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An exploration of mechanism of high quality and yield of Gastrodia elata Bl. f. glauca by the isolation, identification and evaluation of Armillaria
BMC Plant Biology volume 22, Article number: 621 (2022)
Gastrodia elata Bl. f. glauca, a perennial herb of G.elata Bl. in Orchidaceae, is one of the most valuable traditional Chinese medicines. G. elata Bl. is a chlorophyll-free myco-heterotrophic plant, which must rely on the symbiotic growth of Armillaria, but not all Armillaria strains can play the symbiotic role. Additionally, Armillaria is easy to degenerate after multiple generations, and the compatibility between the strains from other areas and G. elata Bl. f. glauca in Changbai Mountain is unstable. Therefore, it is incredibly significant to isolate, identify and screen the symbiotic Armillaria suitable for the growth of G. elata Bl. f. glauca in Changbai Mountain, and to explore the mechanism by which Armillaria improves the production performance of G. elata Bl. f. glauca.
Firstly, G. elata Bl. f. glauca tubers, and rhizomorphs and fruiting bodies of Armillaria were used for the isolation and identification of Armillaria. Five Armillaria isolates were obtained in our laboratory and named: JMG, JMA, JMB, JMC and JMD. Secondly, Armillaria was selected based on the yield and the effective component content of G. elata Bl. f. glauca. It was concluded that the yield and quality of G. elata Bl. f. glauca co-planted with JMG is the highest. Finally, the mechanism of its high quality and yield was explored by investigating the effects of different Armillaria strains on the soil, its nutrition element contents and the soil microbial diversity around G. elata Bl. f. glauca in Changbai Mountain.
Compared with commercial strains, JMG significantly increased the content of Na, Al, Si, Mn, Fe, Zn, Rb and the absorption of C, Na, Mg, Ca, Cr, Cu, Zn and Rb in G. elata Bl. f. glauca; it improved the composition, diversity and metabolic functions of soil microbial communities around G. elata Bl. f. glauca at phylum, class and genus levels; it markedly increased the relative abundance of bacteria such as Chthoniobacter and Armillaria in the dominant populations, and enhanced such functions as Cell motility, amino acid metabolism and Lipid metabolism; it dramatically decreased the relative abundance of Bryobacter and other fungi in the dominant populations, and reduced such functions as microbial energy metabolism, translation and carbohydrate metabolism. This is the main reason why excellent Armillaria strains promote the high quality and yield of G. elata Bl. f. glauca in Changbai Mountain.
Gastrodia elata Bl. f. glauca belongs to a variety of G. elata Bl., a perennial herb in Orchidaceae. G.elata Bl. is also called as Tianma in Chinese. It contains such active ingredients as gastrodin, p-hydroxybenzyl alcohol, balisonoside A, parisenoside B, balisonoside C, balisonoside E . It has the efficacy of suppressing hyperactive liver for calming endogenous wind, relieving pain and spasmolysis, calming lung qi and liver-yang, dispelling wind, removing blood stasis and dredging collaterals . The results of modern pharmacological clinical research have showed that G. elata Bl. related drugs can be widely used to reduce chronic hypertension , increase cardiac and cerebral blood flow , modulate the nervous system , and treat such disease as chronic depression , and Alzheimer’s disease . The demand for G. elata Bl. is growing rapidly at home and abroad, especially the high-quality G. elata Bl. f. glauca in Changbai Mountain is in short supply . However, the wild G. elata Bl. is scarce and artificial plantation is required for its supply.
The growth and development of G. elata Bl., as one of the heterotrophic plants without chlorophyll, requires symbiosis with Armillaria [8,9,10], which has a significant impact on the yield and quality of G. elata Bl.. The isolation and identification of excellent Armillaria strains are of great significance . Armillaria is prone to species degradation during multi-generation reproduction, that is, after repeated asexual reproduction, the quality of the offspring of the strain decreases, and the ability to provide nutrients for G. elata Bl. decreases. Thus, it requires continuous renewal.
Armillaria (Fr.) Staude belongs to Basidiomycota [12,13,14]. It is distributed on all continents of the world, and in China, it is mainly distributed in Jilin, Heilongjiang and Inner Mongolia and other provinces [15,16,17]. At present, over 40 species of Armillaria have been described in the world, some of which are serious tree pathogens, some have medicinal value, and some can coexist with G. elata Bl. for its cultivation and production [18,19,20]. There are 15 Armillaria species in China, of which eight are endemic to China . In the 1960s, Xu Jintang used the culture method of Armillaria cords, fresh wood segments and G. elata Bl. seeds to make G. elata Bl. wild and domesticated. In Changbai Mountain, Zhaotong in Yunnan, and Guizhou, a variety of Armillaria species that can coexist with G. elata Bl. have been identified, including A. mellea, A. gallica, A. sinapina and A. cepistipes. Seven phylogenetic branches can coexist with G. elata Bl. [21,22,23,24,25,26]. New Armillaria species in China are constantly being discovered.
As mentioned above, not all Armillaria can coexist with G. elata Bl.. There are few Armillaria species isolated and identified in Changbai Mountain, and fewer strains can be available for symbiosis with G. elata Bl. there . The unstable compatibility between strains from other areas and G. elata Bl. f. glauca affects its yield . It is vital to isolate, identify and screen symbiotic Armillaria, which is beneficial to the growth of G. elata Bl. f. glauca. Studies have shown that many orchid mycorrhizal fungi can promote nutrient absorption in the plant rhizosphere . Meanwhile, they also affect changes in microbial community structures and their functions in rhizosphere soil [30,31,32]. However, the effect of symbiotic fungus Armillaria on the absorption of nutrition elements by G. elata Bl. . And the ecological relationship as well as nutrient cycles among G. elata Bl., Armillaria, soil and soil microorganisms remain unknown [34, 35]. Therefore, it is of great significance for its cultivation to explore the mechanism by which the excellent Armillaria can improve the production performance of G. elata Bl. f. glauca . In this study, Armillaria was first isolated and identified by using G. elata Bl. f. glauca tubers, rhizomorphs and fruiting bodies of Armillaria . Then, Armillaria was selected according to the yield and active component content of G. elata Bl. f. glauca. Finally, the effects of different strains on the nutrition element content and microbial diversity in the soil around G. elata Bl. f. glauca were investigated to explore the mechanism of high quality and yield of G. elata Bl. f. glauca co-planted with Armillaria [38, 39].
