Bacterial Composition and Diversity Associated with Rhizosphere of the Chinese Medicinal Herb Dendrobium


 Background: Dendrobium is a precious herbal belongs to Orchid and widely used as health care traditional Chinese medicine in Asia. Although orchids are mycorrhizal plants, most researches still focus on endophytes, and there is still large unknown in rhizosphere microorganisms. In order to investigate the rhizosphere microbial community of different Dendrobium species during the maturity stage, we used high-throughput sequencing to analyze microbial community in rhizosphere soil during maturity stage of three kinds of Dendrobium species.Results: In our study, a total of 240,320 sequences and 11,179 OTUs were obtained from these three Dendrobium species. According to the analysis of OTU annotation results, different Dendrobium rhizosphere soil bacteria include 2 kingdoms, 63 phyla, 72 classes, 159 orders, 309 families, 850 genera and 663 species. Among all sequences, the dominant bacterial phyla (relative abundance > 1%) were Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria, Firmicutes, Verrucomicrobia, Planctomycetes, Chloroflexi, Gemmatimonadetes. We analyzed the environmental factors of the growth of Dendrobium and found that the environmental factor that affects the rhizosphere soil microorganisms of Dendrobium is the soil factor. Among them, soil factors most closely related to the influence of Dendrobium rhizosphere soil microorganisms include total nitrogen, available phosphorus, ammonium nitrogen and pH value.Conclusions: We found that the rhizosphere bacterial communities of the three kinds of Dendrobium have significant differences, and the main species of rhizosphere microorganisms of Dendrobium are concentrated in the Proteobacteria, Actinobacteria, Bacteroidetes. Moreover, the smaller the level of bacterial, the greater the difference among Dendrobium species. Soil is the most important environmental factor affecting the bacterial communities in the rhizosphere soil of Dendrobium. These results fill the gap in the rhizosphere microbial community of Dendrobium and provide a theoretical basis for the subsequent mining of microbial functions and the study of biological fertilizers.

and fruiting, and its interaction with phytophagous insects, which is of great signi cance for plant growth and yield [9,10].
Dendrobium is the second largest genus in Orchidaceae, with more than 1400 species in the world. There are 74 species and 2 varieties of Dendrobium in Orchidaceae in China, most of which are precious medicinal plants. Dendrobium has many functions, such as bene ting stomach and promoting body uid, clearing heat and nourishing yin, relieving in ammation and relieving pain, clearing eyesight, and enhancing immunity [11]. Currently, arti cial cultivation using non-symbiotic tissue culture does not meet market demand because of slow growth and low survival rates [12]. Therefore, the development of an effective method for propagating these endangered species for both conservation and commercial production is needed. There are three kinds of medicinal Dendrobium in Ta-pieh Mountain area, which are Dendrobium huoshanense, Dendrobium o cinale and Dendrobium moniliforme [13]. All of them are perennial herbs of Dendrobium in Orchidaceae. They mainly contain polysaccharides, alkaloids and bibenzyls. They have antitumor, immunomodulatory, antioxidant, vasodilator and hypoglycemic effects [14].
At present, the research in Dendrobium mainly focuses on the endophytic bacteria. Indeed, there is a complex relationship between Dendrobium and its endophytes. Endophytic fungi can provide nutrients for Dendrobium plants [12,15]. Previous studies have shown that Sphiugomouas and Mycobacterium bacteria isolated from the roots of D. moschatum (Buch. -ham) SW. could signi cantly improve the seed germination rate of D. moschatum (Buch. -ham) SW [16]; and there are many studies using protocorm as material to successfully isolated and obtained effective fungi that promote seed germination [17][18][19].
Therefore, the goal of our work reported here was to characterize the rhizosphere microbial community of different Dendrobium species during the maturity stage. Our speci c objective was to describe taxa associated with each Dendrobium species and determine which the environmental factors were related to microbial diversity and community composition. Speci cally, we hypothesized that: (1) the dominant genus bacteria of Dendrobium and its comparison with other medicinal plants; (2) rhizosphere community composition will differ between three different Dendrobium; and (3) examine the relationship between environmental factors and composition of microbial communities. A better understanding of the difference of soil microbial communities in Dendrobium and the main in uencing factors that affect the rhizosphere soil microbes of the Dendrobium, could further provide a theoretical basis for analyze microbial functions and development of Dendrobium bio-fertilizer.

