Study on the yield and active ingredient content of Gastrodia elata with different Armillaria mellea
https://doi-001.org/1025/17616282597635
Yutao Zhao，Fulai Luo*，Jinling Li，Hualei Wang，Ping Weng
1College of Agriculture, Guizhou University, Guiyang 550025, Guizhou, China
2 Key Laboratory of Medicinal Plant Breeding and Cultivation of Guizhou Province, Guiyang 550025, Guizhou, China
3Guizhou Jiulong Tianma Co., LTD., Bijie 551600, Guizhou, China
1Email：ypp183025539172022@163.com
2Email：luo512658@163.com
3Email：151904742@qq.com
4Email：whlgu@163.com
5Email：wenping8873@163.com
corresponding author：luo512658@163.com
Abstract: Gastrodia elata is a traditional rare medicinal material with important medicinal value and economic value. It is a completely heterotrophic plant in the family Orchidaceae, rootless and leafless. It relies on the symbiotic relationship with Armillaria during its whole growth cycle, and acquires essential nutrients for growth and development by decomposing and absorbing the mycelium of Armillaria. In the production practice, the biological characteristics of Armillaria millaria significantly affect the infection efficiency, substance accumulation and metabolic process of Gastrodia elata, which directly affect the yield and the content of main active components (such as gastrodin, p-hydroxybenzyl alcohol, etc.) of Gastrodia elata. Therefore, screening and cultivation of excellent Armillaria millaria strains has become the key link to improve the level of artificial cultivation and the quality of gastrodiae. In this review, the effects of different sources and species of Armillaria millaria on the yield and active ingredient accumulation of gastrodia elata were systematically reviewed, including the interaction mechanism between Armillaria millaria and gastrodia elata, strain screening and evaluation system, and the correlation between strain characteristics and the quality of gastrodia elata, in order to provide a theoretical basis for the development and application of excellent Armillaria millaria resources. And promote the sustainable development of gastrodia elata industry.
Key words: Armillaria; Gastrodia elata; Yield; Active ingredients
1. Introduction
Gastrodiae gastrodiae (Gastrodiae gastrodiae gastrodiae) is a kind of traditional rare medicinal material. Its dried tubing is used as a medicine, which has the effects of soothing wind and relieving spasm, soothing liver Yang, eliminating wind and dredging collaterals. Modern pharmacological studies have shown that its core active components gastrodiae gastrodiae and p-hydroxybenzyl alcohol have significant pharmacological activities on the central nervous system and cardiovascular and cerebrovascular system. With the increasing market demand, the resources of wild gastrodia elata are increasingly exhausted. Artificial cultivation has become the main way to ensure the supply of Gastrodia elata. However, the unique biological characteristics of Gastrodia elata, as a completely heterotrophic Orchidaceae plant that is rootless, leafless and unable to carry out photosynthesis, make its growth and development highly dependent on the symbiotic relationship with fungi, of which Armillaria is an indispensable source of nutrients. Gastrodia elata digests the mycelia of Armillaria invading its tubers to obtain carbon sources and nutrients for material accumulation and growth.
In this symbiotic system, the biological characteristics of Armillaria millardii, such as mycelial growth activity, infection efficiency, metabolite composition, and environmental adaptability, directly determine the efficiency and quality of nutrient supply to Gastrodiae gastrodiae, which profoundly affect the yield, appearance, and synthesis and accumulation of key medicinal components of gastrodiae. In actual production, the growth-promoting effects of Armillaria millaria strains from different sources and species are significantly different, and improper selection of Armillaria millaria strains may even cause cultivation failure. Therefore, screening and cultivation of Armillaria millaria strains with high affinity, strong growth promotion ability, and excellent agronomics traits is the core premise to achieve high quality, high yield, and stable yield of Gastrodiae.
At present, many scholars have carried out a lot of research on the classification and identification of Armillaria millaria, strain screening, symbiotic mechanism, and their effects on the growth of Gastrodia elata, and accumulated rich theoretical and practical results. However, there is still a lack of in-depth summary and comparative analysis of how different Armillaria strains systematically affect the yield and the formation of active components of Gastrodia elata. Based on this, this paper aims to systematically review the related research progress, focusing on the mechanism and existing evidence of different Armillaria millaria strains on the biomass accumulation and active components (such as gastroditis, polysaccharides, etc.) content of gastrodiae gastrodiae, discuss the problems in the current strain screening and application, and put forward the prospects for future research. The aim of this study is to provide theoretical reference for the scientific selection and efficient application of excellent Armillaria millardii strains in gastrodia elata cultivation, and to promote the sustainable development of gastrodia elata industry.