Isolation and identification of Armillaria from G. elata Bl. f. glauca
A total of 11 Armillaria isolates were isolated, including four from rhizomorphs, and four from fruiting bodies of Armillaria, and three from G. elata Bl. f. glauca tubers. During the passaging process, the isolates with vigorous mycelial growth were screened. Finally, five Armillaria isolates were selected for morphological and molecular identification in our laboratory and named JMG,JMA,JMC,JMD and JMG, respectively. Among them, JMG and JMA were isolated from rhizomorphs, JMB and JMC from fruiting body tissue, and JMD from G. elata Bl. f. glauca tubers. The morphological characteristics of the mycelium in the five Armillaria isolates were different. Among them, the hyphae of the isolates JMG, JMC and JMD spores germinate faster compared with JMA and JMB, and the germination time of the former and later was 5–6 and 6-7 days, respectively. Different Armillaria isolates were ranked as follows according to mycelial growth rates from fast to slow: JMG > JMC > JMD > JMB > JMA (Fig. 1B, Table S1). The comparison of microscopic characteristics showed that JMG and JMC had dense hyphae and good growth conditions, while JMA, JMB and JMD had sparse hyphae and relatively poor growth conditions (Fig. 1A). This indicated that there are indeed morphological differences between these five isolates.
PCR amplification of the ITS rDNA region yielded five single bands ranging in size from 750 to 1000 bp (Fig. 1C). Through The Basic Local Alignment Search Tool (BLAST) homology comparison, it was found that the base sequences of the five Armillaria isolates shared over 98% homology with registered Armillaria genes in NCBI database (Accession Nos. MW404994, MW404995, MW404996, MW404997and MW404998), and that the same base sequence coverage was more than 97%. BLAST homology comparison showed that the five isolates shared more than 97% homology with A. gallica and A. sinapina (Table S2). The phylogenetic tree showed that the five isolates were clustered in three main clades, of which JMG (MW404994) and JMD (MW404998) had the shortest clade with A. gallica, JMA (MW404995) and JMB (MW404996) had the shortest branches with A. mellea，and JMC (MW404997) had the shortest branches with A. sinapina (Fig. 1D).
Optimization of Armillaria from G. elata Bl. f. glauca
The results of field experiments showed that four Armillaria isolates could promote the growth of G. elata Bl. f. glauca tubers except JMC, and that different isolates had significant effects on the yield of G. elata Bl. f. glauca (P < 0.05). The yield of G. elata Bl. f. glauca co-planted with each isolate was all significantly higher, compared with the commercial strain control check (CK) (P < 0.05). The yield of G. elata Bl.f. glauca co-planted with the commercial strain CK was significantly higher, compared with JMA and JMD (P < 0.05), and the yield of G. elata Bl. f. glauca co-planted with JMA and JMD was both markedly higher, compared with JMB (P < 0.05) (Fig. 2A, Table S3).
G. elata Bl. f. glauca co-planted with each isolate all contained six active components, gastrodin, p-hydroxybenzyl alcohol, parishin E, parishin B, parishin C and parishin A, respectively (Fig. S1A-F). There were significant differences in the content of the six active components among G. elata Bl. co-planted with different Armillaria isolates (P < 0.05). The gastrodin content in the JMA group was significantly higher than that in the other groups, respectively, but with no significant difference among the other groups (Fig. 2B). The content of p-hydroxybenzyl alcohol in JMG and JMD groups were significantly higher than that in the other groups, respectively, but with no significant difference among the other groups (Fig. 2C). The content of parishin E in JMG group was significantly higher than that in the other groups, respectively (P < 0.05), but with no significant difference among the other groups (Fig. 2D). The content of parishin B in JMG and CK groups was significantly higher than that in JMD group, respectively (P < 0.05), and the content of parishin B in JMD and JMB groups were significantly higher than that in JMA group, respectively (P < 0.05) (Fig. 2E). The content of parishin C in JMG group was significantly higher than that in CK, JMB and JMD groups, respectively and the content of parishin C in CK, JMB and JMD groups was significantly higher than that in JMA group, respectively. There was no significant difference among CK, JMB and JMD groups (Fig. 2F). The content of parishin A in JMG and JMD groups was significantly higher than that in CK group, respectively, and the content of parishin A in CK and JMB groups was significantly higher than that in JMA group (P < 0.05). There was no significant difference among the other groups (Fig. 2G). The additive value of gastrodin and p-hydroxybenzyl alcohol in the JMA group was significantly higher than that in CK, JMB and JMD groups, respectively (P < 0.05), but with no significant difference among the other groups (Fig. 2H). The sum value of six active ingredients in the JMG group was significantly higher than that in the other groups (P < 0.05), but with no significant difference among the other groups (Fig. 2I). It can be concluded that both the yield and effective component content of G. elata Bl. f. glauca co-planted with JMG are better compared with the other isolates. Thus, JMG is more suitable for the cultivation of G. elata Bl. f. glauca.
