New technique for observing mycorrhizal fungi
T. Ishii a and K. Amanai

National Institute of Technology, Tokuyama College, Yamaguchi, Japan.

Abstract
Mycorrhizal fungi, particularly arbuscular mycorrhizal fungi (AMF) that infectalmost all of the plants, play a very important role for the maintenance of natural
ecology and the construction of safe, secure and sustainable (3S) crop production. The
standard procedure to observe root colonization, however, is cumbersome, and various
light microscopical techniques are required to clearly distinguish between AMF androot tissues. Recently, the fluorescein-labeled specific antibody against 24 kDa protein
related to mycorrhizal symbiosis was developed. The new antibody was successfully
used to detect various kinds of mycorrhizal fungi in fluorescence microscopic
observation. In this study, we report on a new technique for observing mycorrhizal
fungi by using a newly developed portable fluorescence microscope with the detection
reagent.
Keywords: mycorrhizal fungi, mycorrhizal fungus-detection reagent, newly developed
portable fluorescence microscope

INTRODUCTION
Mycorrhizal fungi form a symbiotic relationships with plants and have been known tocontribute to environmental conservation and 3S food production (Ishii, 2014). Therefore, theobservation of mycorrhizal infection is important for the evaluation of plant productivity.
Generally, ectomycorrhizal infection is easily observed by the naked eye, but the observationof other mycorrhizal fungi, such as AMF, ericoid mycorrhizal fungi (ERMF) and orchid
mycorrhizal fungi (OMF), is cumbersome. Conventional methods introduced by Phillips andHayman (1970) require preparations before using light microscopical observation.
Additionally, it is difficult to discriminate between the mycorrhizal fungi and root tissues.
Recently, Matsubara and Ishii developed a new reagent to detect a specific 24 kDaprotein related to mycorrhizal symbiosis (patent pending). The 24 kDa protein has beenreported to exist in various mycorrhizal fungi, including AMF (Ishii et al., 1999; Matsubaraand Ishii, 2014), OMF (Matsubara et al., 2012; Matsubara and Ishii, 2014), ECMF (Matsubaraand Ishii, 2014) and ERMF (Matsubara and Ishii, unpublished). For isolation and masspurification of the specific 24 kDa protein, we developed new preparative chromatographicaltechniques (Matsubara and Ishii, 2015).
In this study, we developed not only a portable fluorescence microscope that is possible
to use even in the field for the observation of mycorrhizal fungi, but also a new technique forobserving mycorrhizal fungi using the new portable fluorescence microscope with thedetection reagent. Additionally, since only Rhizoctonia solani, which is the congeneric fungus
of OMF, in typical plant pathogenic fungi reacted by the detection reagent, we investigatedwhether it is possible to distinguish between R. solani and AMF, using this technique.

MATERIALS AND METHODS
Since a specific 24 kDa protein related to mycorrhizal symbiosis exists in AMF, ECMF,
ERMF and OMF, the fluorescein-labeled antibody against the 24 kDa protein is possible todetect every mycorrhizal fungus (Figure 1). Therefore, rootlets of various plants were cut in
1-2 cm from the apex. Then, 10 μL of the detection reagent was dropped onto each root, andthen mycorrhizal root colonization was observed at wavelength of 472 nm using the newly
developed portable fluorescence microscope (Joint development with Soma Optics, Ltd. andaE-mail: aguaish@hotmail.com

Acta Hortic. 1267. ISHS 2020. DOI 10.17660/ActaHortic.2020.1267.44
Proc. Int. Symp. on Survey of Uses of Plant Genetic Resources to the Benefit of Local Populations
Eds.: R. Ramananarivo et al.

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Molecular Devices Corporation Japan) (Figures 1 and 2).

Figure 1. A newly developed portable fluorescence microscope and mycorrhizal fungusdetection reagent.

Figure 2. Mycorrhizal root colonization was easily observed even in the field and had been
photographed using camera-equipped mobile phones.

RESULTS AND DISCUSSION
Using the portable fluorescence microscope and the mycorrhizal fungus-detection
reagent, mycorrhizal root colonization by AMF and ERMF was clearly observed (Figures 3-8).
Although R. solani shows the fluorescence using the detection reagent, the distinction of AMF
and R. solani is very easy because no septa in AMF hyphae were observed (Figure 3).
Interestingly, it was clearly demonstrated that even the roots of Brassica rapa and Cyperus
microiria that have been known to be non-mycorrhizal plants (Wang and Qiu, 2006) can form
AMF symbiosis (Figure 7). Under environmental stress conditions or after herbicide
application, however, AMF symbiosis in these plants is frequently observed (Ishii, 2014).

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Figure 3. AMF symbiosis in chrysanthemum roots. Right (enlarged view): there are no septa
in AMF hyphae (see arrows). Therefore, the distinction of AMF and R. solani wasvery easy.

BAFigure 4. AMF symbiosis in several kinds of plant roots. A: corn, B: soybean, C: cucumber, D:lettuce.

CD

Figure 5. AMF symbiosis in several kinds of plant roots. A: carrot, B: garlic, C: peppermint, D:
Pelargonium sp.
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Figure 6. AMF symbiosis in several kinds of plant roots. A: Leucaena leucocephala, B:
Duchesnea chrysantha, C: trifoliate orange.

Figure 7. AMF symbiosis in several kinds of plant roots. A: Brassica rapa var. perviridis, B:
Cyperus rotundus.

Figure 8. ERMF symbiosis in blueberry roots. Above (enlarged view): hyphal complexes
occupying the cell.
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In blueberry roots, a typical feature of ERMF such as hyphal complexes occupying thecell or coiled hyphae in the cell was clearly detected (Figure 8).
Thus, our new technique for observing mycorrhizal fungi is very simple; the only
operation required before observation is the addition of 10 μL of the detection reagent.
Moreover, mycorrhizal root colonization can be easily observed even in the field and wasphotographed using camera-equipped mobile phones. Therefore, the technique willcontribute to the promotion of nature conservation, and 3S crop production or organic
farming which does not hold good condition without the presence of mycorrhizal fungi.
ACKNOWLEDGEMENTS
This work was supported by JSPS KAKENHI (Grant-in-Aid for Challenging Exploratory
Research) Grant Number JP15K14642.
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