2. Research status of Armillaria mellea
2.1 Research progress on biological characteristics of Armillaria mellea
Armillaria is a plant pathogenic fungus in the subphylum Basidiomycota, which includes about 70 known species and is widely distributed in Europe, Asia, Africa, North America and other places [13]. Armillaria is mainly diploid during its vegetative growth stage, rather than maintaining a binucleate state like most basidiomycotes [33]. The spores of Armillaria are haploid, and the monuclear vegetative hyphae are diploid. The vegetative hyphae of Armillaria undergo diploidization. When two haploid monocytes mate, a binucleate cell is formed first, and the binucleate cell fuses to a monucleate cell. In hyphal cells without lock-like union, after the fusion of two haploid nuclei, two diploid nuclei will be formed, and the septum will be formed in the middle to form two monocytes. In the vegetative growth stage, the binuclear stage will not appear again [33]. According to the morphological characteristics of Armillaria at different growth and development stages, Armillaria can be divided into two parts: mycelium and fruiting body. Mycelium is the basic structure of nutrients in the growth of Armillaria, which absorbs nutrients from the external environment. Mycelium is composed of mycelium and cordage. The mycelium is a white slender linear body, and when the growth is dense, it appears white villous. When grown in pure culture on solid medium, the mycelium will grow into a milky white villous shape. The mycelium is like a fine root of a plant, the epidermis is reddish-brown or reddish-brown. The young mycelium is shiny and brown red, and the tip part is white. As the mycelium grows slowly, it becomes brown. In the process of pure culture, the cords change from white when they are young to tan when they are old. The young cords are flexible and brittle after aging. The epidermis of the cords is composed of compact and keratinized hyphae, which have strong adaptability and resistance to facilitate the growth of the cords in adverse environments. The mycelium has a high degree of differentiation and regeneration ability. The treated mycelium can grow mycelium under suitable conditions, and mycelium can grow into mycelium. The corycets can differentiate into fruiting bodies, infect hosts, transmit nutrients, grow between xylem and phloem, and attach to the trunk, tuber surface, and root of gastrodia elata.
The fungi of this genus can live in parasitism, saprophytism and vegetative symbiosis. Some species are tree pathogens [34], some can coexist with gastrodia, and some have important food and medicinal values [35]. Armillaria has a wide host range and can parasitize more than 500 gymnosperms and gametosperms worldwide to cause root rot, leading to plant tissue death before being colonized by mycelium, and seriously affecting agricultural and forestry production. Most Armillaria species are facultative parasites. After colonization and killing of the root cambium, they transform into saprophytic forms and decompose necrotic tissues of their hosts. As saprotrophic bacteria, Armillaria can effectively break down all components of plant cell wall, including lignin, (hemicellulose) and pectin [36]. However, Armillaria mellea, which is symbiotic with gastrodia, is a weak pathogenic species with fast growth rate, abundant rhizoid cords and uniaxial branches [17,37]. The pathogenicity and preferential saprogenesis of Armillaria may affect its symbiotic relationship with Gastrodia. Virulence testing of the five Armillaria species showed that Armillaria tabescens was the least virulent and Armillaria mellea was the most virulent, Armillaria tabescens was the most virulent, followed by Armillaria ostoyae, Armillaria gallica and Armillaria cepistipe[13, 38, 39]. Weakly pathogenic and preferential saprotrophic Armillaria millaria (e.g., A.gallica and A. epistipes) are easy to establish a symbiotic relationship with gastrodia [40]. However, the most virulent Armillaria mellea can also establish a symbiotic relationship with Gastrodia, possibly as a result of the variation in virulence within the species [13,41]. Different Armillaria mellea have different growth characteristics. Compared the growth characteristics of 4 local strains of Armillaria mellea isolated from Zhaotong, Yunnan and the exotic excellent Armillaria mellea Jing-234, the local SNA04 strain showed better growth characteristics [42]. We isolated four Armillaria millardii strains symbiotic with Radix aconitum radix from Zhaotong, and there was no significant difference in the utilization of beef extract, peptone, urea, yeast extract and ammonium chloride nitrogen sources among these Armillaria strains [43]. Although physiological studies showed that these four Armillaria strains had similar utilization of organic and inorganic nitrogen sources, the molecular mechanism of how they absorb and metabolize these two nitrogen sources remains to be studied.
2.2 Progress in genome research of Armillaria
At present, the genome of Armillaria has been analyzed and studied. However, the pathogenic genes of Armillaria have been studied more and more because they are serious plant pathogenic fungi. Researchers have analyzed the genomes of four Armillaria millaria, such as Armillaria audensis and Armillaria gallic [33], and found that Armillaria has a relatively large and expanded genome compared with the genomes of related saprotrophic fungi [33,44]. The expanded gene family in Armillaria includes pathogenic genes, lignocellulosic degradation-related genes and Armillar-specific genes with unknown functions [33,45]. However, the lack of knowledge of the specific recombination environment and the complete reference genome hinders the further understanding of the genetic basis of the genome evolution and phenotypic traits of Armillaria [46], as well as the study of the mechanism of nitrogen metabolism in Armillaria.
At present, there have been several reports of whole genome sequencing of Armillaria, but most of these reports were devoted to analyzing plant cell wall degrading enzymes and some secreted proteins related to the pathogenicity of Armillaria [33,47,48]. Moreover, from the reported Armillaria genomes, it is known that there are differences in genome size, gene number and structure even among closely related Armillaria species [33,49]. The introns and mobile genetic elements in the mitochondrial genome of Armillaria play an important role in the formation of its genome structure [49]. Mobile repeat elements (RE) play an important role in the replication and formation of nucleoprotein complexes and affect gene expression [50]. However, most RE are derived from transposable elements (tes) [50]. Fungal genomes are rich in TEs[51,52], and tes may play an important role in the adaptation of fungi to new niches [53]. In magnaporthe oryzae, TE was found to be associated with genes involved in host specialization [54]. However, there are few studies on the genome of Armillaria mellea in symbiosis with gastrodia. Zhan et al. [55] found that there were about 23.6% repetitive sequences in the genome of diploid A. galica cords isolated from gastrodia tuber, but most of the repetitive sequences have not been studied clearly [45], and there is still a lack of relevant research on the molecular mechanism of the symbiotic relationship between Armillaria and Gastrodia. Therefore, genomic data are essential to understand the complex characteristics of Armillaria species and the symbiotic relationship between Armillaria and Gastrodia.