Plant Material
Dendrobium plants were arti cially cultivated in the greenhouse of Anhui Tongjisheng Biotechnology Company, Lu'an, China. The original source was collected by the company from the wild after obtaining local permission. The growth of protocorm-like bodies protocols and the condition of planting were described by our previous study [25].

Soil sampling
In order to obtain rhizosphere soil, plants were removed from owerpots and large soil aggregates were removed by hand; soil rmly attached to roots was collected with sterile brushes and regarded as rhizosphere soil. The rhizosphere soil was sampled and sieved to remove plant debris. A part of soil samples was put into sterile centrifuge tubes, frozen in liquid nitrogen immediately, and then stored at − 80 ℃ until the soil microbial composition was analyzed. The other part was air dried for chemical analysis.

Soil properties and climate factors
Soil pH was measured with a soil/deionized water ratio of 1/2.5 [26]. Soil total nitrogen (TN) was determined by Kjeldahl method [27]. Soil ammonium nitrogen (AN) was extracted using 2 M KCl solution followed by the method of detection using colorimetric-indophenol blue [26]. Available phosphorus (AP) of soil samples was extracted with ammonium uoride (NH 4 F, 0.03 M) and hydrochloric acid (HCl, 0.025 M), and measured by UV-Vis spectrophotometer [28]. Available potassium (AK) of soil samples was determined by the extraction with CH 3 COONH 4 (1 M), and measured by a ame photometer [29].
There were four replicates for each sample and each indicator. The climate data has been used in our previous experiments, and the collection method has been reported in the previous study [30]. The measurement results of all environmental factors are shown in Table 1. For bacterial diversity analysis, V4 hypervariable regions of 16S rRNA genes were ampli ed with universal primers 515 F (5′-GTGCCAGCMGCCGCGG-3′) and 806 R (5′-GGACTACHVGGGTWTCTAAT-3′) [31], and then PCR products were sequenced by IonS5 TM XL sequencing technique platform. Quality ltering on the raw reads were performed under speci c ltering conditions to obtain the high-quality clean reads according to the Cutadapt [32] (V1.9.1, http://cutadapt.readthedocs.io/en/stable/) quality-controlled process.
The phylogenetic taxonomy was assigned according to the Ribosomal Database Project (RDP) classi er at an 80% con dence threshold (Version 2.2) [36] using the Silva databases for bacteria. Alpha-and Betadiversity analyses were calculated in QIIME software (Version 1.7.0). Alpha-diversity was described for each sample using the metrics observed species (OTU numbers), Chao1, Shannon and Simpson index, ACE and Good-coverage were generated to compare the level of bacterial OTU diversity. Beta-diversity was estimated by computing weighed/unweighed UniFrac and Bray-curtis distances followed by principal coordinate analysis (PCoA).

Statistical analysis
Statistical analysis of p < 0.05 was performed using SPSS 19.0 (IBM, USA). The WGCNA software package, stat and ggplot2 packages in R software (Version 2.15.3) were used for PCoA analysis. The Spearman correlation index between species and environmental factors was calculated, and its signi cance was tested by the corr.test in the psych package of R, and then the visualization was performed by the pheatmap function in the pheatmap package.

