Cultivable Endophyte Resources in Medicinal Plants and Effects on Hosts

1. Introduction

Medicinal plants refer to plants used in medicine to prevent and treat diseases [1]. All or part of its plants are used for medicine and will also be used as raw materials for the pharmaceutical industry, which has a wide range of medicinal and economic values [2]. Especially in the field of traditional medicine represented by traditional Chinese medicine and Indian folk medicine, medicinal plants, as the main source of natural drugs, provide very important health care services for the population of developing countries[3,4,5]. With the rapid development of modern medicine, many clinical drugs still come from natural products extracted from medicinal plants [2]. Although many kinds of medicinal plants have been used in clinical treatment, due to environmental stress, overexploitation, low reproductive capacity, and other factors, some rare, high-demand, and wild medicinal plant resources can not meet the market demand, so how to improve the germplasm resources of medicinal plants has become an urgent problem to be solved.

However, in recent years, researchers have gradually realized that endophytes can play an important role in affecting the quality and yield of medicinal plants through special microbe-plant interactions [6,7]. Plant endophytes are microbial groups that widely exist in healthy medicinal plant tissues and coexist harmoniously with host plants, and will not cause significant damage to hosts [8]. They are also an important part of plant micro ecosystem. It is rich in species, mainly including endophytic fungi, endophytic bacteria and endophytic actinomycetes [7,9,10]. At present, endophytes have been isolated from a variety of medicinal plants, and many endophytes have been verified to secrete plant hormones, growth factors, etc., which are conducive to plant growth and development, and can also regulate the accumulation and production of active ingredients in medicinal plants [10,11]. They increase the active ingredients of the host by producing the same or similar active products as the active ingredients in the host[11,12,13]. The most interesting thing is that these endophytes can convert the original active ingredients of the plant into new compounds. In 1993, Stierle et al isolated an endophytic fungus from Taxus brevifolia and found that it can produce paclitaxel, an anti-tumor substance similar to the host plant, which inspired researchers to find bioactive substances from endophytes of medicinal plants [14]. Endophytes provide a treasure house of new bioactive metabolites, especially alkaloids, saponins, quinones, flavonoids, terpenoids, etc., which have a lot of biological activities and have also become research hotspots in the composition and production of natural drugs [8].

In order to improve the quality of medicinal plants, we need to know more about the special relationship between endophytes and medicinal plants. In this paper, the cultivable endophyte sources and functions of Endophytes in medicinal plants were reviewed by searching the research in the past five years (2019-2023), and the development and utilization of endophytes in medicinal plants were prospected, to provide references for the development of endophyte products and improving the quality of medicinal plants.

2. Medicinal plants and their cultivable endophyte resources

2.1. Culturable endophytic bacteria diversity in medicinal plants

Atractylodes macrocephala, called Baizhu in Chinese, is a medicinal plant used in traditional Chinese medicine theoretical systems to treat gastrointestinal dysfunction, cancer, osteoporosis, obesity and other symptoms, and has various pharmacological activities. [15] Wu et al. [16] explored the cultivable endophytic bacteria in the stems, leaves, roots, and rhizomes of Atractylodes macrocephala in four different regions and their potential correlation with plant bioactives. A total of 118 endophytic bacteria belonging to 3 phyla, 5 classes, 11 orders, 26 families and 48 genera were identified from 4 Atractylodes macrocephala tissues. Among them, Bacillus sp. is the most widely distributed. Dendrobium is one of the largest genera of Orchidaceae, with more than 1500 species distributed all over the world [17]. As a medicinal plant, Dendrobium has contributed greatly to the medical industry by its anticancer, antifatigue, and gastrointestinal protective effects [18]. In addition, there are also many microbial resources in Dendrobium. Wang et al. [19] isolated and cultured endophytic bacteria from Dendrobium officinale samples of six different sources and cultivars. A total of 165 cultivable endophytic bacteria were isolated from sterilized Dendrobium officinale stems and classified into 43 species based on 16S rRNA gene sequence analysis. Mulberry, which belongs to the genus Morus of the Moraceae family, is an aggregated berry that is oval-shaped, palatable, and also rich in nutrients, it is regarded as a very important medicinal and edible plant due to its rich, effective chemical composition and wide range of biological activities[20,21,22]. Xu et al. [23] isolated a total of 608 endophytic bacteria from four mulberry cultivars, belonging to 4 phyla and 36 genera.