2.3 Progress in genetic transformation of fungi and Armillaria
In 1973, the DNA transformation of Neurospora crassa by Mishara and Tatum was the earliest fungal genetic transformation experiment. Subsequently, the research on fungal genetic transformation was developed rapidly, and many methods such as PEG-CaCl2 transformation, electroshock mediated genetic transformation and agrobacterium-mediated genetic transformation were established on the basis of protoplasts [56]. Among these methods, agrobacterium-mediated genetic transformation has the advantages of high transformation efficiency and good heredity, and is widely used in the genetic transformation of fungi. Screening markers such as hygromycin and herbicides used in plants can also be used for screening of fungal transformants, and promoters that work in transgenic plants also work in most fungal transformants [57].
Studies reported that green fluorescent protein (GFP) was used as a reporter gene to evaluate the pGREENII dual expression vector and a series of promoters, and the GFP gene was successfully expressed in Aspergillus dispora [58]. Using the plant dual expression vector pCAMBIA0380, a series of vectors for transformation of Armillaria were constructed. GFP and mRFP were stably expressed in mycelium, and the corresponding fluorescence could be observed in the transgenic fruity body and in the invasive plant of Armillaria. The use of these vectors can successfully make the fluorescent proteins GFP and mRFP expressed in Armillaria, establishing an experimental basis for the molecular level research of Armillaria [57].
Agrobacteria-mediated genetic transformation of Armillaria millaris is to transfer the plasmid with the target gene into agrobacterium, and agrobacterium integrates the target gene into the genome of Armillaris under the induction of acetosyringone. PCR analysis and fluorescence observation are used to determine whether the transferred foreign gene has been stably integrated into the genome of Armillaris [57]. In the early stage of this study, an Armillaria millaria strain YN1 was isolated from Zhaotong Radix gastrodiae, which has a good affinity with Zhaotong Radix gastrodiae. It can effectively promote the growth of Zhaotong Radix gastrodiae and meet the quality requirements of Radix gastrodiae. Hygromycin and herbicides can inhibit the growth of this strain, which can be used for screening markers. Using hygromycin as a screening marker, mRFP gene was transferred into Armillaria millardii by Agrobacterium-mediated method. The 35S promoter could activate the expression of mRFP in Armillaria millardii, and the red fluorescence of mRFP protein was observed under fluorescence microscope, indicating that mRFP was successfully expressed in Armillaria millardii. The optimization of the transgenic method of Armillaria mellea provides a basis for the study of gene function in Armillaria.
2.4 Research progress of nitrogen metabolism in fungi and Armillaria
arbuscular mycorrhiza (AM) fungi, belonging to the phylum Glomeromycota, are one of the important soil microbial groups as plant symbiotic microorganisms [59,60]. The establishment of mycorrhiza relies on nutrient exchange between mycorrhiza fungi and host plants. Nitrogen (N) and phosphorus (P) are the basic mineral nutrients required for plant growth. However, in terrestrial ecosystems, N is often fixed in humus in the form of nitrate, ammonium, free amino acids, and nucleic acids. Therefore, low N availability in soil is one of the common abiotic stresses limiting plant growth. After forming mycorrhizae with plants, AM fungi can promote the absorption of phosphorus and nitrogen and other mineral elements in the soil by plants [61], and can also help plants absorb water and other trace mineral elements. The nutrient exchange between AM fungi and plants is the basis for maintaining the symbiotic relationship between them. Plants provide the carbon required by AM fungi [62,63], and AM fungi provide the mineral nutrients mainly including phosphorus and nitrogen to plants in exchange [64]. At present, researchers have studied inorganic nitrogen metabolism-related genes in ectomycorrhizal fungi and AM fungi, among which the study of nitrogen metabolism-related genes in AM fungi is more in-depth. AM fungi can absorb soil nitrogen, mainly helping plants to absorb inorganic nitrogen (NO3 – and NH4+). Extraradicular hyphae take up soil NH4+ through NH4+ transporters [65] or NO3 – through NO3 – transporters. Inside the mycelium, AM fungi reduce NO3 – to NH4+ by reductase. Then, NH4+ and plant-provided glutamate are synthesized into glutamine by glutamylamine synthetase [66]. Glutamine forms arginine through a series of biosynthesis [67], which is transported in hyphae. Finally, arginine is degraded to form NH4+ through the urea cycle, and NH4+ is released into the plant plasma membrane ectosome through the nitrogen transport ion pump, which is absorbed by plants [68]. There are transporters that can absorb inorganic nitrogen, amino acids, peptides and other nitrogen compounds in mycelium. At present, studies on the ammonium salt transporter genes of different mycorrhizal fungi have been reported [69,70], and these transporters include AMT1, AMT2, and AMT3 types, which all belong to the Mep/Amt family. Researchers have also isolated transporter genes TbNrt2[71] and GiNT[72] related to NO3 – transport from ectomycorrhizal fungi and AM fungi, and NO3 – can induce the expression of these genes. Amino acid transport system has been extensively and deeply studied in yeast [73] and filamentous fungi [74,75]. Cappellazzo et al. [76] recently isolated an amino acid transporter gene, GmosAAP1, from AM fungi. Other researchers have even isolated a transporter gene for transporting urea and peptides from ectomycorrhizal fungi [77,78], and these results provide some molecular basis for studying how mycorrhizal fungi use nitrogen nutrition.