Analysis of absorption Laws of nutrition elements in G. elata Bl. f. glauca co-planted with JMG
The inorganic elements in the surrounding soil of G. elata Bl. f. glauca and its tubers under the symbiosis of it with different Armillaria (the Commercial strain CK, JMG, respectively) were detected by ICP-MS. More than 150 inorganic elements were detected, among which there were 28 related to plant growth. There were 14 chemical elements in G. elata Bl. f. glauca (C, N, Na, Mg, Al, Si, P, S, K, Ca, Mn, Fe, Zn, Rb). The chemical elements (Na, Al, Si, Mn, Fe, Zn, Rb) of G. elata Bl. f. glauca co-planted with JMG were higher, compared with commercial Armillaria strains. There were significant differences in 10 soil chemical elements (C, N, Na, Mg, Al, Si, S, K, Ca, Fe). Among them, the soil chemical elements of G. elata Bl. f. glauca co-planted with JMG were all higher, compared with commercial Armillaria strains. The enrichment coefficients of 10 elements (B, C, Na, Mg, P, Ca, Cr, Cu, Zn, Rb) were significantly different. Among them, the enrichment elements (C, Na, Mg, Ca, Cr, Cu, Zn, Rb) of G. elata Bl. f. glauca co-planted with JMG were all higher, compared with commercial Armillaria strains. It can be seen that JMG significantly increased the content of chemical elements (Na, Al, Si, Mn, Fe, Zn and Rb) and the absorption of chemical elements (C, Na, Mg, Ca, Cr, Cu, Zn and Rb) in G. elata Bl. f. glauca co-planted with it. This is one of the reasons for high quality and yield of G. elata Bl. f. glauca (Table S4).
Microbial sequence analysis of the soil around G. elata Bl. f. glauca co-planted with JMG
Sequence analysis showed that the number of high-quality sequences of fungi was 453,881, and that the number of high-quality sequences of bacteria was 475,153. With the number of read sequences increasing, the number of OTUs increased rapidly and then slowly, and when the number of sequences exceeded 10,000, the number of OTUs tended to flatten, indicating that the sample sequences can cover most of the species in soil samples (Fig. 3A and B). The V-enn diagram of each sample showed that the OTU number in CK and JMG groups was 2124 and 2170 respectively, with a total of 2117, 7 unique bacteria in the CK group and 53 unique bacteria in the JMG group (Fig. 3C). The OTU number in CK and JMG groups was 493,553, respectively. There were 354 fungi in the CK group and 199 fungi in the JMG group (Fig. 3D). It can be seen that the sample sequences in both JMG and CK groups covered most of the same species, but the OUT number in the JMG group was larger, compared with the CK one, and the OTU number of bacteria and fungi unique to the JMG group was larger, compared with the CK one. The results showed that the co-planting of different Armillaria isolates with G. elata Bl. f. glauca had significant effects on the soil microbiota around its tubers.
The composition of soil microbial communities around G. elata Bl. f. glauca co-planted with JMG
Firstly, it was observed that the relative abundance of soil bacteria and fungi around G. elata Bl. f. glauca under the symbiosis of it with different Armillaria strains at the phylum level. Among them, the dominant bacteria in the soil around G. elata Bl. f. glauca tubers were mainly from Proteobacteria, Acidobacteria, Actinobacteria, Verrucomicrobia, Bacteroidetes, Chloroflexi, Gemmatimonadetes, Planctomycetes, Patescibacteria and Nitrospirae. The relative abundance of Acidobacteria, Verrucomicrobia, Planctomycetes and Patescibacteria increased by 0.77, 0.73, 0.12 and 1.02% respectively in the soil around G. elata Bl. f. glauca co-planted with JMG, compared with the commercial strain CK, while the abundance of Chloroflexi and Gemmatimonadetes decreased by 1.79 and 0.75%, respectively (Fig. 4A). The dominant fungi in the soil around G. elata Bl. f. glauca tubers mainly came from Basediomycota, Ascomycota, Mucoromycota, Mortierellomycota, Rozellomycota, Chytridiomycota, Zoopagomycota, Aphelidiomycota, Monoblepharomycota and Olpidiomycota. The relative abundance of Basidiomycetes, Ascomycetes, Mucoromycota and Mortierellomycota increased by 6.05, 2.52, 2.48 and 1.67% respectively in the soil around G. elata Bl. f. glauca tubers co-planted with JMG, compared with the commercial strain CK, while the relative abundance of Rozellomycota decreased by 3.58% (Fig. 4B).
Secondly, the differences in the relative abundance of soil bacteria and fungi around G. elata Bl. f. glauca under the symbiosis of it with different Armillaria strains at the genus level were observed. Among them, the dominant bacteria in the soil around G. elata Bl. f. glauca showed a variety of uncultured-bacterium. The relative abundance of Chthoniobacter and uncultured_bacterium_f_Burkholderiaceae in the soil around G. elata Bl. f. glauca co-planted with JMG increased by 0.94 and 0.88% respectively, compared with the commercial strain CK, while the relative abundance of Bryobacter and uncultured_bacterium_f_Gemmatimonadaceae decreased by 0.62 and 0.61%, respectively (Fig. 4C). The dominant fungi in the soil around G. elata Bl. f. glauca mainly came from Trichoderma, Agrocybe, Mortierella, Mycena, Fusarium, Leptodontidium, Lecanicillium, Mycofalcella, Armillaria and Cylindrosympodium. The relative abundance of Armillaria, Mortierella, Fusarium and Agrocybe in the soil around G. elata Bl. f. glauca co-planted with JMG increased by 3.00, 1.53, 0.46 and 0.01%, respectively, compared with the commercial strain CK, while the relative abundance of Lecanicillium, Cylindrosympodium and Mycena decreased by 0.95, 0.03 and 0.01%, respectively (Fig. 4D). The results showed that JMG improved the composition of bacterial and fungal communities in the soil around G. elata Bl. f. glauca.