Bacteria, as the largest group of plant endophytes, have been isolated from many kinds of medicinal plants and widely studied due to their biocontrol functions[24,25,26,27,28,29,30,31,32]. By reviewing the recent literature, most of the endophytic bacteria of medicinal plants Bacillus sp., Pseudomonas sp., Enterobacter sp., Agrobacterium sp., etc., and a large number of endophytic bacteria exist in the roots, stems, and leaves, we collate some of the relevant data of the endophytic bacteria isolation as Table 1.

2.2. Culturable endophytic fungal diversity in medicinal plants

Aconitum heterophyllum is an alkaloid rich medicinal plant that is widely used clinically in tradition Chinese medicine [33,34]. A total of 328 fungal isolates were found in leaf, stem and root tissues of plants by Hafeez et al., and identified 12 endophytic fungal species by molecular characterization [34]. Crocus sativus L. (family Iridaceae) has been widely used as an antimicrobial, antidepressant, digestive, anticancer, and anticonvulsant medicine due to its abundant natural products as well as antioxidant activity [35,36]. Lu et al. [37] isolated endophytic fungi from five different locations, five Crocus sativus different tissues (corm, scape, leaf, petal, and stigma) and identified a total of 32 endophytic fungal groups, assigned to 7 orders within 4 classes. Wang et al. [38] isolated 34 endophytic fungi from Salvia miltiorrhiza, a traditional Chinese medicine, belonging to 10 genera and 16 species. Ogbe et al. [30] isolated a total of 11 endophytic fungi from the roots and leaves of a drought tolerant mint species Endostemon obtusifolius. Similarly, five endophytic fungi were isolated from the leaf segments of wild Dendrobium nobile and identified as Colletotrichum tropicicola, Fusarium keratoplasticum, Fusarium oxysporum, Fusarium solani, and Trichoderma longibrachiatum [39]. Fan et al. [40] obtained 205 strains of endophytic fungi from the roots of Codonopsis pilosula, a traditional medicinal plant collected from six regions in Gansu Province, China, of which Fusarium sp., Aspergillus sp., Alternaria sp., Penicillium sp., and Plectosphaerella sp. were the dominant genera. Niu et al. [41] isolated more than 30 kinds of Endophytic Fungi from the flowers of the medical plant Vernonia anthelmintica.

From the researches in recent years, it can be seen that Fusarium sp., Aspergillus sp., and Penicillium sp. can be isolated from most medicinal plants, and because of their many biological functions, they are regarded as the key research objects of endophytic fungi in medicinal plants. The recent research results are summarized in Table 2.

2.3. Culturable endophytic actinomycetes diversity in medicinal plants

Dioscorea has powerful medicinal functions and is a potential source of bioactive substances for combating various diseases [42]. Zhou et al. [43] isolated 116 actinomycetes from Dioscorea opposita Thunb. tissue and found a new Streptomyces sp. with strong biocontrol function. As a traditional Chinese medicine, Eucommia ulmoides Oliv. has been used to treat various diseases since ancient times [44]. The research group led by Mo et al. [45,46] isolated two new species of Nocardia sp. from the leaves and roots of Eucommia ulmoides Oliv. Musa et al. [47] isolated 128 strains from the roots, stems and leaves of the Chinese medical herb Thymus roseus schipcz, with a predominence of Streptomyces sp., followed by Nocardiopsis sp., Micrococcus sp., Kocuria sp., and others. Salwan et al. [48] isolated Streptomyces with antibacterial activity from the unique medicinal plant Viola odorata in the Himalayas. Hu et al. [49] isolated two new Streptomyces strains from healthy leaves and seeds of Xanthium sibiricum.

