Quantification of Arbuscular Mycorrhizal Fungi Colonization Rate in the Study of Invasive Alien Plants

Published: December 01, 2023

doi:

¹State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection,Chinese Academy of Agricultural Sciences

Summary

This protocol provides a simple and easy-to-use approach for determining the colonization rate of Arbuscular mycorrhizal fungi (AMF) in the roots of invasive plants.

Abstract

Arbuscular mycorrhizal fungi (AMF) are widely distributed soil fungi in ecosystems and can form symbiotic associations (mycorrhizae) with the roots of most terrestrial plants. Plants provide carbon sources to AMF through mycorrhizal associations, while AMF hyphae can expand the range of nutrient absorption by roots and promote plant nutrient uptake. There are many different species of AMF, and the symbiotic relationships between different species of AMF and different plants vary. Invasive plants can enrich AMF species with better symbiotic capabilities through root exudates, promoting their growth and thereby increasing their colonization in invasive plant roots. At the same time, invasive plants can also disrupt the symbiotic relationship between AMF and native plants, affecting the local plant community, which is one of the mechanisms for successful plant invasion. The colonization rate of AMF in the roots of invasive and native plants indirectly reflects the role of AMF in the process of invasive plant invasion. In this method, collected plant roots can be processed directly or saved in a fixative for later batch processing. Through decolorization, acidification, staining, and destaining treatment of roots, the hyphae, spores, and arbuscular structures of AMF in the root system can be clearly observed. This method can be completed in a basic laboratory to observe and calculate the colonization rate of AMF in the root systems of invasive plants.

Introduction

Mycorrhizal fungi are prevalent in natural ecosystems and establish symbiotic relationships with the roots of most plants, forming mycorrhizae¹. These associations are mutually beneficial, as plants provide photosynthetically fixed carbon compounds such as fatty acids and sugars to support the growth of mycorrhizal fungi, while the fungi reciprocate by supplying mineral nutrients like phosphorus and nitrogen to the host plants, thereby promoting plant growth². Based on their mycorrhizal types formed with plant roots, mycorrhizal fungi can be divided into four main types: ectomycorrhizal (ECM) fungi, ericoid mycorrhizal (ERM) fungi, orchid mycorrhizal (ORM) fungi, and arbuscular mycorrhizal (AM) fungi¹. Among them, arbuscular mycorrhizal fungi (AMF) have the widest distribution and can form mycorrhizal associations with over 80% of plant species³^,⁴.

AMF play a crucial role in enhancing soil nutrient cycling⁵, improving plant nutrient uptake⁶, and regulating plant competition and succession. They play an important role in the process of invasive plant species invasion⁷^,⁸. AMF, classified under the phylum Mucoromycota⁹, encompasses more than 250 species¹⁰. The specific symbiotic relationships between different species of AMF and different plants may vary. Invasive plant species have the potential to alter AMF diversity and promote the enrichment of AMF species with better symbiotic capabilities that contribute to their competitive advantage during growth and colonization⁸^,¹¹^,¹²^,¹³. Understanding the dynamics of AMF and their interactions with invasive plant species is essential for comprehending the mechanisms underlying plant invasions and their ecological impacts.

Qualitative studies of AMF species typically involve two main methods. One is morphological identification, such as collecting AMF spores from soil using methods like wet sieving-sucrose centrifugation, followed by classifying and quantifying the spores based on their morphology¹⁴. The other method involves molecular techniques, amplifying conserved regions of AMF genes and sequencing them for identification¹⁵. However, these methods often require extensive morphological identification experience or higher financial resources. On the other hand, quantitative studies of AMF colonization rates, while unable to determine changes in AMF species and composition, still provide a comprehensive assessment of the symbiotic relationship between AMF and plants. Such studies are indispensable in both basic research and subsequent validation work for inoculation experiments.