Finally, the cluster differences in soil bacterial and fungal species abundance around G. elata Bl. f. glauca under the symbiosis of it with different Armillaria strains at the class level were investigated. The soil bacteria around G. elata Bl. f. glauca co-planted with JMG were clustered into Acidimicrobiia, Acidobacteria, Oxyphotobacteria, Alpha proteobacterium, Fimbriimonadia, Thermoleophilia, Coriobacteriia and Nitrospirae (Fig. 4E). The soil bacteria around G. elata Bl. f. glauca co-planted with the commercial strain CK were clustered into Anaerolineae, Bacilli, Armatimonadia, Clostridia, Sericytochromatia and Ktedonobacteria. Meanwhile, the soil fungi around G. elata Bl. f. glauca co-planted with JMG were clustered into Orbiliomycetes, Rhizophydiomycetes, Saccharomycetes, Laboulbeniomycetes, Pucciniomycetes, Tritirachiomycetes, Tremellomycetes, Lobulomycetes, Eurotiomycetes, Monoblepharidomycetes, Mortierellomycetes, Cystobasidlomycetes and Olpidiomycetes. The soil fungi around G. elata Bl. f. glauca co-planted with commercial strains were clustered into Dacrymycetes, Lecanoromycetes, Dothideomycetes, Glomomycetes and Pezizomycetes (Fig. 4F). There were significant differences in clustering between the JMG and CK groups at the class level. The results showed that JMG improved the composition of bacterial and fungal communities in the soil around G. elata Bl. f. glauca at the phylum, class and genus levels. This may be one of the reasons why JMG promoted the absorption of soil nutrients by G. elata Bl. f. glauca and increased its yield.
Alpha and Beta diversity of soil microbial communities around G. elata Bl. f. glauca co-planted with JMG
Although the above results demonstrated that JMG affects the composition of soil microbial communities around G. elata Bl. f. glauca, its effects on the community diversity have yet been unclear. In order to further investigate whether JMG affects the soil microbial community diversity around the G. elata Bl. f. glauca, the soil microbial α-diversity around G. elata Bl. f. glauca tubers was firstly analyzed. In the 0–5000 range, Shannon index increased sharply with the continuous increase of sequence number. When the number of sequences exceeded 5000, the curve tended to flatten, and Shannon index would no longer increase with the continuous increase of sequence number (Fig. 5A and B). The number of sequencing data was large enough to cover the information related to most microorganisms and their species. It was found that there was no significant change in soil bacterial α-diversity around G. elata Bl. f. glauca in the JMG and CK groups. (Fig. 5C-F), but the the Ace, Chao1, Shannon and Simpson indexes ones of fungi in the soil around G. elata Bl. f. glauca co-planted with JMG were higher, compared with the CK (Fig. 5G-J). The data coverage of the sequencing library of the samples in this study exceeded 99.9 and 98.5% respectively, indicating that the sample sequencing results fully reflect the real situation of various microorganisms in each sample.
Secondly, the soil microbial β-diversity around G. elata Bl. f. glauca tubers was analyzed. It was found that the soil microbial population around G. elata Bl. f. glauca under the symbiosis of it with JMG and the commercial strain CK was clustered on their own branches (Fig. 6A and B). The clustering heatmap showed that the rank abundance of both soil bacterial and fungal samples around G. elata Bl. f. glauca co-planted under the symbiosis of it with the commercial strain CK and JMG were both clustered into class1 (Fig. 6C and D). The results showed that there were different microbial diversities in the soil around G. elata Bl. f. glauca under the symbiosis of it with different Armillaria strains. The symbiosis of JMG and G. elata Bl. f. glauca increased the microbial diversity in the soil around G. elata Bl. f. glauca, which may be another mechanism by which JMG promoted the soil nutrient absorption by G. elata Bl. f. glauca and increased its yield.
The analysis of soil microbial differences around G. elata Bl. f. glauca co-planted with JMG
Based on the significant differences in the soil microbial composition and diversity around G. elata Bl. f. glauca under the symbiosis of it with JMG and the commercial strain CK, the differences between the two groups of samples were further investigated. First, through ANOVA analysis, there were significant differences among Actinobacteria, Chloroflexi, Gemmatimonadetes, Planctomycetes, Patescibacteria and Cyanobacteria at the level of bacteria (P < 0.05). Among them, the relative abundance of Actinobacteria, Patescibacteria and Planctomycetes in the soil around G. elata Bl. f. glauca co-planted with JMG was higher (Fig. 7A). At the fungal phylum level, there were significant differences among Ascomycota, Basediomycota, Mortierellomycota, Rozellomycota, Mucoromycota, Chytridiomycota and Zoopagomycota in different groups. Among them, the relative abundance of Mucoromycota, Mortierellomycota, Chytridiomycota, Basediomycota and Ascomycota in the soil around G. elata Bl. f. glauca co-planted with JMG was higher, compared with the commercial strain CK (Fig. 7B). Second, Linear discriminant analysis Effect Size (LEfSe) analysis was used to test the difference in the relative abundance. Among these bacteria, only p-Chloroflexi contributed the most to the soil around G. elata Bl. f. glauca (Fig. 7C). Among the fungi, Lecanicillium_fusisporum contributed the most to the soil around G. elata Bl. f. glauca and Bolbitiaceae contributed the most to the soil around G. elata Bl. f. glauca tubers. (Fig. 7D). The results showed that there were significant differences in the relative abundance of bacteria and fungi between the two groups, with fungi contributing more significantly to the two groups, which confirmed that there are significant differences in the microbial composition and diversity between the two groups.
