Fritillaria Cirrhosae Bulbus is an antitussive and expectorant traditional Chinese herbal medicine that has been widely used for more than 2000 years. It is primarily used to treat coughs with less phlegm, sputum with blood, bronchitis with a dry cough, and pulmonary carbuncle .
Fritillaria Cirrhosae Bulbus is derived from bulbs of source plant species, including Fritillaria cirrhosa D. Don, F. unibracteata Hsiao et K. C. Hsia, F. przewalskii Maxim., F. delavayi Franch., F. taipaiensis P. Y. Li, and F. unibracteata Hsiao et K. C. Hsia var. wabuensis (S. Y. Tang et S. C. Yue) Z. D. Liu, S. Wang et S. C. Chen . These species are primarily distributed at an altitude of 3,200-4,600 m in areas in the eastern Himalaya–Hengduan Mountains, Qinghai–Tibet Plateau, and they inhabit a narrow geographic range and restricted habitat [2,3]. Excessive exploitation of Fritillaria Cirrhosae Bulbus source plants and deterioration of the ecological environment have resulted in continuous reduction and gradual depletion of the limited plant resources and there are difficulties in artificial propagation of these plant species [4,5]. As a result, conflict between exploitation and protection of these plant resources increases.
Endophytic fungi of medicinal plants are generally a significant origin of active ingredients in medicinals [6,7], and it was revealed that a few Fusarium isolates in bulbs of F. unibracteata and F. unibracteata wabuensis were able to produce the same imperialine 3β-D-glucoside, peiminine, and peimisine as their host plants [8-11]. Additionally it was reported that the alkaloid content reduced when the diversity of rhizospheric fungi community increased in F. taipaiensis . Bulb-associated fungi communities may represent a critical origin of the active alkaloid substances in bulbs of Fritillaria Cirrhosae Bulbus source plants. Thus, increasing conflict between natural resource conservation and demand for Fritillaria Cirrhosae Bulbus source plants as medicinal material may be relieved by the exploitation of their bulb-associated fungi resources in the future.
Previous studies conducted by cultural method revealed diversity of cultured endophytic fungi in bulbs of Fritillaria Cirrhosae Bulbus source plants. Yan et al.  isolated 90 fungal species assigned to one class, six orders, and 30 genera from F. cirrhosa in four different growth periods, which included Stachylidium, Curvularia, Verticillium, Cephalosprium, and Fusarium species. Chen  and Chen et al. [14,15] cultured fungal species of Gliocladium, Myrothecium, Bionectria, Nectria, and Fusarium from F. unibracteata, F. przewalskii, F. cirrhosa, and F. delavayi. Pan et al.  obtained 53 fungal isolates of Fusarium, Plectosphaerella, Nectria, and Bionectria from F. wabuensis. We acquired 362 fungal isolates that belonged to 57 genera in Ascomycotina, Basidiomycotina, and Zygomycotina in bulbs of Fritillaria cirrhosa, F. unibracteata, F. przewalskii, and F. delavayi (unpublished data).
The cultural method is commonly used to identify fungal species associated with plants, but alone it cannot systematically reveal the fungal diversity. A combination of cultural and molecular methods may provide more comprehensive results that reveal species diversity of plant-associated fungi, because both culturable and non-culturable fungi can be identified Additionally, community composition of plant-associated fungi should be influenced by genotype, geographic area and phenology of host plants. As a result, fungal diversity and community composition associated with bulbs of Fritillaria Cirrhosae Bulbus source plants were studied relative to host plant species, primary geographic areas, and crucial plant phenology stages by the internal transcribed spacer (ITS) rDNA sequencing technique in this research, which can provide insight into exploitation of potential fungal resources, and conservation and sustainable development of Fritillaria Cirrhosae Bulbus source plant natural resources in the eastern Himalaya–Hengduan Mountains, Qinghai–Tibet Plateau, China.