The colonization of AMF plays a crucial role in determining the distribution of resources among coexisting plant species⁷. It reflects the establishment and strength of the symbiotic relationship between AMF and host plant roots. In the same habitat, invasive plant species often exhibit higher colonization rates compared to native plants¹⁶^,¹⁷. This enhanced colonization of AMF contributes to the successful invasion of invasive species such as Ambrosia artemisiifolia¹⁸, Solidago canadensis¹⁹, Sapium sebiferum²⁰, Ageratina adenophora²¹, Sphagneticola trilobata²², and Flaveria bidentis⁷. Understanding the colonization rate of AMF in the roots of invasive plants provides a foundation for unraveling the soil microbial mechanisms underlying the successful invasion of these species. Investigating the colonization rate of AMF in invasive plant roots sheds light on the ecological implications of plant-microbe interactions and contributes to our understanding of the mechanisms driving plant invasions.

The determination of AMF colonization rate involves a staining microscopy technique, which includes several steps: root preservation, clarification, acidification, staining, destaining, and microscopic examination (Supplementary Figure 1). Over the past decades, researchers have explored various observation methods for AMF and have developed various staining techniques. In the early stages, Trypan blue staining was widely used²³^,²⁴. However, this method has limitations due to the toxicity of Trypan blue. Acid fuchsin staining, on the other hand, is a commonly used method that provides bright colors, and shows reliable, and stable staining results²⁵. Additionally, the staining solution can be reused, making it more cost-effective. The colonization rate is determined using the gridline intersect method, which provides more objective statistical results compared to other approaches²⁶. This method is characterized by its simplicity, low cost, and minimal equipment requirements, making it feasible to be performed in basic laboratory settings. It offers a practical and accessible approach to assess the colonization rate of AMF and contributes to our understanding of the symbiotic associations between AMF and plant roots.

Protocol

We conducted experiments using one invasive plant F. bidentis and one native plant Setaria viridis. Both plants were grown in experimental plots at the Langfang Scientific Research Pilot Base of the Chinese Academy of Agricultural Sciences (CAAS), Hebei, China. Each plant species was individually planted in separate plots, with each plot measuring 2 m x 3 m and a 1-meter gap between plots. The plants were left to grow naturally, and after approximately two months, root samples were collected. <p cla…

Representative Results

The staining results of the invasive plant roots using this method are shown in Figure 1. The structures (hyphae, arbuscules, spores, and vesicles) of AMF are stained red, the root cortex cells are stained light red after destaining, and the central cylinder is stained red. This staining result is sufficient to distinguish the fungal structures as AMF mainly exists in the plant's cortex. From the staining result, clear structures such as arbuscules (A), intraradical hyphae (IH), extrarad…

Discussion

The interactions between invasive plants and AMF are complex and diverse. Studying these interactions is crucial for understanding the success of invasive plants and their ecological effects. They can influence the invasive ability of plants, the structure and function of soil ecosystems, and the competitiveness of native plants. The colonization rate serves as an important indicator for studying the relationship between invasive plants and AMF. It provides a quantitative measure for establishing symbiotic relationships,…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

This work was funded by the National Key R&D Program of China (2021YFD1400100, 2021YFC2600400 and 2022YFC2601100), and by the National Science Foundation of China (42207162).

Materials

70% Alcohol	Shanghai Aladdin Biochemical Technology Co., Ltd	R433197
Acetic acid solution	Shanghai Aladdin Biochemical Technology Co., Ltd	A116166
Acid fuchsin	Shanghai Aladdin Biochemical Technology Co., Ltd	A104917
Formaldehyde solution, Formalin	Shanghai Aladdin Biochemical Technology Co., Ltd	F111941
Glycerol	Shanghai Aladdin Biochemical Technology Co., Ltd	G116203
Hydrochloric acid, HCl	Shanghai Aladdin Biochemical Technology Co., Ltd	H399657
Lactic acid	Shanghai Aladdin Biochemical Technology Co., Ltd	L432769
Manual System Microscope BX43	Olympus (China) co., Ltd
Potassium hydroxide, KOH	Shanghai Aladdin Biochemical Technology Co., Ltd	P112284

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