PICRUSt functional prediction analysis of the soil around G. elata Bl. f. glauca co-planted with JMG
The above research results demonstrated that JMG affects the composition and diversity of soil bacteria communities around G. elata Bl. f. glauca, and that there are significant differences between the two soil around G. elata Bl. f. glauca co-planted with JMG and the commercial strain CK. In order to further analyze the effect of microorganisms on their metabolic function, PICRUSt was used to predict and analyze the metabolic function of the soil bacteria community around G. elata Bl. f. glauca. The results showed that there were 42 secondary metabolic functions in the KEGG metabolic pathway of predicted genes at the secondary level, among which 17 secondary metabolic functions were different between the JMC and CK groups (Fig. 8). Among them, the copy number of seven secondary function predicted genes in the JMG group was greater compared with the CK group. They included these genes of Cell motilit, Signal transduction, Membrane transport, “Xenobiotics Biodegradation and Metabolism”, Amino acid metabolism, Lipid metabolism, other amino acid metabolism, etc. (Fig. 8). The copy number of 10 secondary metabolic function predicted genes in the JMG group was smaller, compared with the CK group. They included the genes of Transcription, Folding, sorting and degradation, Replication and repair, Global and overview maps, Glycan biosynthesis and metabolism, Nucleotide metabolism, Energy Metabolism, Metabolism of cofactors and vitamins, Translation, Carbohydrate metabolism, etc. (Fig. 8). The results showed that soil microorganisms around G.elata Bl. f. glauca under the symbiosis of it with different Armillaria strains had different metabolic functions.
In summary, JMG can improve the composition and diversity of the soil microbial community, increase the ability of G. elata Bl. f. glauca to absorb nutrients, and the metabolic function of soil microorganisms around it, which promotes its growth and development, thus increasing its yield. This may be one of the mechanisms for high yield of G. elata Bl. f. glauca.
The growth and development of G. elata Bl. are inseparable from the nutrition provided by Armillaria, and its isolation and identification have become a research hotspot . A total of 15 Armillaria species were isolated and identified in China, among which eight endemic species were found to be widely distributed only in China . However, not all Armillaria can provide nutrition for G. elata Bl. . There are diverse symbioses between different Armillaria strains and G. elata Bl. , which has different effects on the yield of G. elata Bl. . After multiple generations of Armillaria, its activity and ability to provide nutrients decreased, thus resulting in a decrease in G. elata Bl. production [27, 43, 44]. Therefore, it is necessary to isolate and identify more Armillaria strains, which are beneficial to the production of G. elata Bl.. In this study, five Armillaria isolates were obtained from the tubers and fruiting bodies of Armillaria officinalis in Changbai Mountain. Based on phylogenetic analysis, the amplified ITS regions of the five isolates were sequenced, conserved nucleotide regions are used for identifying different strains . The five isolates were found to be more closely related to A. gallica, A. sinapina and A. mellea, which is consistent with previously reported Armillaria near Changbai Mountain and also confirms the M. strains that can be symbiotic with Amanita in previous studies . These findings provided better Armillaria for improving the yield of endemic G. elata Bl. f. glauca cultivars in Changbai Mountain [46, 47].
Armillaria plays a crucial role in the growth of G. elata Bl. f. glauca. The strain quality directly affects the yield and quality of G. elata Bl. f. glauca, which are important indexes for screening high-quality Armillaria. Liu et al. compared the yield and effective components of G. elata Bl. co-planted with four different Armillaria strains in 2019. They found that the yield of G. elata Bl. co-planted with M1 strain is 1.905 times higher, compared with M3 one, and that the total amount of gastrodin and p-hydroxybenzyl alcohol in G. elata Bl. co-planted with the M1 strain is 2.4 times the standard of Pharmacopoeia, indicating that the yield and quality of G. elata Bl. co-planted with Armillaria are important indexes for screening high-quality Armillaria . Additionally, the mycelial germination time, morphology and growth rate of different Armillaria stains are not the same, which directly affects their own biomass, and in turn exerts influence on the production of Armillaria, as well as the yield and quality of G. elata Bl.. This showed that they are also important indexes for screening high-quality Armillaria . In this study, a multi-index evaluation method was adopted to comprehensively evaluate the strengths and weaknesses of Armillaria, which can help screen high-quality Armillaria strains more accurately, with more important guiding value for the production practice of G. elata Bl. f. glauca. Among the five Armillaria isolates, JMG has faster growth rates, and the yield and effective components of G. elata Bl. f. glauca co-planted with it are better, compared with the other isolates. Therefore, JMG is more suitable for forming a symbiosis with G. elata Bl. f. glauca in Changbai Mountain.
The vast majority of terrestrial plants on earth form a nutritional relationship of mycorrhizal symbiosis with fungi, which can provide plant bodies with various nutrients required for their normal growth and development . Mycorrhiza can effectively improve the absorption of carbon, nitrogen, phosphorus, potassium, etc. by plants, thus promoting their growth and development . Mycorrhizal fungi can directly provide varieties of nutrients and minerals required for the growth of orchid plants . Mycorrhizal fungi such as Cymbidiumhybridum  and D.nobile  can not only markedly increase leaf and stem dry weights but also promote the absorption of N, P, K and other chemical elements by plants. Compared with commercial strains, JMG, promoted the absorption of various nutrition elements by G. elata Bl. f. glauca, which is one of the mechanisms by which this isolate increased the yield of G. elata Bl..
Soil microorganisms, an important component of soil, exist in the form of communities. And they are the drivers of the cycling and transformation of soil nutrients and organic matter , with an impact on soil quality and plant growth . The species affecting the soil microbial community can be detected by investigating the diversity of soil microorganisms . The growth and development of Orchidaceae are highly dependent on symbiotic mycorrhizal fungi. The impacts of symbiotic mycorrhizal fungi on the composition and diversity of soil microbial communities may lead to changes in the ecological environment of soil microgranisms around plants, thus improving the growth status and plant yield . In this study, JMG could change the composition and diversity of soil microbial community around G. elata Bl. f. glauca . For example, the relative abundance of beneficial fungi such as Armillaria in the soil around G. elata Bl. co-planted with JMG was higher, compared with commercial strains, while the relative abundance of Cylindrosympodium was lower. This may be one of the reasons why JMG isolate increased its yield by improving the soil microbial environment as well as the absorption and utilization of soil nutrients and disease resistance by G. elata Bl. f. glauca . This result was consistent with that of the research on the effect of mycorrhizal microorganisms in some other plants on antagonizing soil pathogens and promoting beneficial soil fungi .
