Discovery of seed germinating fungi (Mycetinis scorodonius) from Gastrodia elata Bl. f. glauca S. chow in Changbai Mountain and examination of their germination ability

Multi-generational asexual reproduction of Gastrodia elata Bl. will cause seedling species degeneration. Sexual reproduction of Gastrodia elata Bl. seed is an effective method to solve the problem of degeneration. The development of Gastrodia elata Bl. seeds cannot be separated from the germination fungus. However, there are few strains of germination fungus in production, and there is also the problem of species degradation in application for many years. It is very important for the sexual reproduction of Gastrodia elata Bl. to isolate more new strains of excellent germination fungus from the origin. This study used the Gastrodia elata Bl. f. glauca S. chow seeds germination vegetative propagation corms capture method to isolate its symbiotic germination fungus, and comprehensively identified the species of germination fungus by colony morphology, ITS, sporocarps regeneration and germination function, and compared the growth characteristics and germination ability with other germination fungus (Mycena purpureofusca, Mycena dendrobii and Mycena osmundicola). The germination fungus was isolated from the vegetative propagation corms of Gastrodia elata Bl. f. glauca S. chow seeds and named GYGL-1. After comprehensive identification, GYGL-1 was Mycetinis scorodonius. Compared with other germination fungus, GYGL-1 has fast germination speed, vigorous growth, and high germination ability for Gastrodia elata Bl. f. glauca S. chow seeds. Innovated the isolation method of Gastrodia elata Bl. seeds germination fungus, obtained the regenerated sporocarps of the germination fungus, and discovered that Mycetinis scorodonius has a new function of germinating Gastrodia elata Bl. f. glauca S. chow seeds, enriching the resource library of Gastrodia elata Bl. germination fungus.

Gastrodia elata Bl. is a bulk medicinal material, which is well-known in Jilin Province, China.It has traditional effects such as relieving wind and convulsions, calming the liver, dispelling wind, and dredging collaterals 1 .G elata Bl. also has modern pharmacological effects, such as antidepressant 2,3 , anticonvulsant 4,5 , anti-aging 6,7 , and neuroprotective effects 8,9 , with a thousand years of medicinal use.In 2019, the Chinese government announced the categorization of G elata Bl. as a "food and drug substance, " and the demand is increasing 10 .However, there is a serious shortage of wild G elata Bl. resources 11 .An effective way to solve the contradiction between supply and demand of G elata Bl. is artificial cultivation, and reproduction is the key to artificial cultivation.
Methods for reproduction of G. elata Bl. includes asexual and sexual reproduction.Asexual reproduction takes a short time to produce immature tuber seedlings, but multiple generations of asexual reproduction will cause degradation of immature tuber seedlings, resulting in thin, deformed and yellowish-brown G. elata Bl., slow growth, poor hyphal ability, easy to rot, yield reduction or even no harvest, etc. 12,13 .Compared to asexual reproduction, sexually reproducted immature tuber seedlings have strong resistance to adversity and vigorous vitality, and its cultivation of high quality and high yield of G. elata Bl., is currently an effective means of solving the degradation of G. elata Bl. 14 .As shown in Fig. 1, sexual reproduction involves at least nine steps: bolting, flowers, pollination, capsules, seeds, protocorms, vegetative propagation corms, immature tubers, mature tubers, and other growth and development stages 15 .In the process of sexual reproduction, the germination ability of seed is the most critical, but G. elata Bl. seeds are tiny, without endosperm and other nutrient storage organs, and must rely on nutrients supplied by germination fungus (Mycena osmundicola, Mycena dendrobii) to provide nutrients after infesting the embryo cells in order to germinate 16,17 .
Gastrodia elata Bl. f. glauca S. chow originates from northeast China and is one of the six varieties of G. elata Bl.It is a perennial heterotrophic herbaceous plant of the Orchidaceae family and is also an obligate fungal heterotrophic plant 18 .G. elata Bl. has different living habits in different producing areas, and the suitable germination fungus for the germination of G. elata Bl. seeds in different producing areas are also different.Therefore, it has become inevitable to isolate excellent strains suitable for the germination of local G. elata Bl.However, because the germination fungus are weak saprotrophs, their slow growth and difficult culture make isolation and identification more difficult 19 .Few strains were isolated and identified, mainly including six types of Mycena osmundicola, Mycena dendrobii, Mycena anoectochila, Mycena purpureofusca, Mycena abramsii and Mycena citrinomarginata [20][21][22][23][24][25] .After being used for many generations, the viability of fungi such as germination fungus were significantly reduced, resulting in a reduction in the germination rate of G. elata Bl. seeds or even no germination at all 26 .Obviously, it has become inevitable to isolate more and better germination fungus resources.In particular, the isolation of strains of origin suitable for germination of the G. elata Bl. f. glauca seeds is more important to ensure the yield and quality of G. elata Bl.
Therefore, the following studies were carried out: firstly, based on the characteristic that the germination of G. elata Bl. f. glauca seeds to grow into vegetative propagation corms depends on the symbiosis of germination fungus, the germination fungus were captured using the vegetative propagation corms germinated from G. elata Bl. f. glauca seeds sown in the wild as carriers.Secondly, the germination fungus was isolated and purified