Materials and Methods
Fritillaria cirrhosa (Fc), F. unibracteata (Fu), F. przewalskii (Fp), and F. delavayi (Fd), four representative Fritillaria Cirrhosae Bulbus source plants, were collected in 2014‒2016 from the eastern Himalaya-Hengduan Mountains, southwestern China at Weixi (WX), Yunnan (alt. 3,200 m); Songpan (SP), Sichuan (alt. 3,300 m); Yuzhong (YZ), Gansu (alt. 3,500 m), and Kangding (KD), Sichuan (alt. 4,500 m). The average annual temperature is 3℃, the highest temperature is 25.6℃, and the lowest temperature is -25.8℃ in these areas; average annual precipitation is 784.9 mm [18,19]. Dominant flora in these areas are north temperate plant communities composed of Rhododendron, Spiraea, Sibiraea, Saussurea, Potentilla, Salix, and Anaphalis species . Three populations of each plant species in the same geographic area and at the same plant phenology stage were sampled, and one plant was sampled from each population, which resulted in a total of 24 samples for the four plant species (Supplementary Table S1).
Then, F. cirrhosa samples were collected in four geographic areas where it is primarily distributed: WX, Yunnan (alt. 3,200 m); Huadianba (HDB), Yunnan (alt. 3,200 m); Qiaojia (QJ), Yunnan (alt. 3,800 m); and Leiwuqi (LWQ), Xizang (alt. 3,900 m). Three populations of each geographic area at the same plant phenology stage were sampled, and one plant was sampled from each population, which resulted in a total of 24 samples for the four geographic areas (Supplementary Table S1). Among them, F. cirrhosa samples from WX were collected at four representative phenology stages with rapid metabolism, which included 2-yr-olds in the mature period (2-MP), 2-yr-olds in the germination period (2-GP), 4-yr-olds in the flowering period (4-FP), and 4-yr-olds in the germination period (4-GP). Plants of each phenology stage were sampled from three constant populations in the same geographic area, and one plant was sampled from each population, which resulted in a total of 12 samples for the four plant phenology stages (Supplementary Table S1). Bulbs of plants were excavated and stored in the original soil at 4℃ for further treatment as soon as possible.
Molecular identification of bulb-associated fungi
Bulb samples were surface cleaned before molecular identification, which was based on sequence analysis of the ITS rDNA region. DNA was extracted following the procedures described by Gao and Yang . ITS rDNA was amplified using primer combinations of ITS1F×ITS4, ITS5×ITS4, and ITS1F×LR1 . Amplified products were purified and ligated into TaKaRa pMD18-T plasmids (TaKaRa Bio Inc., Shiga, Japan), the cloned plasmids were transformed into Escherichia coli strain DH5α. For each bulb, about 100 clones were arbitrarily selected for PCR amplification and automatically sequenced with an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA; Sangon Biotech Co., Ltd., Shanghai, China).
Sequences were sorted into operational taxonomic units (OTUs) at a sequence similarity of ≥97% across ITS rDNA  using DOTUR . BLASTn analysis of the representative sequence of each OTU was carried out against the GenBank database for preliminary identification. ITS matches for taxa in National Center for Biotechnology Information’s GenBank were defined as corresponding to at least 90% of the sequence length with a sequence similarity of 97-100% and e-values below e-100 at the species level, 90-97% sequence similarity and e-values below e-90 at the genus level, and 80-90% sequence similarity and e-values below e-80 at the order level.
Generated sequences were deposited in GenBank under accession numbers MK405809‒MK405831, MK405833‒MK406272, MK406275‒MK406310, MK406312‒MK407528, MK407530‒MK407667, MK407669‒MK407694, and MK407696‒MK408161.