The metabolic function of microbial communities, such as the existence or deletion of functional genes, and their abundance, can be reliably predicted by PICRUSt analysis . In this study, the functional genes of soil microorganisms around G. elata Bl. f. glauca co-planted with different Armillaria strains were predicted. Among them, compared with the CK group, the copy number of functional genes of amino acid and lipid metabolism and so on increased, while the copy number of functional genes of glycan biosynthesis and metabolism, nucleotide metabolism, cofactor and vitamin metabolism, energy metabolism, carbohydrate metabolism,etc. decreased. These changes in metabolic functions associated with plant growth  may also regulate the growth and development of G. elata Bl. f. glauca . The increased copy number of functional genes of cell movement, signal transduction, membrane transport and so on may be correlated with the regulation of the nutrient absorption by G. elata Bl. f. glauca by JMG [63, 64]. The decreased copy number of functional genes of transcription, folding, classification and degradation, replication and repair and translation and so on may be correlated with the improvement in disease resistance in G. elata Bl. f. glauca by JMG [65,66,67]. These results indicated that JMG improves soil microbial metabolic functions of G. elata Bl. f. glauca, which differs from commercial strains. This may be another mechanism by which JMG increased its yield by improving soil microbial metabolic function, as well as soil nutrient absorption, utilization and disease resistance of G. elata Bl. f. glauca. At the same time, it will be a new research paradigm to see whether Armillaria can directly contribute to the enhancement of G. elata Bl. f. glauca production.
In addition, some Armillaria strains can form a good symbiosis with G. elata Bl. f. glauca, which is closely related to the yield and the active component content of G. elata Bl. f. glauca. In this study, the five Armillaria strains were isolated and screened, and the most suitable symbiosis of Armillaria JMG with G. elata Bl. f. glauca was obtained. The following was found in this study: First, JMG could affect the nutrient absorption of G. elata Bl. f. glauca, especially it could increase the content of sodium and other chemical elements; second, JMG improved the microbial community in the soil around G. elata Bl. f. glauca, especially it increased the relative abundance of fungi such as Armillaria; finally, JMG also changed the metabolic function of microorganisms, especially Glycanbiosynthesis and metabolism, Nucleotide metabolism,etc. This preliminarily demonstrated how JMG can improve the yield and effective components of G. elata Bl. f. glauca. And further studies are certainly required for its molecular mechanism. These results provide new ideas for the research on the symbiosis of Armillaria and G. elata Bl. f. glauca.
Materials and methods
Isolation and identification of Armillaria
In this study, G. elata Bl. f. glauca tubers, rhizomorphs and fruiting bodies of Armillaria collected from Jingyu County, Baishan City, Jilin Province in 2019 were used to isolate and identify Armillaria strains. First, the tubers of G.elata Bl. f. glauca, and the rhizomorphs and fruiting bodies of Armillaria were washed with sterile water. After absorbing excess water with the sterile filter paper, G.elata Bl. f. glauca tubers were cut into 0.5 cm long sections on a sterile operation table, with each section of epidermis cut off (thickness 2 mm). Second, plates of corn-wheat bran medium(a solid medium prepared by sieving corn flour and boiling wheat bran with distilled water, filtering the solution with gauze, and adding agar powder) were inoculated one piece per plate. Meanwhile, take the rhizomorphs around the G. elata Bl. f. glauca tuber, rinse with sterile water 2–3 times, absorb the water on filter paper, pinch the two ends of the rhizomorphs, pull it off slowly and forcefully next to the flame of an alcohol lamp, cut the white mycelium 3-5 mm at the break with scissors, inoculated into potato dextrose agar (PDA) medium (culture 5 sections of mycelium per plate), then, 3–5 mm of thalli at the stipe and cap of Armillaria fruiting bodies were also inoculated into PDA medium plates, and each plate was inoculated with one piece. Third, the two plates were placed in a constant temperature incubator at 25 °C for 7–10 days in the dark. After the mycelium grew, the white mycelium was transferred to the plate of the corn flour and wheat bran medium for continuous culture. After the mycelium germinated, the tip of the mycelium was taken and inoculated onto the PDA medium for storage in the dark. Then, the Armillaria isolates were inoculated onto a new corn-wheat bran medium test tube, and a vernier caliper was used to measure the length of the mycelium every day. Then, the growth rate was calculated, and the morphology of the mycelium was observed and photographed through a microscope. Next, ITS genes of Armillaria was amplified by universal fungal-specific primers ITS 4 (F:5′-TCCTCCGCTTATTGATATGC-3′) and ITS 5 (R:5′-GGAAGTAAAAGTCGTAACAAGG-3′) . After that, the amplified products were sent to Bioengineering (Shanghai) Co., Ltd. for sequencing. And these sequences were deposited in NCBI GenBank and compared with the sequences in GenBank by BLAST search. Phylogenetic analysis was performed by Mega7.0, and the prepared sequences were aligned by Clustal-W software [69, 70]. Finally, the phylogenetic tree was constructed by neighbor-joining (NJ) methods to analyze and compare the interspecific and intraspecific genetic distances of Armillaria sequences (the bootstrap test of 1000 times) [11, 45].