Filamentous fungi are able to use many compounds as sole nitrogen sources, but as long as preferential nitrogen sources are present in the medium, filamentous fungi will preferentially utilize these energetically favorable nitrogen sources, such as NH4+ and glutamine. In the absence of these preferential nitrogen sources, less absorbable nitrogen sources such as nitrate, urea, uric acid, amines, amides, purines and pyrimidines can also be utilized [79,80]. The regulatory mechanism by which easily absorbed nitrogen sources can be preferentially utilized in the presence of preferential nitrogen sources, and these secondary nitrogen sources can be selectively utilized in the absence of preferential nitrogen sources, is called nitrogen metabolite inhibition. This regulatory mechanism ensures transcriptional activation of structural genes encoding enzymes that are required for uptake and degradation of less energetic nitrogen sources [79,81]. In ascomycete yeasts and filamentous fungi, nitrogen metabolite repression is mediated by transcription factors belonging to the GATA family. Ammonium and glutamine nitrogen sources can inhibit the expression of GATA transcription factors, and the transcription of most structural genes is regulated by GATA transcription factors. GATA transcription factors contain a DNA-binding domain with one or two cysteines and a Cys2/ Cys2-zinc finger structure. They recognize the 5 ‘-HGATAR-3’ sequence (where H stands for A, T, C, and R for A or G) in the promoter sequence of the target gene, and their activity is regulated by the level of nitrogen-generating substances in the cell.
The standard model for nitrogen metabolite dererepression is that the GATA transcription factor AreA/NIT2 and its orthologs mediate the activation of a number of genes involved in the utilization of secondary nitrogen sources in the absence of preferential nitrogen sources such as glutamine and ammonium salts in several other ascomycetes [82]. Under nitrogen starvation, AreA binds to GATA sites in the promoter region and mediates histone H3 acetylation by recruiting histone acetyltransferase to mediate chromatin remodeling [83,84]. However, it activates the transcription of catabolic genes only when substrates are available to it. For this substrate-specific gene activation, additional pathway-specific transcription factors are required to mediate the induction of a range of genes in response to specific inducers. In the nitrate assimilation system of Aspergillus nesteriformis, synergistic interactivity of AreA and pathway-specific transcription factor NirA is required to ensure the use of nitrate as the sole nitrogen source [84,85]. The activity of AreA itself is regulated by several signaling processes that report on the availability of extracellular nitrogen and the status of intracellular nitrogen [86]. Fungi can provide mineral nutrients to plants. Arbuscular mycorrhizae (AM) are symbiotic relationships between fungi (Balloonobacteria spp.) and plant roots [59,60,87]. The establishment of symbiotic relationships relies on nutrient exchange between mycorrhizal fungi and plants. Nitrogen and phosphorus are the basic mineral nutrients for plant growth, and certain fungal species can promote plant uptake of mineral elements in the soil, such as nitrogen and phosphorus [88,89], helping plants to absorb water and other trace mineral elements. The exchange of nutrients between AM fungi and plants is fundamental for maintaining their symbiotic relationship. Plants provide a carbon source for AM fungi to grow [62,63,90], and AM fungi provide mineral nutrients, mainly phosphorus and nitrogen, to plants in exchange [64,91].
2.5 Auxin promotes the growth of Armillaria
Studies have shown that trees such as pterosandalwood, oak and European beech can significantly promote the growth of Armillaria, while lilac is not conducive to the growth of Armillaria [92,93]. The addition of potato to the culture medium is beneficial to the growth of A.mellea [94]. Plant growth regulators, such as NAA[95], indole-3-acetic acid [91], 2, 4-D[96], tannins [97], tristanol and inositol [98], also have certain regulatory effects on the growth of some Armillaria species. Recent studies have shown that some plant hormones also play important roles in regulating arbuscular development in mycorrhizae. Abscolic acid, gibberellin and strigolactone have important functions in arbuscular maintenance and formation [99-102]. Both abscolic acid and strigolactone contribute to arbuscular mycorrhizal fungal infection of plant roots and promote arbuscular mycorrhizal formation, while gibberellin inhibits the formation of arbuscular mycorrhizal symbiosis and reduces the number of arbuscular. Although the mechanism of auxin in this plant-microbe interaction remains unclear, studies have shown that auxin also plays a role in arbuscular mycorrhizal symbiosis [103-105]. Auxin content varies in different mycorrhizal roots; in tobacco and leve mycorrhizae [106,107], its content is maintained at a certain level throughout the symbiotic process, while it increases in maize and soybean mycorrhizal roots [108-110]. In nark soybean mycorrhizae, a mutant with defects in nodule autoregulation, IAA content increased less, suggesting that IAA may be indispensable in the self-regulation of mycorrhizal cosymbiosis [109]. Auxin signaling within host plant roots is required for the early stages of arbuscular mycorrhizal symbiont formation, such as the recognition of symbiotic signals before arbuscular mycorrhizal fungi come into contact with the host [103,111]. Auxin sensing or auxin signaling is also critical for arbuscular development after mycorrhizal symbionts are formed [112,113]. A.mellea grows vigorously on medium supplemented with auxin and is capable of producing abundant corycetes [114,115]. In the medium, addition of a certain concentration of 2, 4-dichlorophenoxyacetic acid and NAA can promote the growth of A.mellea mycelium [95,96,115]. Although previous studies have shown that NAA can promote the growth of Armillaria, the mechanism of action has not been investigated at the molecular level. In the production of Gastrodia, the growth characteristics of Armillaria directly affect the yield and quality of Gastrodia. To study the mechanism of NAA promoting the growth of Armillaria at the molecular level, we will lay the foundation for the molecular breeding of Armillaria.