α-and β-diversity of soil microbial communities
The rhizosphere soil alpha diversity indexes of different Dendrobium species are different (Fig. 1). The Good's coverage index of Dendrobium library was greater than 98.5%, indicating that the sequencing results re ect the real situation of the bacterial population in the sample. We found that Dh had the highest Shannon diversity index (10.29), while Do had the lowest (9.16), which indicated that Dh rhizosphere soil has the highest diversity of bacterial communities. The index of ACE and Chao1 have the same trend, Dh is the highest, respectively 5225 and 4877, indicating that Dh samples have the highest community richness. Further analysis revealed that the Shannon index and Simpson index of soil bacteria of Dh was signi cantly different from Do, and Do was signi cantly different from Dm.
Principal coordinate analysis (PCoA) was performed at the operational taxonomic unit (OTU) level (Fig. 2). The analysis of PCoA using the weighted UniFrac distance, indicated a distinct pattern in the rhizosphere bacterial communities associated with the two axes explaining 29.58% and 19.62% of the total variation in the Dendrobium rhizosphere soil. Using the unweighted UniFrac distance, the two axes explaining 17.64% and 11.79% of the total variation in the Dendrobium rhizosphere soil. Bacterial communities associated with the rhizosphere soil were clustered in three regions according to a PCoA using the unweighted UniFrac distance, corresponding to Dh, Do, and Dm. However, no clustering was detected using the weighted UniFrac distance from different Dendrobium species.

Taxonomic classi cation and abundance
Rarefaction curves for bacterial communities suggested that changes in OTU density within the different Dendrobium species was su ciently captured, and the sequencing was relatively comprehensive in covering the microbial communities (Fig. 3a). After quality ltering and processing according to a 97% similarity, a total of 240,320 sequences were obtained from these three Dendrobium species  (Fig. 3b).
Among all sequences, the dominant bacterial phyla (relative abundance > 1%) were Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria, Firmicutes, Verrucomicrobia, Planctomycetes, Chloro exi, Gemmatimonadetes, and with contributions of 45.41%, 17.58%, 9.94%, 7.77%, 3.93%, 3.03%, 2.88%, 1.98% and 1.14%, respectively. The difference at the phylum level of different Dendrobium species is not large, but there were more and more obvious differences in the level of the class afterwards. At the order level, the relative abundance of Alteromonadeles on Dh and Dm was low, while the relative abundance on Do was relatively high. Pseudoalteromonadaceae at family level and Pseudoalteromonas at genus level also have the same relative abundance pattern. At the species level, although the top ten species of bacteria in the rhizosphere soils of the three Dendrobium species were the same, the relative expression abundances were very different.

LEfSe analysis based on the relative abundance of different Dendrobium species
To further elucidate the possible interactions between identi ed bacterial dependencies in rhizosphere soil samples, linear discriminant analysis (LDA) effect size (LEfSe) method was used for quantitative analysis of biomarkers in different species. We detected signi cant differences in the abundance of bacterial biomarkers from different groups and identi ed a total of 13 biomarkers from all rhizosphere soil samples as shown in the branching diagram (Fig. 5b). The signi cant taxa in the Dh were a liated with diverse phylogenetic groups, including the family Sphingomonadaceae, and order Sphingomoadales. In the Dm, signi cantly abundant taxa were the genus Flavobacterium, family Flavobacteriaceae and order Flavobacteriales. In the Do, the signi cant taxa belonged to the phylum Actinobacteria, class unidenti ed Actinobacteria and Acdobacteriia, order Pseudonocardiales and Acidobacteriales, family Pseudonocardiaceae and unidenti ed Acidobacteriales, genus Crossiella, were abundant.

Relationship between ecological factors and bacterial communities
To obtain further evidence for this relationship between environmental factors and bacterial communities, the contributions of soil and climate factors to variations in the soil bacterial community were quanti ed by performing variance partitioning canonical correspondence analysis (Fig. 4a). Soil factors and climate factors were signi cantly related to variation in bacterial communities based on forward model selection (p < 0.05). Moreover, soil factors explained a higher proportion (25.31%) of the variation than climate factors (5.3%). It can be seen from the results that soil factors are considered to be one of the most important environmental factors affecting the rhizosphere microorganisms of Dendrobium.
Environmental factors in uenced the percentage abundance of soil bacterial communities. The correlation of bacterial genus with environmental factors was analyzed by Spearman's rank correlation (Fig. 4b). Among these environmental factors, soil factors are the most important factors affecting the rhizosphere microorganisms of Dendrobium, especially TN, AP, AN and pH value. However, among climate factors, the most in uential factors are only concentrated in the three factors including solar radiation, maximum monthly average relative humidity and temperature. Moreover, relative humidity has no signi cant effect on any bacterial genus.