Co-cultivation and harvest of Armillaria and G. elata Bl. f. glauca
The field experiment was conducted in the farmland base of Baishan Jingzhen Gastrodia Development Co., Ltd. Commercial strains and G. elata Bl. f. glauca seeds for cultivation were provided by Baishan Jingzhen Gastrodia Development Co., Ltd. The commercial strain, as the control check (CK) in this study, belongs to A. gallica and has been widely used for cultivating G. elata Bl.. As the main nutrient material, soybean straws were used for Armillaria bag culture medium, and the distribution ratio of raw materials was wheat bran(0–20%), corn(0–40%), and soybean straws with a particle diameter of 1–30 mm(40–100%). The materials were mixed evenly, followed by the addition of water to prepare a wet culture material with the final moisture content of 60–70. After autoclaving, it was put into a double-layer bag. The inner polypropylene bag measures 16.2 cm × 31 cm × 0.04 cm, and the outer polyethylene bag measures 16.5 cm × 34 cm × 0.04 cm. The five Armillaria isolates were inoculated into sterilized Armillaria bags, the bag mouth sealed, cultured at 22–25 °C for 15–20 min. and saved for later use. In May 2020, the field cultivation experiment was conducted, Several cultivation holes of G. elata Bl. f. glauca with a length of 50 cm, a width of 30 cm and a depth of 60 cm were first dug on the ground. Then, four tree sticks 6–9 cm in diameter and 40 cm in length, a fungus bag of different Armillaria isolates and 300 g G. elata Bl. f. glauca seeds were simultaneously placed in the cultivation holes. With each Armillaria isolate as a treatment unit, 10 cultivation holes were included in each treatment unit. In November of the same year, G. elata Bl. f. glauca in the experimental site was harvested, with the performance of random harvest for five holes per treatment unit. The fresh G. elata Bl. f. glauca was weighed and the data recorded.
Determination of effective components of G. elata Bl. f. glauca co-planted with different Armillaria isolates
Standard samples of gastrodin, p-hydroxybenzyl alcohol, balisonoside A, parisenoside B, balisonoside C and balisonoside E were purchased from Shanghai Yuanye Biotechnology Co., Ltd. The active ingredients were determined separately from G. elata Bl. f. glauca co-cultivated with different isolates of Armillaria. First wash and dry the sample with water, and it was steamed for 30 min, dried in an oven at 60 °C to a constant weight and finally made into powder. Firstly, 4 mg of each of gastrodin standard, p-hydroxybenzyl alcohol standard, balisonoside A standard, parisenoside B standard, balisonoside C standard and balisonoside E standard were weighed and fixed in a 5 mL volumetric flask with acetonitrile-water (3:97) mobile phase to make 0.800 mg-mL-1 of the control solution. The standards of gastrodin, p-hydroxybenzyl alcohol, balisonoside A, balisonoside B, balisonoside C and balisonoside E were precisely aspirated according to 2:1:2:2:1:2, and prepared as a mixed control solution containing 20 μg, 10 μg, 20 μg, 20 μg, 10 μg and 20 μg per 1 mL. The concentrations of the mixed controls were increased in a sequential gradient. The standard solutions were determined by high performance liquid chromatography (HPLC), the peak areas were calculated and the standard curves were plotted using the concentration (μg-mL-1) as the horizontal coordinate and the peak area as the vertical coordinate. Then, 2.000 g G. elata Bl. f. glauca powder was accurately weighed, and put in a conical flask with a stopper, followed by the addition of 50 mL of 50% dilute ethanol and weighed. The powder solution was extracted for 30 min (power 120 W, frequency 40 kHz) at room temperature and weighed after cooling, and dilute ethanol was used to make up for the lost mass. 15 mL solution was absorbed into the centrifugal tube with a liquid transferring gun and centrifuged at 3000 r/min for 20 min. And 10 mL supernatant was precisely absorbed into the evaporating dish, concentrated at 60 °C until no alcohol flavor, followed by the addition of acetonitrile-water (3:97) mixture to dissolve the residue. Next, the dissolved residue was transferred to a 25 mL volumetric flask, with its volume made up to scale with acetonitrile-water (3:97) mixture, shaken well, passed through a 0.220 μ m filter membrane and set aside. After that, the content of each active component was measured by HPLC, with a mobile phase of acetonitrile (A) and 0.05% phosphoric acid solution (B) (3:97). The detected wavelength was 220 nm, with a column temperature of 35 °C. The volume flow was 1.0 mL/min, with gradients of 98 to 90% B (0–10 min), 90 to 88% B (10–15 min), 88 to 82% B (15–25 min), 82% B (25–40 min), 82 to 5% B (40–42 min), 60% B (42–47 min), 40% B (47–52 min), and 97% B (52–62 min). The injection volume was 20 μ L, and the Century SIL C18 column (250 mm × 4.6 mm,5 μ m) was adopted. The content of each active ingredient in the sample was calculated from the standard curve.
Analysis of absorption Laws of nutrition elements of G. elata Bl. f. glauca co-planted with JMG
0.5 g tubers of G. elata Bl. f. glauca co-planted with each Armillaria isolate and its surrounding soil were weighed with an accuracy of 0.001 g, respectively, followed by the addition of 5.0 mL HNO3 and 1.0 mL H2O2 to the microwave digestion tank, and mixed well. And the tank lid was tightened. Then, the mixture was digested at 120 °C for 4 h. After the digestion, it was cooled and taken out. Next, the tank lid was slowly opened, the acid injected to about 0.5 mL, and then1% nitric acid was used to make up the volume to 50 mL, shaken well for later use. 1% HNO3 was utilized as the blank control solution. The processed samples were measured by inductively coupled plasma mass spectrometry (NexIONTM350X)--0.60 V; Atomization gas flow(0.90 L/min); Cooling gas flow(1.50 L/min); Auxiliary gas flow(0.80 L/min).