Bl. f. glauca seeds
Took the vegetative propagation corms of G. elata Bl. f. glauca collected in 2.2, washed them three times with sterile water, and blotted them dry with sterile filter paper.Soaked the vegetative propagation corms in 75% ethanol for 1 min and then washed them three times with sterile water.Cut the collected vegetative propagation corms into 1 × 1 mm small pieces, put a small piece of vegetative propagation corm together into a 1.5 ml centrifuge tube filled with sterile water, shook at 120 rpm for 10 min, mixed well, and diluted it into 10 3 , 10 4 , and 10 5 times of the extraction solution respectively.Used an inoculating loop to dip the extracts of different dilution ratios inoculate into PDA slant medium and cultured them at 25 °C.Colonies will grow out in about 1-3 days.

Molecular identification of isolated germination fungus from G. elata Bl. f. glauca seeds
Took the purified germination fungus GYGLs, used the Fungal DNA Extraction Kit (Solarbio, Beijing, China) to extract strain DNA, and stored the extracted DNA at − 20 °C for the following experiment.PCR amplification and analysis of the ITS region of GYGLs DNA were conducted using fungal universal primers ITS1 (5ʹ-TCC GTA GGT GAA CCT GCG G-3ʹ) and ITS4 (5ʹ-TCC TCC GCT TAT TGA TAT GC-3ʹ).The PCR conditions were described as follows: a total of 25 μL volume including Taq PCR Master Mix, 12.5 μL; DNA template, 1 μL; ITS1, 1 μL; ITS4, 1 μL; ddH 2 O, 9.5 μL.The PCR conditions included initial denaturation (94 °C, 4 min), followed by 30 cycles of denaturation (94 °C, 30 s), annealing (55 °C, 30 s), extension (72 °C, 50 s), and the final extension was performed at 72 °C for 10 min.The amplified PCR product was detected by electrophoresis on a 1% agarose gel, and samples with a single, clear, bright band were subjected to sequencing (Sangon, Shanghai, China).To determine the biological classification status, the sequencing results were compared with NCBI data to identify the fungus with the highest similarity.The top 50 sequences with the highest homology were selected for phylogenetic tree construction using MEGA 7.0 software and the Maximum Likelihood method with 1000 bootstrap repeats.
After inoculation, placed it in a culture room at a constant temperature of 22-25 °C with light shielding for about 25-30 days (Fig. S2).After the GYGL-1 mycelium grew all over the bag, stimulated it at low temperature (8-10 °C) for 14 h.Then cut a 10 cm small opening in the plastic film of the bag to expose the mycelium and cultured it at 25 °C for 15 days to observe the morphology of the sporocarps and determine the species of GYGL-1.