According to the research on endophytic actinomycetes of medicinal plants in recent years, actinomycetes are mainly distributed in the roots of medicinal plants, and their number is greater than that of other tissues of plants. Streptomyces sp. is the main research object of actinomycetes, and Streptomyces sp. has received extensive attention because of its strong biological activity [50]. Actinomycetes of other genera can also be isolated from medicinal plants, but the number is relatively small compared with Streptomyces. Many studies have isolated new species of bioactive endophytic actinomycetes from medicinal plants, which greatly expanded microbial resources and laid a foundation for the industrial application of actinomycetes[45,46,49,51,52,53]. The results of endophyte isolation from medicinal plants in recent years are shown in Table 2.

Table 3. Endophytic actinomycete resources isolated from medicinal plants in recent years.

Host plant	Tissue	Endophytic actinomycetes	Reference
Dioscorea opposita		Streptomyces sp.	[43]
Eucommia ulmoides Oliv.	Roots, leaf	Nocardia sp.	[45,46]
Thymus roseus	Root, stem, leaf	Nocardiopsis sp., Micrococcus sp., Kocuria sp., and etc.	[47]
Viola odorata	Root	Streptomyces sp.	[48]
Xanthium sibiricum	Leaf, seed	Streptomyces sp.	[49]
Kandelia candel	Root	Nocardioides sp.	[51]
Mentha haplocalyx	Bark	Nakamurella sp.	[52]
Acacia mangium	Root	Fodinicola sp.	[53]

Table 3. Endophytic actinomycete resources isolated from medicinal plants in recent years.

Host plant	Tissue	Endophytic actinomycetes	Reference
Dioscorea opposita		Streptomyces sp.	[43]
Eucommia ulmoides Oliv.	Roots, leaf	Nocardia sp.	[45,46]
Thymus roseus	Root, stem, leaf	Nocardiopsis sp., Micrococcus sp., Kocuria sp., and etc.	[47]
Viola odorata	Root	Streptomyces sp.	[48]
Xanthium sibiricum	Leaf, seed	Streptomyces sp.	[49]
Kandelia candel	Root	Nocardioides sp.	[51]
Mentha haplocalyx	Bark	Nakamurella sp.	[52]
Acacia mangium	Root	Fodinicola sp.	[53]

3. Beneficial effects of Endophytes from medicinal plants on the host

There are abundant endophytes in medicinal plants. In recent years, many studies have shown that endophytes have made important contributions in promoting the growth of medicinal plants, enhancing the stress resistance of medicinal plants and biological control of plant diseases.

3.1. Promoting the growth of medicinal plants

Khan et al. [54] isolated an endophytic fungus Acremonium sp. Ld-03, which has antibacterial activity and can produce IAA and siderophores, from the medicinal plant Lilium davidii. Application of 40% culture dilution of Acremonium sp., the root and bud length of Allium tuberosum can be significantly increased. Zou et al. [55] isolated a Bacillus subtilis strain from the medicinal plant Aconitum carmichaelii DEBX., which can produce gluconase, cellulase, protease, indole acid, siderophore, antifungal lipopeptides and polyketides, and significantly increased the fresh weight and dry weight of the host stem, main root and lateral root. Tao et al. [56] isolated four strains of endophytic bacteria with indole acid production, phase solubilization, and nitrogen fixation abilities from the precious traditional Chinese medicine Pairs polyphylla var. yunnanensis, and significantly increased the host's biomass. The metabolite of endogenous Aspergillus niger isolated from Albizia lebbeck (L.) Benth by Mathur et al., effectively promoted seed germination of many plants [57]. Purushotham et al. [58] isolated an endophytic actinomycete, Nocardia sp. TP1BA1B, which can solubilize phosphate and produce siderophores from New Zealand native medical plant Pseudowitera colorata (horopito), can promote the growth of seedlings.