Analysis of Interweek variations in soil microbial community diversity around G. elata Bl. f. glauca co-planted with JMG
The commercial strain Armillaria and Changbaishan G. elata Bl. f. glauca used as the CK samples were provided by Baishan Jingzhen Gastrodia Development Co., Ltd. JMG samples are the dominant strains screened in our previous research work. Each Armillaria isolate and G. elata Bl. f. glauca were weighed and take a uniform sample of 5–10 g of soil in the same position around the circumference, three times for each sample, and put in a sterile zip-lock bag. After natural air-drying, impurities such as animal and plant residues and gravel were removed, with large samples smashed. After passing through a 2 mm sieve, the samples were aliquoted into 2 mL EP tubes, with a soil content of 0.25–0.5 g in each tube, and the sample volume was guaranteed to be 1-2 g. After aliquoting, the samples were stored in liquid nitrogen, with the collection of three replicates for each soil treatment.
Total DNA extracted from soil served as a template to amplify bacterial 16 s rDNA and fungal ITS, respectively. The PCR products were purified, quantified and homogenized by bacteria, with 338F (5 cm-ACTCCTACGGGAGGCAGCA-3′) and 806R (5 mm-GGACTACHVGGGTWTCTAA Tmer3’) as universal primers, and by fungi, with ITS 1 (F: 5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS 2 (R: 5′-GCTGCGTTCTTCATCGATGC-3′) as universal primers. PCR products were sequenced by Illumina HiSeq 2500 and USEARCH software (version 10.0)  was used to cluster high-quality sequence data with a 97% quality control level, with the adoption of Venn diagrams for displaying the number of common and unique ones among samples . The RDP classifier Bayesian model algorithm was adopted to perform the species- type classification and sequence annotation for each sample in the species sequence information database. The bacterial 16S rRNA Silva database (Release132, http://www.arb-silva.de), and the Unite fungal ITS one (Release 8.0, https://unite.ut.ee/) were utilized. QIIME software was used to generate species abundance tables at different taxonomic levels, followed by the adoption of R language tools to draw community structure diagrams and species clustering heat maps of the samples at each taxonomic level, and Python language tools were utilized to draw the phylogenetic diagram at the taxonomic level of the genus . Finally, α-diversity indexes of the samples were evaluated by QIIME2 software, and the Ace, Chao1, Shannon and Simpson indexes were calculated, respectively . The curves of the dilution, rank abundance and species accumulation reflected by Shannon diversity indexes were plotted by Mothur software and R language tools . The Unweighted paired average method (UPGMA) was adopted to cluster the samples, and plot their heatmap, and then the similarity of species composition among them was judged through R language tools . ANOVA and Linear discriminant analysis Effect Size (LEfSe)  were applied to the differential analysis between groups. The PICRUSt2  software was adopted for annotating the species in feature sequences to be predicted and the existing phylogenetic tree. And the IMG microbial genome database was used for functional information output to infer the composition of functional genes in the samples, and in turn analyze functional differences between different samples or groups.
Statistical analysis of all data was performed on significance at a significance level of 0.05 by one-way ANOVA and LSD and Duncan’s test.
Availability of data and materials
The sequence data generated in this study can be obtained in NCBI database through login numbers MW404994(Armillaria gallica isolate 1 internal transcribed spacer 1, partial se - Nucleotide - NCBI (nih.gov)), MW404995(Armillaria sinapina isolate 2 small subunit ribosomal RNA gene, partia - Nucleotide - NCBI (nih.gov)), MW404996(Armillaria sinapina isolate 3 small subunit ribosomal RNA gene, partia - Nucleotide - NCBI (nih.gov)), MW404997(Armillaria sinapina isolate 4 internal transcribed spacer 1, partial s - Nucleotide - NCBI (nih.gov)) and MW404998(Armillaria sinapina isolate 5 internal transcribed spacer 1, partial s - Nucleotide - NCBI (nih.gov)). All data analyzed during this study were included in this manuscript and its additional files.
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We are grateful to Zhaochun Li, the manager of Baishan Jingzhen Gastrodia Development Co., Ltd. for experimental materials. Also, we thank BMK Cloud (www.biocloud.net) for providing an analysis platform.
This study was supported by the Science and Technology Development Program of Jilin Province (Grant No. 20210401093YY, 20190301079NY). These funders had no role in study design, data collection and analysis, and interpretation of data or in writing the manuscript.
Ethics approval and consent to participate
The Armillaria isolated and identified in this study was provided by our laboratory, and the author has the right to use it without ethical and legal issues. The commercial strain Armillaria used was purchased from Jingzhen Gastrodia Development Co., Ltd. (Baishan City, Jilin Province, China). The laboratory experiments were conducted in accordance with relevant legislations and under permissions. All methods were performed in accordance with relevant guidelines and regulations in this study.
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The authors declared that there is no conflict of interest.
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Additional file 1. Table S1.
Growth characteristics of five Armillaria isolates under the same culture conditions.
Additional file 2. Table S2.
Homology alignment of five Armillaria isolates in GenBank.
Additional file 3. Table S3.
Yield of different Armillaria isolates co-planted with Gastrodia elata Bl. f. glauca.
Additional file 4. Table S4.
Element contents and enrichment factors of Gastrodia elata Bl. f. glauca and soil under different treatments.
Additional file 5.
Table S5. GenBank accession numbers of different species.
Additional file 6.
Table S6. Table of fungal and bacterial alpha diversity indices.
Additional file 7.
Fig. S1. Detection of 6 sample solutions by high performance liquid chromatography.
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Yu, E., Gao, Y., Li, Y. et al. An exploration of mechanism of high quality and yield of Gastrodia elata Bl. f. glauca by the isolation, identification and evaluation of Armillaria. BMC Plant Biol 22, 621 (2022). https://doi.org/10.1186/s12870-022-04007-8
- Gastrodia elata Bl. f. glauca S. chow
- Separation and identification
- Nutrition elements
- Microbial diversity