Germination function identification of GYGL-1
First, put some sterilized soil (20 g, thickness 1-1.5 cm) into the culture bottle (6 cm × 9.5 cm × 3.5 cm), and the G. elata Bl. f. glauca seeds (1 mg) were stirred well with the germination fungus (10 g) cultivated in the previous step and spread on the soil, and the germination fungus mixed with G. elata Bl. f. glauca seeds were covered with a layer of soil (20 g), and the process was carried out under aseptic conditions, and repeated in 6 bottles.Cultivated at a constant temperature (25 °C) for 8 weeks, to observe whether the G. elata Bl. f. glauca seeds had germinated into vegetative propagation corms.

Comparison of growth rate between the GYGL-1 and other germination fungus
In order to screen for excellent germination fungus, different strains were cultured comparatively.First, the M. purpureofusca, GYGL-1, M. dendrobii and M. osmundicola, which had been frozen at − 80 °C, were inoculated on PDA culture medium and cultured at a constant temperature of 25 °C.The time required for germination was calculated from the day of inoculation until the appearance of tiny mycelium.Each treatment was repeated 6 times.Secondly, statistical analysis was performed on the growth rates of different germination fungus on PDA culture medium and fungal bag culture medium.On the PDA medium, starting from the day of germination of the germination fungus, a vernier caliper was used to to measure the spreading distance (s) at the edge of the colony at the same time every day (t), and the growth rate (v = s/t) was calculated.On the fungal bag culture medium, the time until the mycelium grew over the fungal bag was recorded starting from the day of inoculation of the germination fungus.Each treatment was repeated 6 times.
Finally, 1 ml of fungal liquid in different logarithmic growth phases of the above four germination fungus (the fungus ball was broken with a magnetic stirrer, and the OD value were adjusted to 0.6) was taken.Inoculated into 30 ml of PDB medium, incubated at 120 rpm and 25 °C in the dark for 15 days, and the fungus ball was weighed after 15 days.Each treatment was repeated 6 times.

Effect of GYGL-1 and other germination fungus on the germination ability of G. elata Bl. f. glauca seeds
In order to evaluate the germination ability of four different strains on G. elata Bl. f. glauca seeds, the M. purpureofusca, GYGL-1, M. dendrobii and M. osmundicola were cultured with G. elata Bl. f. glauca seeds respectively.The culture method was the same as 2.6 and was repeated for twelve bottles.Six culture bottles were selected at the fifth week of culture, and five randomly selected fields of view under a dissecting microscope were used to record the number of protocorms that had germinated from G. elata Bl. f. glauca seeds and the number of seeds that did not germinate, and to calculate the G. elata Bl. f. glauca seed germination rate (the number of germinated www.nature.com/scientificreports/seeds as a percentage of the total number of seeds).In the eighth week of cultivation, the number and weight of vegetative propagation corms germinated from the remaining six bottles of G. elata Bl. f. glauca seeds were recorded and measured to evaluate their germination ability.

Effect of GYGL-1 and other germination fungus on the germination ability and seedling growth of G. elata Bl. f. glauca seeds sown in farmland
Took the M. purpureofusca, GYGL-1, M. dendrobii, M. osmundicola and G. elata Bl. f. glauca seed, mixed them at a ratio of 1 mg of seeds and 10 g of germination fungus, and then sowed them in farmland according to conventional production methods.Each treatment was designed with six replicates, each with an area of 45 m 2 (Fig. S3).The amount of sowing mixed seeds and germination fungus was 1.5 kg/m 2 , and the amount of A. gallica was 2.5 kg/m 2 .On the fifth week after planting, 60 g of soil where G. elata Bl. f. glauca seeds had been sown was randomly collected from each replicate, and five fields of view were randomly selected under a dissecting microscope to calculate the germination rate of G. elata Bl. f. glauca seeds as described above.In the eighth week after planting, 60 g of soil sown with G. elata Bl. f. glauca seeds was collected in each repetition, and the vegetative propagation corms were collected and tested for number and weight to evaluate the germination abality of each strain on G. elata Bl. f. glauca seeds.At the 18th month after planting, the soil sown with G. elata Bl. f. glauca seeds was collected in 1 m 2 of each replicate, and immature tuber seedlings were collected, and tested for number and weight of immature tuber seedlings to evaluate the effect of each strain on immature tuber seedlings.Farmland sowing G. elata Bl. f. glauca seed specific steps: in the farmland, dug a length of 30 m, 1.5 m wide, 7-8 cm deep long pit, in the bottom of the pit laid a layer of cut through the mouth of the fish scale mouth of the Quercus mongolica (diameter 5-6 cm, length 15 cm), spacing 2-3 cm, filled the gaps with soil.Placed A. gallica block at both ends and at the mouth of the fish scale (medium formula: 50% wood chips (Quercus mongolica), 25% corn, 10% cottonseed husk, 8% bran, 5% soybean meal, 1% gypsum and 1% lime, autoclaved at 121 °C for 2 h and then cooled naturally).Covered the A. gallica blocks with a layer of soil 2 cm thick.Evenly placed the germination fungus mixed with G. elata Bl. f. glauca seeds on top.Covered the germination fungus with a layer of 2 cm thick soil, and evenly placed the Quercus mongolica (3-4 cm in diameter, 15 cm long) on top of the soil, and placed A. gallica blocks in the gaps, filled with a layer of soil with a thickness of 7 ~ 8 cm, and compacted the soil slightly.Covered it with 7-8 cm thick Quercus mongolica leaves and covered it with a shade net.