3. The symbiosis between Gastrodia and Armillaria millardii
3.1 Gastrodia elata cultivation and Armillaria mellea
Gastrodia elata Blume is a rare traditional Chinese medicinal material. It is a rootless and leafless perennial heterotropic orchid plant. It is widely used for the prevention and treatment of various diseases because of its high medicinal value [1], mainly produced in Yunnan, Sichuan, Guizhou, Hubei and other places. Gastrodia elata Blume was first recorded in Shennong Bencao Jing (Shennong’s Classic of Materia Medica). It has the effect of soothing the liver, soothing the wind and relieving pain, and is mainly used to treat headache, dizziness, convulsions and other diseases in children. Modern studies have shown that gastrodia. tianma also has the effects of anti-depression, anti-convulsion, delaying aging, improving memory and promoting brain development [2]. Gastrodin (4-hydroxymethylbenzene-β-D-glucopyranoside) and 4-hydroxybenzyl alcohol are the main active ingredients in gastrodiae gastrodiae, and the pharmacopoeia (2015 edition) stipulates that the content of gastrodiae gastrodiae used as medicinal materials cannot be less than 0.25%[3]. Wild Gastrodin has a high content of active components and good efficacy, but its yield is low. In addition, wild gastrodin is currently on the verge of extinction, so it needs artificial cultivation to meet the market demand.
Gastrodia elata is a highly degenerate, leafless and rootless orchid that can neither photosynthesize nor absorb nutrients from the soil through its roots. Its life cycle requires symbiosis with two types of fungi, namely germinata and Armillaria millaria. The seeds of Gastrodia elata are very small, and the nutrients such as polysaccharides and fats in the embryo are not enough to support seed germination. Therefore, Gastrodia elata must digest the germinating bacteria that invade its cells to obtain nutrients to promote seed germination to form protocorms.
During most of its life history, Gastrodia elata needs to be symbiotic with Armillaria millaria to digest and absorb the invading mycelia of Armillaria, so as to normally grow and develop into rice hemp and white hemp without flower buds and commercial hemp (commercial hemp) with flower buds [4]. Although Gastrodia elata has no root or leaf differentiation and is difficult to be cultivated artificially, after a lot of research on the cultivation technology of Gastrodia elata, the cultivation technology of asexual reproduction and sexual reproduction of Gastrodia elata have been formed [5]. After a large number of previous studies on cultivation techniques, the unified cultivation method of three lower nests has gradually formed in China. Since then, several cultivation methods have been developed, such as the live-growing material with culture method, the culture bed culture method, and the culture material with fuelwood method. The method has the advantages of high inoculation rate and stable yield, but the wood consumption is high and the utilization rate is low. In the fixed bed cultivation, Armillaria millaria is less damaged, which makes the gastrodia elata faster and more susceptible to bacteria, and is conducive to the acquisition of sufficient nutrients. The fuelwood embedding method can improve the germination rate of seeds and solve the degradation problem of Armillaria millaria planted with gastrodia [6]. These new cultivation methods can solve the problems of poor stress resistance, decreased quality and yield of Gastrodia elafillea caused by the spread of diseases and pests, the degradation of Armillaria millaria species and the degradation of Gastrodia elafillea species.
Although recycling or ecologically guaranteed planting techniques have been developed, they still cannot completely solve the ecological problems existing in the cultivation process of gastrodia. that is, they cannot solve the problem of resource waste caused by the low efficiency of using wood and the low utilization rate of wood by Armillaria [7]. Therefore, it is urgent to study the absorption mechanism of Armillaria millaria on nutrient elements, so as to cultivate high-quality Armillaria strains with efficient utilization of nutrient elements, so as to improve the utilization efficiency of bacteria materials and the yield and quality of gastrodia [8].