The growth of medicinal plants is related to various environmental factors, such as light, temperature and microorganisms, among which endophytes are very important environmental factors to participate in the growth process of the host [59]. Endophytes promote the growth and development of plants in different ways, such as secreting iron carriers to improve the utilization rate of iron in plants. Endophytes with nitrogen fixation, phosphate solubilization and potassium solubilization promote the growth of medicinal plants by promoting the absorption of nitrogen, phosphorus and potassium. Endophytes can also promote plant growth by providing growth hormone to the host [10,60,61].

3.2. Enhance the stress resistance of medicinal plants

Li et al. [62] isolated a strain of Streptomyces from Glycyrrhiza uralensis and confirmed through inoculation that this strain can enhance the strain can enhance the tolerance of the host to drought, salt and drought salt conditions. Studies have found that under drought stress, the growth of Helianthus tuberosus L. (Jerusalem artichoke) can be better promoted by the inoculation of endophytic bacteria [63]. Sphingomonas paucimobilis, an endophytic bacterium in the rare medicinal plant Dendrobium officinale, has good resistance to stresses of salt, drop and cadmium, and this strain is the only one with birth ability reported in this species [64]. Some researchers isolated Endophytes from Astragalus mongholicus and co-inoculated them with Trichoderma Strains under drought conditions, which significantly improved the root biomass, root length, calycosin-7-O-β-D-glucoside content of the host, and activities of nitrate reductase and soil urease [65].

Endophytes can enhance the environmental adaptability of host plants by enhancing the expression of stress resistance related genes in host plants and increasing the activity of related enzymes [66]. In addition, some endophytes may also produce antibiotic compounds, antimicrobial peptides or alkaloids to help the host resist pests and diseases [67,68].

3.3. Promoting the accumulation of secondary metabolites in medicinal plants

Endophytic fungus Cladosporium tenuissimum DF11 isolated from Salvia miltiorrhiza by Chen et al. [69], promoted the biosynthesis and accumulation of Tanshinone in roots by up regulating the expression of HMGR, DXS, DXR, GGPPS, CPS, KSL and CYP76AH1, which are key enzyme genes of tanshinone biosynthesis pathway. Other researchers used endophytes of Salvia miltiorrhiza to prepare elicitors, which affected the accumulation of metabolites in hairy roots of Salvia miltiorrhiza by inducing the expression of key genes (SmAACT, SmGGPPS, and SmPAL) [70]. Researchers have also isolated two strains of fungi from Salvia abrotanoides that can increase host cryptotanshinone and tanshinone IIA production [71]. Ye et al. [72] isolated three Endophytes from houttuynia cordata, named ilyonectria liriodendra, unidentified fungal sp., and Penicillium citrinum, which can respectively increase the phenolic compounds of the host, increase the components such as afzelin, decanal, 2-undecanone, and increase the biomass of the host. Xie et al. [73] isolated an endophytic fungus Schizophyllum commune from Panax ginseng and significantly enhanced the expression of key enzyme genes involved in ginsenoside biosynthesis pathways such as pgHMGR, pgSS, pgSE, and pgSD under co-culture conditions, promoting the accumulation of specific ginsenosides.

Endophytes can directly participate in the synthesis of secondary metabolites of medicinal plants, and can also induce the formation of secondary metabolites of medicinal plants [74]. Selecting appropriate endophytes to act on medicinal plants can improve the content of secondary metabolites, which is of great significance for improving the quality of medicinal plants, protecting endangered medicinal plants, and synthesizing and developing new drugs.

3.4. Helping the host resist pathogens

Streptomyces dioscori isolated from Glycyrrhiza uralensis exhibited inhibitory effects on three pathogenic fungi: Rhizoctonia solani, Fusarium acuminatum, and Sclerotinia scrotiorum [62]. An endophytic fungus, Diaporthe sp., was isolated from the leaves of the Indian medicinal plant Chloranthus elator Sw., and its camphor odor volatiles showed inhibitory effects on eight fungal pathogens in vitro [75]. Burkholderia gladioli, an endophytic bacterium from Crocus sativus Linn., can reduce corm rot and increase endogenous jasmonic acid (JA) level and expression of JA-regulated and other plant defence genesthrough antibacterial effect, and improve the host's resistance to Fusarium oxysporum [76].