Statistical analysis of fungal diversity and community distribution
The evaluation was conducted on species diversity and community distribution of bulb associated fungi. Rarefaction curve analysis of the number of fungal individuals and fungal OTUs relative to sample size was carried out for the entire fungal community of all plant species from different geographic areas and different plant phenology stages using R (Version 2.15.3, )  with observed index and Chao1 index. OTU frequency was defined as the number of ITS rDNA sequences of the OTU. Diversity measures including Chao1 and ACE estimators, and Shannon and Simpson indexes were calculated for fungal OTUs associated with each host plant species, geographic area, and plant phenology stage using Qiime (Version 1.7.0, ) . A Kruskal–Wallis (KW) rank-sum test of diversity indexes based on Chao1, Shannon, and Simpson distances, and permutational multivariate analysis of variance (PerMANOVA) of OTU frequency based on Bray–Curtis and Jaccard distances were performed using Mothur (, mothur.org/wiki/Download_mothur/) ; diversity indexes and OTU frequency used as dependent variables, and “Host plant species,” “Geographic area,” and “Plant phenology stage” as independent variables. The significance level was set to p=0.05.
Diversity of bulb-associated fungi of Fritillaria Cirrhosae Bulbus source plants
A total of 1,486 fungal sequences (excluding host plant-contaminated sequences and failed sequences) generated from the four plant species sampled in different geographic areas and at different plant phenology stages were assigned to 251 OTUs and belonged to 12 classes, 32 orders, 51 families, and 99 genera based on primary molecular identification (Supplementary Table S2 and Table S3). Of the OTUs, 33% (84) were preliminarily recognized at the species level, with reference sequences of taxa with ≥97% similarity found in GenBank. Most of the fungal OTUs (96.41%) were Ascomycotina, 3.52% were Basidiomycotina, and 0.07% were Zygomycotina, and were assigned to 90, nine, and three lineages, respectively. OTU richness was high in Fusarium (33 OTUs), Ilyonectria (17 OTUs), Tomentella (14 OTUs), Tetracladium (12 OTUs), and Leptodontidium (10 OTUs), followed by Cladosporium (seven OTUs), Trichoderma (six OTUs), Neonectria (five OTUs), Gibberella (four OTUs), and Phoma (four OTUs) (Supplementary Table S2 and Table S3). For relative frequency, Sordariomycetes (74.53% of the total sequences) dominated at the class level, Hypocreales (64.88%) dominated at the order level, Nectriaceae (46.21%) dominated at the family level, and Fusarium (39.91%) and Ilyonectria (14.83%) dominated at the genus level (Fig. 1 and Supplementary Fig. S1); Fusarium sp. 03 (frequency = 516), Ilyonectria rufa (187), Fusarium sp. 08 (222), and Ilyonectria sp. 03 (137) were the most frequent OTUs (Fig. 2). The fungal community primarily comprised frequent OTUs and fewer rare OTUs, with 16% of the total OTUs having a cumulative frequency ≥10, 53% having a frequency ≥3, and 39% detected as singletons (Supplementary Table S2 and Table S3). The rarefaction curve did not approach an asymptote with increasing sequencing size, which revealed that a saturated sequencing depth that would cover all fungal OTUs in the samples was not reached (Supplementary Fig. S2); this indicates a need for enhanced sequencing.
Community distribution of bulb-associated fungi of Fritillaria Cirrhosae Bulbus source plants
For the four host plant species, 92 fungal OTUs were detected in Fc, 15 OTUs were unique for this plant, OTU richness was highest in Tetracladium, and three Fusarium OTUs were the most frequent. Thirty four of the 74 OTUs were unique in Fd, OTU richness was highest in Fusarium and one Ilyonectria OTU was the most frequent. Two unique OTUs were detected out of 27 in Fp, OTU richness was highest in Fusarium, and one Truncatella OTU was the most frequent. Nine of the 20 OTUs were unique for Fu, OTU richness was highest in Fusarium, and two Fusarium OTUs were the most frequent (Supplementary Fig. S1 and Table S3). Eight OTUs were shared by two plants, and one Fusarium OTU was shared by four plants (Fig. 3 and Supplementary Table S2).