Approval for experiments
Experimental and field research on plants (cultivated or wild) in the manuscript, including the collection of plant materials, in accordance with relevant institutional, national, and international standards and legislation.

Results and analysis
Results of the isolation of seed germination fungus from G. elata Bl. f. glauca seeds Eleven vegetative propagation corms that were symbiotic with the germination fungus were successfully obtained using G. elata Bl. f. glauca seeds (Fig. 3).Eleven strains with single colonies were isolated and screened from the vegetative propagation corms, and three strains were selected that conformed to the morphological characteristics of germination fungus (the germination fungus colonies were concentric circles, the mycelium was white and fluffy, the aerial roots were developed, and the middle was dense, with sparse edges and neat outlines), early germination (mycelium began to germinate 48-72 h after inoculation), and fast growth rate, and were named GYGL-1, GYGL-2, and GYGL-3 respectively.

Results of the regenerated sporocarps identification of GYGL-1
After cultivation in a fungal bag, GYGL-1 regenerated sporocarps were successfully obtained (Fig. 5A).As depicted in Fig. 5B, the regenerated sporocarps of GYGL-1 were relatively small, with a cap diameter ranging from 0.5 to 3 cm.The cap had a flat hemispherical to spreading shape, initially with rolled-in margins that later stretched out.The surface appeared dry and could range from smooth to slightly undulating, displaying shades of yellowish-brown to brownish coloration.The fungus flesh was thin, the gills were straight to nearly free, the density varies from dense to sparse, and the lengths were also different.The stipe was 1.5-6 cm long, 0.05-0.8cm thick, light yellow to brown, tapering at the lower part, cylindrical or flattened, dry, smooth, brittle to hard, consistent with the characteristics of the fruiting body of Mycetinis scorodonius.
The PCR amplified fragment of the DNA of the regenerated sporocarps of GYGL-1 was about 750 bp (OR418367) (Fig. S6), and its base sequence was the same as that of GYGL-1 (Fig. S7).It was further confirmed that the isolated GYGL-1 was My. scorodonius.

Identification results of the germination function of GYGL-1
After eight weeks of co-culture between GYGL-1 and G. elata Bl. f. glauca seeds, the vegetative propagation corms of G. elata Bl. f. glauca could be sprouted, proving that GYGL-1 had the function of promoting the germination of G. elata Bl. f. glauca seeds (Fig. 6).

Comparative results of GYGL-1 and other germination fungus on seed germination ability and seedling growth of G. elata Bl. f. glauca
Comparing GYGL-1 with M. purpureofusca, M. dendrobii, and M. osmundicola, there were significant differences in the effects of GYGL-1 on the protocorms (germination rate) and vegetative propagation corms (number and weight) of indoor cultured G. elata Bl. f. glauca seeds.GYGL-1 was significantly higher than the other three   germination fungus (P < 0.05) (Table 2, Fig. 8-1).There were significant differences in the effects of GYGL-1 on the protocorms (germination rate), vegetative propagation corms (number and weight) (Table 2, Fig. 8-2) and immature tuber seedlings (number and weight) cultured in the field of G. elata Bl. f. glauca seeds.GYGL-1 was also significantly higher than the other three germination fungus (P < 0.05) (Table 2, Fig. 8-3).It indicated that GYGL-1 was more suitable for the germination of G. elata Bl. f. glauca seeds in the Changbai Mountain region.