Many endophytes can inhibit the occurrence of plant diseases caused by pathogenic bacteria. Endophytes can inhibit the activity of pathogens by inducing host resistance to resist the infection of pathogens and competing with pathogenic bacteria to produce antibiotics, hydrolytic enzymes, alkaloids and other secondary metabolites and signal interference, to resist the disease caused by pathogens in host plants [77].

4. Medicinal components produced by endophytes in medicinal plants

An endophytic fungus Xylaria feejeensis, derived from the medicinal plant Geophila repens in Sri Lanka. Integrated acids, derived from fungal metabolites, has strong antibacterial activity and is a potential resource of antibiotics [78]. Saikosaponin d (SSd) is an important medicinal component of the medicinal plant Bupleurum scorzonerifolium Willd.. Some researchers isolated two endophytic Fungi from Bupleurum scorzonerifolium Willd., which can produce saikosaponin through UPLC/Q-TOF-MS detection [79]. The metabolite 7-methoxy-13-dehydroxypaxilline of Penicillium sp., an endophytic fungus isolated from the leaves of the traditional medicinal plant Baphicacanthus cusia (Nees) bremek., is a new indole diterpenoid, which exhibits anticancer activity [80]. Gu et al. [81] isolated a new compound phomopolide G from the fermentation broth of endophytic fungi of Artemisia argyi and showed a wide range of antibacterial activities. Cochliobolus sp., an endophytic fungus of the Indian medical herb Andrographis paniculata, can metabolize the alkaloid aziridine, 1- (2-aminoethyl) -, and can be antibacterial and insect resistant [82]. There is a new crystalline compound 5- (1-hydroxybutyl) - 4-methoxy-3-methyl-2h- pyran-2-one (c-hmmp) in the endophytic fungus Colletotrichum acutatum in the medicinal plant Angelica sinensis and shows antibacterial, antimalarial, anticancer, antioxidant and other activities [83]. The antibacterial compound 1,4-dihydroxy-2-methyl-anthraquinone was also isolated from the endophytic bacteria of archidendron pauciflorum [32]. The structures of the above compounds are shown in Figure 1.

At present, many alkaloids, flavonoids, phenolic acids, terpenoids, coumarins and other substances with antioxidant activity have been isolated from endophytes and their secondary metabolites of medicinal plants [10,11,32,84,85,86]. These natural antioxidant active substances often have anti-inflammatory, antioxidant, antibacterial, anti-tumor, Antiviral and other functions [10,11,87]. Through searching the literature in the past five years, it was found that the bioactive natural products of Endophytes in medicinal plants were metabolized by fungi, while the metabolites of bacteria and actinomycetes were mainly antibiotics [10]. More and more studies have found that the ‘Phytochemistry components’ produced by host plants may be produced by or closely related to endophytes. More and more studies have found that natural product produced by host plants may be produced by endophytes or closely related to endophytes [88]. Further research on the metabolites of endophytes will be of great significance for the development of medicinal plants and clinical drugs.

5. Discussion

In recent years, people have gradually realized that endophytes play an important role in affecting the yield and quality of crude drugs by interacting with the host in a specific way. The traditional method of endophyte research is to use the artificial medium to culture, isolate and purify microorganisms to obtain pure culture strains, and use microscopic technology to observe and classify their morphology (Figure 2). According to the physiological and biochemical characteristics of the strain, 16S rRNA, its gene sequencing, and other molecular biological methods were used for gene identification [10].