3.2 Effects of Armillaria millardii on the vegetative growth of Gastrodia elata
Gastrodia elata grows through five growth and development stages: seed, protobulb, rice hemp, white hemp and arrow hemp [9]. Kusano first reported the symbiotic relationship between Gastrodia elata and the pathogenic bacterium Armillaria millaria [10]. Gastrodia elata acquires nutrients required for growth and development through digestion and absorption of invading Armillaria, while nutrients of Armillaria come from wood [11,12]. Thus, a tree-Armillar-gastrodia nutrient transfer pathway is formed, and Gastrodia elata relies on this pathway to obtain nutrients and grow and reproduce continuously. However, when Armillaria cannot obtain sufficient nutrients from the rhizome or is under stress, it will use gastrodia as a nutrient and become the main host of Armillaria. In addition, the symbiotic relationship between Gastrodia and Armillaria is not equal. The survival of Gastrodia depends on Armillaria, but the survival of Armillaria does not depend on gastrodia, which is mainly reflected in the parasitics of gastrodia to Armillaria [13]. Excellent Armillaria millaria strains can provide enough nutrients for gastrodia, improve the yield of gastrodia, and the quality of gastrodia also depends on the growth of Armillaria [14,15]. Subsequent studies have shown that Gastrodia seeds need to digest germinating bacteria (such as Mycena) invading germ cells in the process of germination, so as to obtain enough nutrients to germinate and form protocorms [16]. After the formation of the procorm, Gastrodia will begin the first stage of asexual reproduction, and the germinating bacteria can no longer provide nutrients for the procorm. Thereafter, the vegetative reproduction stem of Gastrodia must form a symbiotic relationship with Armillaria, and complete its vegetative growth stage by digestion of the invading mycelia of Armillaria as a source of nutrients [17]. However, in the protocorms, few protocorms could establish symbiotic relationship with Armillaria millardii. Armillaria can invade into the cortical cells of Gastrodia through the form of cord-forming bacteria, thus establishing a symbiotic relationship to provide nutrients for Gastrodia [18]. Therefore, the ability of Armillaria to absorb nutrients and its growth status are extremely important for the growth of gastrodia.
At present, researchers have clarified the mechanism of seed germination of Gastrodia elata after inoculation by transcriptome analysis [19]. The symbiotic mechanism between Gastrodia elata and Armillaria mellea has been revealed [20,21]. It was found that Gastrodia elata has a relatively complete phenylpropanoid compound anabolic pathway [22]. Up to now, the physiological morphological characteristics of the symbiosis between Armillaria millaria and Gastrodia elata have been studied thoroughly, but the underlying mechanism of nutrient metabolism is still unclear.
3.3 Effects of different Armillaria millardii strains on the yield and active ingredient content of Gastrodia elata
The species and physiological characteristics of Armillaria mellea affect the yield and active ingredient content of cultivated gastrodia elata. There are significant differences in the contents of gastrodin and 4-hydroxybenzyl alcohol among different cultivars and different variation types [23] in different tissues of the same cultivar [24,25]. In the production process of gastrodia elata, with gastrodia elata on many generations after planted, even superior strains of armillaria also can be degraded and mutation, thus, gastrodia tuber yield and quality serious decline, cause serious economic losses [26]. A large number of studies have shown that even Armillaria mellea and Armillaria gallic, which are commonly used in production, have great differences in the yield and active ingredient content of gastrodia when different strains of Armillaria mellea are planted with gastrodia [14,27,28], and the gastrodin content will be greatly different when different Armillaria mellea strains are planted with gastrodia [15]. The growth status and growth rate of the strains are closely related to the yield, quality and agronomic traits of Gastrodia. The Armillaria mellea strains with fast growth rate, thick and more branches of the cords have better infection of the rhizoma sinensis. The gastrodia elata cultivated with this strain not only has the highest yield and quality, but also has the highest ethanol-soluble extract content and the total content of gastrodin and p-hydroxybenzoyl alcohol.
Armillaria millaria is the pathogen of root white rot [13,14], but a few Armillaria millaria can establish a symbiotic relationship with Gastrodia elata. Previous studies have shown that the growth of Armillaria is closely related to the activities of extracellular enzymes such as laccase, cellulase, xylanase, pectinase and amylase secreted by it. Extracellular enzymes play a decisive role in the ability of wood-decaying fungi to degrade nutrients, and their types and activities are related to the species of fungi [15]. The secretion of these extracellular enzymes provides the material basis for the infection of Armillaria mellea on the epidermis of Gastrodia. Studies have also found that different extracellular enzymes play different roles in the growth of Armillaria. Laccase can degrade lignin and phenols, and its activity affects the ability of Armillaria to degrade lignin [16,17]. Cellulase hydrolyzes cellulose to produce glucose, which provides a carbon source for the growth of the strain [15,18]. Xylanase can break down xylan, a polysaccharide structure located in the secondary wall of plants [19]. Amylase mainly hydrolyzes plant polysaccharide starch to provide nutrition. Pectinases degrade pectin in plant cell matrices and primary cell walls [16,19]. It is important to study the activities of Armillaria extracellular enzymes for the growth of Armillaria and Gastrodia elata.
In the production process of Gastrodia elata, it is necessary to not only increase the yield of gastrodia elata, but also increase the content of its active components. Lei Youcheng et al. [29] found that the content of gastrodin was different in different grades of gastrodin. Different Armillaria mellea have important effects on the yield, quality and agronomic traits of Gastrodia elata, and affect the grade formation of Gastrodia elata. The Armillaria millaria strains with strong cords and more branches and good growth can significantly improve the yield and quality of Gastrodia elata [17,30]. Studies have shown that the promotion of Armillaria millaria on gastrodin synthesis mainly comes from the nutrients of Armillaria cells, which are digested and degraded by gastrodia into small molecular substances that can be used as the substrates of gastrodin synthesis and nutrients of gastrodia [31]. Armillaria secretes extracellular enzymes to degrade lignocellulose into small molecular substances such as glucose, which are then used as nutrients for its own growth. Different Armillaria species have different ability to degrade lignocellulose and other substances. Therefore, understanding the nutrient metabolism mechanism of Armillaria is conducive to the cultivation of excellent Armillaria strains.