Discussion
G. elata Bl. has an extremely high medicinal value; therefore, its demand shows an upward trend.Due to overexploitation, the number of wild G. elata Bl. in China is declining, and the species is currently classified as "vulnerable" by the International Union for Conservation of Nature.Environmental conditions are the basis of plant reproduction and critical factors that control seed germination 27 .Environmental factors are the most important factors influencing the growth of G. elata Bl., including altitude and precipitation 28 .Therefore, different regions cultivate G. elata Bl. in different geographical locations and climates 28,29 , resulting in different varieties of germination fungus.In the cultivation of G. elata Bl., the commonly used germination fungus includes M. osmundicola and M. dendrobii, but they were all isolated and identified in central and southern China.These strains are unstable and severely degraded after multiple generations of reproduction in northeast China.It is particularly important to isolate new strains that promote the germination of G. elata Bl. seeds.The germination fungus isolated and identified in northeast China that can promote the germination of G. elata Bl. f. glauca seeds are more conducive to the production of G. elata Bl. f. glauca 25 .The Changbai Mountain region, where our experimental materials were collected, has significant regional characteristics because it is located at mid-to-high latitudes and is very sensitive to global climate change 30,31 .The affinity of the isolated germination fungus to G. elata Bl. f. glauca is more stable.Over the past two decades, isolation methods for germination fungus have mainly consisted of finding sporocarps, wild protocorms isolation, and seed-capture protocorms, each with its advantages and disadvantages.Finding the sporocarp method, the technical steps are simple and quick 32 .However, the strains found in this way are accidental.Most strains do not have the function of germinating seeds and are inefficient.The results showed that few germinated seeds were found, which is not satisfactory for downstream production.Most fungi isolated by identifying wild protocorms can be used for seed germination.Compared with the the method of searching for sporocarps, the method of searching for wild protocorms is used to isolate germination fungus.Most of the isolated strains have the function of seed germination 33 .However, the protocorms are small and difficult to find, and the search process is very difficult.The method of capturing protocorms from seeds with well-defined targets has been widely used 34 .However, few strains isolated by this method can development of protocorms into vegetative propagation corms.This study uses a new method, which is to use seed-germinated vegetative propagation corms to capture germination fungus.This method is easy to find the vegetative propagation corms and has a high success rate in isolating germination fungus.Protocorms can develop into vegetative propagation corms, indicating that the germination fungus isolated by this method have higher germination ability for G. elata Bl.
Mycena (Pers.)Roussel, with approximately 600 species distributed worldwide, is one of the largest genera in Agaricales 35 .To date, fewer than 100 species of Mycena have been documented in China 36 ,among them, only a few strains can be used for G. elata Bl. cultivation, including M. osmundicola, M. dendrobii, M. purpureofusca, etc.At present, our understanding of germination fungus is limited, and we have not yet identified any strains that can be used for the cultivation of G. elata Bl., with the exception of Mycena spp.In this study, we identified an effective germination fungus.Through morphology, molecular biology, sporocarps regeneration and functional identification, we identified the strain as My.scorodonius.This strain is reported for the first time to have the function of germinating G. elata Bl. f. glauca seeds.It is of great significance to the production of G. elata Bl.
In this study, My. scorodonius regenerated sporocarps were obtained using fungal bag culture.The germination fungus was identified through the regenerated sporocarps, which is not common among other isolated and identified strains.At the same time, due to the strong garlic odour of My. scorodonius sporocarps, it is useful for the development of My. scorodonius spices 37,38 .Compared with other current G. elata Bl. seed germination fungus, My. scorodonius requires the shortest germination time after cryopreservation, has a fast mycelium growth rate, high mycelium biomass, and high germination ability for G. elata Bl. f. glauca seeds, indicating that My. scorodonius can shorten the growth time of G. elata Bl. and increase the yield of G. elata Bl.
Some studies have shown the dynamics of the fungal community during the growth of G. elata Bl.It is believed that the growth of G. elata Bl. is also related to a variety of fungi other than Mycena 39 .These fungi change with the growth stages of G. elata Bl., such as Resinicium and Campanella 40,41 .However, these studies only analyzed the correlation between microbial species and G. elata Bl. germination, which are possible results of speculation, and there is still a lack of direct evidence.And there are many kinds of strains in the Mycetinis family.Just like the Mycena family, not all strains have the function of germination G. elata Bl. seeds.The specific strain has not been identified, and isolation and identification and germination function verification are still needed.
Although this study has done some meaningful work, there is still a lot of related and interesting research that needs to be carried out.For example, through multi-omics analysis, the interaction mechanism between G. elata Bl. seeds and germination fungus is analyzed, and how G. elata Bl. seed germination sends out signals for interaction with fungi.By comparing the germination fungus and non-germination fungus of the Mycena family, we can find out the substances that make G. elata Bl. seeds germinate from the germination fungus.To elucidate the degradation mechanism of multi-generation asexual reproduction of germination fungus and to study the regulation technology by comparing different generations of germination fungus in artificial culture.Through microbial isolation and identification and tieback experiments, we will verify whether the germination fungus is endophytic fungi of G. elata Bl. and whether they can colonize G. elata Bl.Isotope tracer techniques and molecular labelling techniques are used to investigate how germination fungus are spread in nature (spores, mycelium and G. elata Bl.).In this study, the combination of germination fungus and A. gallica promoted the growth of vegetative propagation corms of G. elata Bl.It inspired us to screen the different family of Mycena and Mycetinis that can be symbiotic strains by confrontation experiments, and to evaluate whether the effect of their combination on the germination of G. elata Bl. seeds can be enhanced by the germination seed experiments.The same method can also be used to study the effect of mixing germination fungus of the same family on the germination of G. elata Bl. seeds.All are good research directions.