Discussion
Rhizosphere microorganisms can co-exist with plant roots, colonize and maintain in roots, and play an important role in promoting plant growth and development [37]. Among them, the utilization rate and sensitivity of bacteria to root exudates are far higher than that of fungi, and bacteria are the most active and dominant microorganisms in rhizosphere [38,39]. In addition, the number and species of rhizosphere microorganisms have a direct impact on soil biochemical activity and nutrient transformation. Under the in uence of various complex factors of natural conditions, there are great differences in rhizosphere microbial ora of different plants and even different genotypes of the same plant [40,41]. So, in this study, bacterial communities associated with the rhizosphere of three different Dendrobium species were characterized by high-throughput sequencing during the maturity stage.  [44,45], due to the fact that Proteobacteria contains a large level of physiological, morphological and metabolic diversity, and that Proteobacteria is of great signi cance to the C and N cycles [46]. Proteobacteria reproduce fast, have good adaptability to unstable carbon sources, and are widely distributed in the global soil environment [47]. In general, the abundance of Proteobacteria or Acidobacteria in soil samples is the most abundant. These bacterial groups rich in rhizosphere microorganisms of Dendrobium are also found to be the dominant communities of other plant rhizosphere microbiomes [48,49].

Differences of bacterial communities in rhizosphere soils of three different Dendrobium species
According to the diversity index of alpha and beta, there were signi cant differences among the three Dendrobium species. The relative abundance of Acidobacteriales, Pseudonocardiaceae, Pseudoalteromonas and Peseudomonadales in Dendrobium o cinale were higher than those in the other two species. Acidobacteriales is the dominant bacteria in the common plant rhizosphere bacterial community. Because Acidobacteriales can degrade complex root exudates such as cellulose and lignin, it plays a major role in the plant rhizosphere carbon cycle [50]. Pseudoalteromonas sp. secretes a variety of extracellular active substances, including proteins, polysaccharides, brominated compounds, extracellular enzymes, extracellular toxins, antibiotics and so on [51]. These substances have antibacterial, algicidal, bactericidal and cellulose degrading activities [52,53]. Peseudomonadales is an important group of biocontrol microorganisms, and is also one of the most widely distributed microorganisms in nature. Its rapid reproduction, strong colonization ability and simple nutrition requirements have been widely studied for its inhibition of plant diseases and promotion of plant growth. In the existing studies, Pseudonocardiaceae is mainly associated with cellulose degradation and antibiotic synthesis [54][55][56]. It can be seen from the above that the function of the bacterial community in the rhizosphere of Dendrobium o cinale may be stronger than that of the other two kinds of Dendrobium. In Dendrobium moniliforme, the relative abundance of Bacteroidetes is relatively high, and Bacteroidetes is a poor nutrient bacterium, which is suitable for growth in the environment with less absorbable nutrients such as organic matter and available nitrogen [57].
In bacterial species, we found that Lysobacter soli have a high relative abundance in Dendrobium moniliforme, this fungus has previously been isolated from the soil where ginseng grown [58], and has been found in other plants to promote plant activity [59]. Psychrobacter ibarius has been found in Dendrobium o cinale. Some researchers have isolated this bacterium from the root plane of Angelica sinensis [60]. This is a fungus related to polysaccharide synthesis. This is consistent with our previous research results [13]. Our previous research also found that among these three kinds of Dendrobium, the polysaccharides of two-year-old Dendrobium are higher than the other two kinds of two-year-old Dendrobium.