3.4 Research progress on the molecular level of the symbiotic relationship between Armillaria millaria and Gastrodia elata
Whole genome and transcriptome sequencing technology has become an important means to excavate important trait genes and study gene functions in medicinal plants. In the method of studying gene expression level, transcriptomics can systematically excavate important functional genes expressed in different time and space. In the absence of whole genome information, it can promote the research on the molecular mechanism of the synthesis of effective ingredients in medicinal plants [32]. By analyzing the genome of Gastrodia elata, Yuan Yuan et al. [20] revealed the mechanism of the symbiosis between Gastrodia elata and Armillaria millaria, and found that strigolide was an important signal for the establishment of the symbiotic relationship between Gastrodia elata and Armillaria millaria. The increase in the number of key genes for strigolide synthesis and transport in Gastrodia elata helped to promote the establishment of the symbiotic relationship between Gastrodia elata and Armillaria millaria. These findings revealed that totally heterotrophic plants can remodel their genomes to complete their unique life history. Wen Huan et al. [22] found a large number of functional genes related to the synthesis of gastrodia-phenylpropanoid products through comparative analysis of transcriptome sequencing. The previous study of our research group took Radix elata from Zhaotong, Yunnan and Armillaria mellea as materials, and carried out reference free transcriptome high-throughput sequencing analysis on samples of Armillaria, oryzae symbiont region of Armillaria and Paratractonia, which revealed that laccase, xylanase, cellulase, cell wall degrading enzyme and polygalacturonic enzyme of Armillaria were down-regulated. These results indicated that after the formation of symbiotic relationship between Armillaria and gastrodia, the life activities of Armillaria invading the cortex of gastrodia were weakened and the formation of gastrodia diseases was avoided. In addition, the expressions of peroxidase, catalase and superoxide dismutase genes were also down-regulated, indicating that GR did not exert biological stress on Armillaria during the symbiotic relationship. Although it was found that the life activity of C. millaria was reduced in G. mellea and its growth was not affected by GR, it is not clear whether GR promotes the nutrient metabolism of A. millaria in vitro and whether GR changes the nitrogen metabolism of A. millaria. Although studies have shown that one-horned lactone in rhizoma gastrodiae gold with armillaria symbiosis plays an important role in the signal, but whether to armillaria plays a role of regulation of nitrogen metabolism mechanism, it remains to be further research.
4.Current problems of strain screening and application and future research directions
4.1 Problems in current strain screening and application
Although remarkable progress has been made in the study of the symbiotic relationship between Armillaria millaria and gastrodia elata, there are still many challenges in the selection and application of excellent strains, which restricts the quality and efficiency improvement of gastrodia elata industry.
(1) The system of strain screening and evaluation is not perfect
At present, the selection of superior strains mainly depends on phenotypic indicators such as growth rate, cord morphology, and extracellular enzyme activity, as well as field performance such as yield and active ingredient content of Gastrodia after cultivation. These methods provide intuitive, but cycle is long, large workload, is significantly influenced by environmental factors, and difficult to from the mechanism level forecast strains with specific varieties of gastrodia elata, such as red gastrodia elata gastrodia elata, symbiotic affinity. The lack of efficient and accurate molecular marker-assisted screening systems directly related to the symbiotic efficiency is the key bottleneck that leads to more effort and less effort.
(2) The degradation of Armillaria was serious and its mechanism was unclear
In the production process, even the initially excellent strains often suffer from strain degradation after multiple generations of cultivation with Gastrodia elata, which is manifested as decreased growth activity of cords and weakened infection ability, and ultimately leads to a significant reduction in the yield and quality of Gastrodia elata. However, whether the underlying mechanism is due to the genetic variation of the strain itself, the nutrient depletion of the strain material, or the adaptive evolution during the long-term interaction with gastrodia. has not been systematically studied. However, there is still a lack of theoretical guidance for the rejuvenation and preservation of the strains, and the strains have to be frequently replaced or purchased during production, which increases the cost and uncertainty.
(3) The research on the interaction mechanism of strain-gastrodia-environment (G×E) is weak
The growth status of Armillaria millaria and its symbiotic efficiency with Gastrodia could be affected by different ecological zones, altitude, climate conditions and material types. Current studies mainly focus on the evaluation of the effects of a single strain or a single environment, and there is a lack of studies on the regional suitability of strains for different main production areas. There is no clear technical standard for which strain can exert the best growth-promoting effect under what environmental conditions, which leads to the blindness of the promotion and cultivation of excellent strains.
(4) The molecular mechanism of nutrient metabolism in the symbiotic system is not well understood
Although it is known that the nitrogen and carbon metabolism and various extracellular enzymes of Armillaria play a central role in the symbiosis, the precise regulatory network of these processes, the functions of key genes, and the signaling pathway of GR to nutrient metabolism of Armillaria remain poorly understood. In particular, the molecular mechanism of how Armillaria efficiently decompress lignocellulose in the material and deliver nutrients to G. mellea is a theoretical high ground for the future molecular breeding of strains.
