the presence of 3,3ʹ-diaminobenzidine (DAB) and H 2 O 2 (DAB staining) revealed that even small colonization events (e.g., hyphopodia) could be detected, whereas the background signals observed in DAB staining were eliminated in the chemiluminescence method. Chemiluminescence thus provides both high sensitivity and high specificity. The chemiluminescence-based colonization levels of roots grown in maize soil were significantly higher than those of roots grown in buckwheat soil, demonstrating the feasibility for high-throughput quantification of the relative colonization rates of L. japonicus seedlings by AM fungi.

show abstract

“…Recently, Kobae and Ohtomo (2016) established a WGA (wheat germ agglutinin)-HRP (horseradish peroxidase)-DAB (3,3ʹ-diaminobenzidine) staining method for the bright-field imaging of AM fungal mycelia within roots of soybean and other plant species. In this method, WGA-conjugated HRP, which specifically targets N-acetylglucosamine polymers and detects AM fungal cell walls, penetrated the cortical layers of 10% potassium hydroxide-treated host roots and stained AM fungal mycelia in the presence of DAB and hydrogen peroxide.…”

Section: Mycorrhizal Intensity (%)mentioning

confidence: 99%

Estimation of the root colonization of soybean by an arbuscular mycorrhizal fungus, Gigaspora rosea, based on specific fatty acid profiles

Sakamoto

Kaji

2017

Soil Science and Plant Nutrition

View full text Add to dashboard Cite

Root colonization by arbuscular mycorrhizal (AM) fungi has traditionally been analyzed by microscopy. However, this method is time consuming and it is often difficult to distinguish between AM and non-AM fungi. In this study, we analyzed the fatty acid profiles in soybean roots colonized by AM fungi to determine if specific fatty acids derived from AM fungi can be used as markers for the intensity of the AM fungal colonization. The wild-type Enrei and hypernodulating Kanto100 soybean cultivars were inoculated with an AM fungus (Gigaspora rosea) alone or with Bradyrhizobium diazoefficiens, which nodulates soybean roots. Fatty acids 20:1ω9, 20:4ω6, and 20:5ω3 were specifically detected in the lateral roots of AM fungus-inoculated and dual-inoculated soybean plants. In the second lateral roots, the percentage of AM-specific fatty acids (i.e., 20:1ω9, 20:4ω6, and 20:5ω3) derived from AM fungi was closely correlated with the intensity of the AM fungal colonization. We propose that the AM-specific fatty acids represent useful markers for estimating the degree of AM fungal colonization. The percentage of AM-specific fatty acids was more than twofold higher in the second lateral roots than in the first lateral roots. Thus, the degree of AM fungal colonization is probably twofold higher in the second lateral roots than in the first lateral roots. ARTICLE HISTORY

show abstract

An improved method for bright-field imaging of arbuscular mycorrhizal fungi in plant roots

Cited by 32 publications

References 9 publications

Impact of Phosphorus Fertilization on Tomato Growth and Arbuscular Mycorrhizal Fungal Communities

Impact of Phosphorus Fertilization on Tomato Growth and Arbuscular Mycorrhizal Fungal Communities

Chemiluminescence-based quantification of the colonization rates of Lotus japonicus roots by arbuscular mycorrhizal fungi

Estimation of the root colonization of soybean by an arbuscular mycorrhizal fungus, Gigaspora rosea, based on specific fatty acid profiles

Contact Info

Product

Resources

About