The rapid development of gene sequencing technology, especially the emergence of high-throughput sequencing and other technical means, has brought unprecedented development to microbiology research [89]. High throughput sequencing technology has been applied to the study of the structure and diversity of a variety of plant endophytes, and avoids the process of endophyte culture, to explore more microbial resources [89,90]. Some researchers conducted 16S rRNA gene based high-throughput sequencing analysis on the root samples of coastal native salt marsh plants, and detected a total of 4 Actinobacteria, 14 orders, 35 families, and 63 known genera, mainly including Arthrobacter sp., Mycobacterium sp., Micromonospora sp., Nocardia sp., and Streptomyces sp. [91]. Pan et al. also used the method of Illumina HiSeq sequencing technology to explore the composition and diversity of Endophytes in the roots, stems and leaves of Huperzia serrata. Total effective reads were assigned to 936 operational taxonomic units, belonging to 12 phylla and 289 general. Sphingomonas sp., Acidibacter sp., Bradyrhizobium sp., Bryobacter sp., Methylocella sp., Nocardioides sp., etc. were the dominant genera, and the microbial enrichment in roots was significantly higher than that in stems and leaves [92]. High throughput sequencing technology can explore the richness of microbial resources in medicinal plants, but only a small part can be isolated, which also means that there is still a great research potential for endophytes in medicinal plants. Therefore, it is necessary to optimize cultivation methods to obtain more cultivable microbial resources. Medicinal plants are the foundation of the development of pharmaceutical industry. With the understanding and utilization of the cultivation, growth and various functions of medicinal plants, the quality of medicinal plants has attracted great attention in society [93]. The quality and yield of medicinal plant raw materials are largely affected by many factors, such as plant genetic background, ecological habitat of plants and soil nutrients [94,95]. Endophytes live in medicinal plants and have the functions of promoting the growth of host plants, enhancing the stress resistance of host plants, and regulating the synthesis of secondary metabolites with important medicinal value [96]. Rational utilization of microbial resources, especially endophyte resources, is conducive to biological control and bioremediation of contaminated soil [97,98], thus reducing the use of chemical fertilizers and pesticides, which is of great significance for sustainable development.

The secondary metabolites of Endophytes from most medicinal plants have medicinal activities, which have great potential in the development of new drugs [11,98]. If endophytes of medicinal plants can be used to produce drugs in the future, it will make up for the demand for some rare medicinal plants to a certain extent [99]. The research methods of secondary metabolites of plant endophytes are usually based on the separation and purification strategy of natural products after endophyte fermentation. However, when the endophyte leaves the host, its biosynthesis of metabolites during fermentation is not as efficient as when it is in the host, and the microbial metabolic ability and biological function will decline after multiple generations of cultivation [100]. Therefore, optimizing the culture method of endophytes, promoting the metabolism of endophytes of medicinal plants, producing more metabolites with medicinal effects, and maintaining the biological function of endophytes are also urgent problems to be solved.

The use of chemical pesticides causes serious pollution to the environment and even endangers human health [101], so it is necessary to put forward more sustainable development strategies for the environment and human health, and find suitable pesticide substitutes. Endophytes come from plants and act on plants, and they are rich in species and have strong biological control functions [56,102]. Therefore, they are the most suitable to replace chemical pesticides in the cultivation and protection of medicinal plants, and are an important environmental protection strategy. The metabolites of Endophytes in medicinal plants are also a huge treasure house for the discovery of medicinal ingredients, which can greatly make up for the shortage of natural resources, which also makes the research of endophytes attract more researchers' attention [8,103]. In order to fully develop the research and application of Endophytes in medicinal plants, the following problems need to be solved urgently: 1. How to make the biocontrol strains survive in the environment outside the plant for a long time? 2. Can the reduced metabolic capacity and biological function of endophytes after multi-generation culture be overcome by optimizing culture regulation? 3. In vitro endophytes can produce a variety of secondary metabolites to antagonize pathogenic bacteria and inhibit their growth. Can the development of new green pesticides be produced in large quantities with high efficiency? 4. How can we improve the fermentation efficiency of Endophytes in medicinal plants, to tap more abundant potential medicinal ingredients? To solve the above problems, exploring the interaction mechanism between endophytic bacteria and hosts, as well as the metabolic mechanism of medicinal components, and conducting more research on the application of endophytes in production, will be of great help to the production of medicinal plants and the development of medicinal components.