4.2 Prospect of future research direction
In view of the above problems, combining molecular biology technologies (genome editing, synthetic biology) and ecological methods, future research can focus on the following directions to provide support for the sustainable development of gastrodiae gastrodiae industry:
(1) A multi-dimensional and cross-scale strain screening and evaluation system should be constructed
To break through the limitation of single index, a three-level index of “molecular characteristics-physiological characteristics-field performance” was established. At the molecular level, molecular markers related to high degradation efficiency (laccase gene *LAC*, cellulase gene *CEL*) and high symbiotic affinity (monotregolactone receptor gene) were screened. At the physiological level, the cord-growth rate, extracellular enzyme activity, and nitrogen use efficiency of the strains under low temperature and low nitrogen stress were evaluated. At the field level, combined with the ecological conditions of Yunnan, Guizhou, Hubei and other main production areas, long-term verification of more than three growth cycles was carried out to screen the excellent strains with “broad adaptability” or “regional specificity”.
(2) In-depth analysis of the molecular regulatory network of symbiosis
Multi-omics technologies (genome, transcriptome and metabolome) were used to explore the key pathways. First, the synergistic mechanism of nitrogen metabolism was analyzed, focusing on the regulation of GATA transcription factors (such as *AreA* orthologue) on nitrogen utilization of Armillaria, and how GR regulates nitrogen transporters expression in Armillaria through secondary metabolites. The second is to clarify the signal transduction pathway of symbiosis, to clarify how strigolactone triggered the down-regulation of cell wall degrading enzymes genes in Armillaria, and to reveal the molecular basis of “non-pathogenic symbiosis”. The third is to explore the mechanism of strain degradation, and to screen anti-degradation genes by comparing genomic variations (transposable element activity and mitochondrial intron changes) between the degenerated strain and the original strain, so as to provide targets for strain improvement.
(3) To carry out the research and development of molecular breeding and efficient application technology of excellent strains
Based on the previous genetic transformation system (Ford et al., 2016; pH2GW7-35S-mRFP vector), we directed the introduction of genes with high extracellular enzyme activity or anti-degradation genes to cultivate “efficient degradation-high symbiosis-anti-degradation” strain. At the same time, the hybrid matrix of “bacteria material-crop straw” should be developed to optimize the cyclic cultivation mode of “GR.
(4) Strengthen the integration of production, education and research and the promotion of industrialization
A collaborative platform of “scientific research institute-enterprise-farmers” was established, and the excellent strains were combined with standardized cultivation techniques (fixed bacterial bed method, bacterial material sandwich fuelwood method) to form regional technical regulations. The strain resource bank and information sharing platform were constructed to record the ecological adaptability and degradation cycle of strains, so as to provide accurate guidance for farmers and promote the transformation of gastrodiae cultivation from “empirical” to “scientific”.
5. Concluding remarks
As a traditional and precious medicinal material, Gastrodia elata is highly dependent on the symbiosis of Armillaria millaria due to its completely heterotrophic biological characteristics. In this paper, we systematically reviewed the biological characteristics of Armillaria millaria (function of mycelium and cord, mechanism of nitrogen metabolism), the process of gastrodia-Armillaria symbiosis (nutrient transfer from seed germination to armillaria formation), and the effects of different Armillaria strains on the yield and active ingredients of Gastrodiae. It was concluded that the growth activity, extracellular enzyme activity and pathogenicity of Armillaria millaria were the core factors regulating the quality and yield of Gastrodiae.
The existing researches have made some progress. On the one hand, the key role of strigolactone in the symbiotic signaling and the extracellular enzyme in the degradation of rhizoma has been clarified. On the other hand, some strains with good regional adaptability, such as SNA04 and YN1 from Zhaotong, Yunnan, were screened out, which provided practical reference for gastrodiae cultivation. However, we need to face up to the current shortcomings: the strain screening system still needs to be improved, the molecular mechanism of symbiosis needs to be deepened, and the problems of degradation and industrialization suitability have not been solved. These bottlenecks not only limit the improvement of cultivation efficiency, but also affect the sustainable development of the industry.
In the future, through the construction of multi-dimensional screening system, the analysis of symbiotic molecular network, the development of molecular breeding and recycling technology, it is expected to overcome the existing limitations. In theory, it will fill the gap in the research of the metabolic mechanism of gastrodia-Armillaria symbiosis and provide a paradigm for the study of complete heterotrophic medicinal plant symbiosis. In practice, the transformation of gastrodia elata cultivation from resource consumption to eco-friendly can not only ensure the quality and supply of traditional Chinese medicinal materials, but also realize the efficient use of forestry resources. In the future, with the help of the rapid development of multi-omics technology, molecular biology and synthetic biology, we will deeply reveal the internal mystery of the symbiosis between Armillaria millaria and gastrodiae, which will promote the leap from traditional experience to scientific theory. Breeding of high yield, high quality and stable yield of armillaria strains for special purpose, and establish and form a complete set of standardized cultivation technique system, will be the necessary way of gastrodia elata industry modernization and sustainable development.
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