For F. cirrhosa collected from four geographic areas, 12 of the 94 detected OTUs were unique for WX, OTU richness was highest in Tetracladium, and three Fusarium OTUs were the most frequent. Twelve of the 53 OTUs were unique in HDB, OTU richness was highest in Fusarium, and one Volutella OTU was the most frequent. For 25 common OTUs in QJ, OTU richness was highest in Fusarium, and two Fusarium OTUs were the most frequent. For seven common OTUs in LWQ, OTU richness was highest in Colletotrichum, and one Debaryomyces OTU was the most frequent (Supplementary Fig. S1 and Table S3). Twelve OTUs were shared by two sites, and four OTUs were shared by three sites (Fig. 3 and Supplementary Table S2).
For F. cirrhosa collected at four plant phenology stages, 10 of the 55 OTUs detected were unique for 4-FP, OTU richness was highest in Leptodontidium, and one Ilyonectria OTU was most frequent. Two of the 22 total OTUs were unique in 2-MP, OTU richness was highest in Ilyonectria, and one Fusarium OTU was the most frequent. In 4-GP, OTU richness was highest in Tetracladium, and two Fusarium OTUs were the most frequent. For the two OTUs in 2-GP, one Fusarium OTU was the most frequent (Supplementary Fig. S1 and Table S3). Three OTUs were shared by two phenology stages, and one OTU was shared by three phenology stages (Fig. 3 and Supplementary Table S2).
It should be noted that Fusarium and several Fusarium OTUs were common fungal lineages and general fungal OTUs associated with Fritillaria Cirrhosae Bulbus source plants in terms of OTU richness and OTU frequency, and they were widely distributed across different host plant species, geographic areas, and plant phenology stages. Fusarium displayed high OTU richness in the four host plant species, three geographic areas, and two plant phenology stages. Fusarium OTUs were frequent in the four host plant species, three geographic areas, and three plant phenology stages; one Fusarium OTU was shared by three plants, three sites, and two phenology stages, and another Fusarium OTU was recovered from two plants, three sites, and one phenology stage.
Diversity measures relative to host plant species, geographic area, and plant phenology stage are illustrated in Supplementary Table S4. KW rank-sum test revealed a statistically significant difference in diversity indexes of fungal communities among the four host plant species (Chao1, p=0.0853, >0.05; Shannon, p=0.0188, <0.05; Simpson, p=0.0249, <0.05), among Fritillaria cirrhosa in four geographic areas (Chao1, p=0.0156, <0.05; Shannon, p=0.0237, <0.05; Simpson, p=0.0237, <0.05), and among F. cirrhosa at four plant phenology stages (Chao1, p=0.0153, <0.05; Shannon, p=0.0156, <0.05; Simpson, p=0.0188, <0.05). PerMANOVA test supported a statistically significant difference in composition of fungal community among the four plants (Bray–Curtis, p=0.002, <0.05; Jaccard, p=0.001, <0.05), among F. cirrhosa in four geographic areas (Bray–Curtis, p=0.001, <0.05; Jaccard, p=0.001, <0.05), and among F. cirrhosa at four plant phenology stages (Bray–Curtis, p=0.001, <0.05; Jaccard, p=0.001, <0.05). Thus, preferences of fungal communities for host plant species, geographic area, and plant phenology were identified.
Fusarium spp. are generally associated with medicinal plants [23,24]. Fusarium tricinctum, F. acuminatum, F. sambucinum, and F. redolens were isolated from bulbs of Fritillaria Cirrhosae Bulbus source plants [8,14,15,25]. Our study confirmed that Fusarium spp. were dominant, general fungal species associated with bulbs of Fritillaria Cirrhosae Bulbus source plants, and were widespread in different source plant species collected from different sites and at different phenology stages. Common Fusarium species that have been isolated from Fritillaria Cirrhosae Bulbus source plants, such as F. acuminatum, and those that have been found in other medicinal plants, such as F. solani, and F. oxysporum, were detected in our study. However, unique Fusarium species that were also first revealed in this research, such as F. lateritium, F. proliferatum, F. lateritium, F. penzigii, and F. delphinoides.