Soil is the most important environmental factor affecting the bacterial communities in the rhizosphere soil of Dendrobium
Due to the differences of research scale and microbial groups, there are no environmental factors that can fully explain the distribution pattern of all soil microorganisms. For different space-time scales and different research objects, the environmental factors that affect the distribution of microorganisms are also different, and there is no single natural factor that affects the microbial community. Various environmental factors work together to make a choice for the spatial distribution of microorganisms [61].
Studies have shown that soil properties, vegetation types and meteorological factors have varying degrees of in uence on soil microbial diversity. Therefore, this study selected a total of 14 factors of soil factors and climate factors, and analyzed their correlation with the rhizosphere bacterial community of Dendrobium, so as to determine the most important environmental factors affecting the rhizosphere bacterial community of Dendrobium.
Among the climatic factors, it has been reported that temperature and precipitation have a driving effect on soil microbial composition [62][63][64]. In this study, the bacterial community of Dendrobium was affected by the maximum monthly average relative humidity and temperature, which was consistent with previous studies [65]. These data indicate that temperature and relative humidity are the key climatic factors affecting the bacterial composition of Dendrobium. Research has shown that the increase of nitrogen input had consistent effects on the richness, diversity and composition of soil bacterial community in two consecutive crop seasons. In our soil factors, total nitrogen explained most of the major variations in bacterial communities, consistent with previous studies [66]. These results indicate that TN is an important soil factor for bacterial community formation. In addition, we also found that solar radiation is also the main climate affecting factor, which was also reported in Panax notoginseng [67]. Zarraonaindia studies showed that as a potential microbial pool of plant-related microorganisms, soil microorganisms have a great impact on grape root related microorganisms [68]. Soil type is an important factor affecting the rhizosphere microbial community, and the main reason for the in uence is the difference of soil microbial community [69]. Therefore, in our previous analysis, we also found that the most important environmental factor affecting the bacterial community of Dendrobium is also soil factor, accounting for 25.31%. Total nitrogen, available phosphorus and available potassium in soil are important indexes to measure soil fertility, and they are also the main sources of nutrients absorbed by plants. Previous studies have shown that plant roots can affect the community structure of root microorganisms by changing the physical and chemical properties of soil. For example, plant existence can change the structure of rhizosphere soil, water holding capacity, nutrients [70] and form environmental conditions that are conducive to the growth of some strains or not conducive to the growth of certain strains, thus changing the community structure of soil microorganisms.

Conclusions
Overall, the chemical properties of rhizosphere soil of Dendrobium and the climatic characteristics of Dendrobium cultivation were rstly analyzed. Then, combined with high-throughput sequencing technology, the rhizosphere soil microbial communities of different species of Dendrobium were studied, and the rhizosphere microorganisms and environmental driving factors of different species of Dendrobium were discussed. The main conclusions are as follows: the dominant bacteria in rhizosphere soil of Dendrobium are as follows: Proteobacteria, Bacteroidetes, Actinobacteria, and Acidobacteria. These are the dominant bacteria in the rhizosphere bacterial community. However, there are some differences in the bacterial communities among different Dendrobium species, and the smaller the bacterial level is, the greater the difference is. We found that there are Psychrobacter ibarius in Dendrobium o cinale, which is a kind of fungi related to the accumulation of polysaccharides. Soil factors and climate factors jointly affect the species diversity and community composition of rhizosphere soil microorganisms of Dendrobium. And soil is considered to be the most important factor that affects the rhizosphere soil microbes of Dendrobium. Among these soil factors, total nitrogen, available phosphorus, ammonium nitrogen and pH value have the greatest impact on the rhizosphere soil microbes of Dendrobium.

Declarations
Ethics approval and consent to participate Dendrobium huoshanense, Dendrobium o cinale and Dendrobium moniliforme used in this study were cultivated by Maoyun Yu's greenhouse from Anhui Tongjisheng Biotechnology Company, Lu'an, China. No permits were required for the collection of the samples.