Figure 1 .
Figure 1.Sexual reproduction life history of G elata Bl.(The red arrow marks the stage that requires germination fungus).

Figure 3 .
Figure 3. Eleven strains with single colonies were isolated and screened from the vegetative propagation corms.(A and B) Vegetative propagation corms grew in the sowing field; (C) eleven strains with single colonies had been isolated and screened from the vegetative propagation corms (the arrows indicated three strains that conformed to the morphological characteristics of germination fungus).

Figure 4 .
Figure 4. ITS sequence alignment and phylogenetic tree analysis.(A) The alignment results between the base sequence obtained from sequencing and the My.scorodonius sequence in NCBI (JN943593; JN943594; JQ272364; KJ817281); (B) The phylogenetic tree of the top 50 sequences with the highest homology.

Figure 7 .
Figure 7.Comparison of GYGL-1 and other germination fungi.1: Compared germination time after cryopreservation; 2: Compared mycelium growth rate on PDA medium; 3: Compared the weight of fungus ball in PDB medium; 4: Compared the time required for the fungus bag medium to be covered with the mycelium (A, GYGL-1; B, M. purpureofusca; C, M. osmundicola; D, M. dendrobii).

Figure 8 .
Figure 8.Comparison of GYGL-1 and other germination fungus on seed germination ability and seedling growth.1: The effect of GYGL-1 and other germination fungus on the germination of indoor-cultured G. elata Bl. f. glauca seeds into vegetative propagation corms; 2: The effect of GYGL-1 and other germination fungus on the germination of field-grown G. elata Bl. f. glauca seeds into vegetative propagation corms; 3: The effect of GYGL-1 and other germination fungus on the germination of field-grown G. elata Bl. f. glauca seeds into immature tuber seedlings.(A) M. purpureofusca; (B) GYGL-1; (C) M. dendrobii; (D) M. osmundicola.

Table 2 .
Comparison of GYGL-1 and other germination fungus on seed germination ability and seedling growth.Data with the different letters in the same row are significantly different (P < 0.05).