It should be noted that many Fusarium species are a source of significant bioactive ingredients in valuable medicinal plants [16,25-27]. It was revealed that Fusarium isolates from Fritillaria unibracteata and F. unibracteata wabuensis were able to produce active alkaloid components of their host plants [8-10]. Therefore, further research on active metabolites of related Fusarium isolates associated with our host plants is needed to exploit potential biopharmaceutical fungi resources, and for resource conservation of valuable Fritillaria Cirrhosae Bulbus source plants in the future.
In the meantime, our results revealed the diversity of bulb-associated fungi of Fritillaria Cirrhosae Bulbus source plants. It was demonstrated that bulbs of the four Fritillaria Cirrhosae Bulbus source plants harbored abundant fungal species distributed across diverse lineages. Endophytic fungi in Fusarium, Penicillium, Paraphoma, Clonostachys, Gibberella, Colletotrichum, Plectosphaerella, Lachnum, Volutella, Cladosporium, Phoma, Paraconiothyrium, Alternaria, Ilyonectria, Nectria, Myrothecium, Rhizoctonia, Tomentella, Inocybe, and Mortierella (20 taxa, 1/5 of the total taxa identified) have been found in bulbs of Fritillaria Cirrhosae Bulbus source plants using the cultural method [13-16,25]. However, a larger variety of fungal lineages (82 taxa, 4/5 of the total taxa identified) was detected from bulbs of Fritillaria Cirrhosae Bulbus source plants for the first time in this study. Among them, isolations of Fusarium, Trichoderma, Penicillium, Epicoccum, Chaetomidium, Paraphoma, Cylindrocarpon, Neonectria, Plectosphaerella, Volutella, Chaetomium, Colletotrichum, and Clonostachys (13 taxa, 13% of the total taxa identified in this study by molecular method) in bulbs of these plant species have been cultured in our culture tests (unpublished data).
We detected several fungal species that have not been revealed in our or other culture tests [13,16] on Fritillaria Cirrhosae Bulbus source plants by ITS rDNA sequencing in this study. The results are somewhat biased because epiphyte fungi were detected on plant bulbs. However, an integrated molecular and cultural approach is indispensable for more comprehensive identification of fungal diversity associated with medicinal plants.
Besides, community distribution of bulb-associated fungi in terms of host plant species, geographic area, and plant phenology was tested in this study. It was revealed that composition and distribution of fungal community are associated with genotype (species), ecological and environmental conditions (geographic area) and phenology of host plants [6,28,29]. Additionally, it was previously reported that fungal species and quantity are most abundant in 3-yr-old stage of Fritillaria cirrhosa . In this research, fungal community composition associated with Fritillaria Cirrhosae Bulbus source plants showed preferences for host plant species, geographic area, and plant phenology. Many fungal lineages and species were unique in specific sampling situations; fungal diversity and specificity were much higher in some plant species (Fc, Fd), some sampling sites (WX, HDB), and one phenology stage (4-FP). This result may be influenced by the small sample size in this study, and sampling-dependent composition of fungal community was indicated; therefore, further research with an increased sample size is needed.
It was revealed that the microbial community of may impact the host , and thus influence the quality of . Therefore, systematic research that characterizes dynamic correlations between the fungal community and the active metabolites in Fritillaria Cirrhosae Bulbus source plants under different sampling scenarios can provide insight into resource exploitation of critical fungal species related to the production of crucial bioactive compounds, artificial propagation, and conservation of valuable Fritillaria Cirrhosae Bulbus source plants in the future, and also application of key fungal species to improve the quality and medical effectiveness of Fritillaria Cirrhosae Bulbus.
We thank Dr. Xinhui Li at Southwest Forestry University, Dr. Guodong Li and Dr. Weijiao Li at Yunnan University of Chinese Medicine for their kind helps, and thank Mallory Eckstut, PhD, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. This study was supported by Yunnan Provincial Science and Technology Department-Applied Basic Research Joint Special Funds of Yunnan University of Chinese Medicine (Grant No. 2017FF116 (-005) and 2018FF001(-038)).