Secondary Metabolite Accumulation Associates with Ecological

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Agricultural and Environmental Chemistry

Secondary Metabolite Accumulation Associates with Ecological Succession of Endophytic Fungi in Cynomorium songaricum Rupr. Jin-Long Cui, Yan-Yan Zhang, Vinod Vijayakumar, Gang Zhang, Meng-Liang Wang, and Jun-Hong Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01737 • Publication Date (Web): 11 May 2018 Downloaded from http://pubs.acs.org on May 12, 2018

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Journal of Agricultural and Food Chemistry

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Secondary Metabolite Accumulation Associates with Ecological Succession of Endophytic

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Fungi in Cynomorium songaricum Rupr.

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Jin-Long Cui,*,† Yan-Yan Zhang,† Vinod Vijayakumar,§ Gang Zhang,*,‡ Meng-Liang Wang,†

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Jun-Hong Wang†

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†

‡

Institute of Applied Chemistry, Shanxi University, Taiyuan, Shanxi 030006 P. R. China. College of Pharmacy, Shaanxi University of Chinese Medicine, Xianyang, Shaanxi

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712046, P. R. China.

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§

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Technology, The Ohio State University, Columbus, OH, 43210, USA.

College of Food, Agricultural and Environmental Sciences, Department of Food Science and

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*

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*

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E-mail: [email protected]

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*

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E-mail: [email protected]

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Keywords: Root-parasitic interactions, SM active ingredients, fungal endophytes, medicinal

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plants

Correspondence: Jin-Long Cui Phone: +86-351-7016101

Gang Zhang

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ACS Paragon Plus Environment


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INTRODUCTION

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Cynomorium songaricum Rupr. belongs to the Cynomoriaceae family, has no chlorophyll

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but tends to parasitize the roots of Nitrariaceae plant.1 It is distributed in the peripheral

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regions of desert with extreme environments of dry, rocky, sandy and large temperature

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differences in Asia, Africa and Europe.2 As a rare folk medicine and healthy food, it is

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traditionally used to treat the conditions of impotence, premature ejaculation, low sexual

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function, spermatorrhea colic, and stomach ulcers.3 While modern phytochemical and

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ethnopharmacological studies have indicated that its effective properties are based on diverse

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chemical components especially active ingredients such as ursolic acid, protocatechuic acid,

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catechins, gallic acid, flavanoids, tannins and polysaccharides (Figure 1),4 which are

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secondary metabolites (SMs) produced during the root-parasitic plants developmental

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process.

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To obtain high content of active ingredients, a medicinal plant needs to be collected at the

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appropriate time of the season which requires experience.5 Further, depending on plant age

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and phenology the content of the active ingredient varies largely, mainly affected by

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extracorporeal environment factors, such as soil pH, temperature and humidity.6 Besides these

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factors, it is noteworthy that the accumulation of SMs are also regulated by plant internal

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factors, such as endophytes.7 Which not only serve as reservoirs of novel bioactive SMs

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themselves, but also as potential candidates for drug discovery.8 However, lots of knowledge

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in this field is largely undetermined including mechanisms of interaction, signal transmission

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and transformation, co-evolution and specificity between endophyte and host, and so on.9

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Endophytes can colonize host but do not cause strong hypersensitive reactions to plant.9 2


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The enormous potential of endophytes especially endophytic fungi affecting the accumulation

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of SMs has received much attention as an integral part of their host’s biology, have been

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proposed to be akin to host organs in and of themselves.10 Though limited data is available to

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date, at least three possible ways are broadly accepted that highlight the effects of endophytes

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on host physiology and metabolism: (ⅰ) secretion of versatile chemical entities including plant

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hormones such as GA, IAA and ethylene,11 antibiotics or other products,12 which promote

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plant metabolite accumulation, alter plant metabolic status or help plants respond to other

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biotic and abiotic stresses. (ⅱ) by “induced effect” or “lateral gene transfer” altering host gene

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expression and metabolism. Upon recognition of elicitors from endophyte, plants produce

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reactive oxygen species (ROS) including H2O2 and O2-; resulting in ion flow of Cl−/Ca2+ and

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K+/H+, signal net cross-talk such as cAMP, NO and SA; transcription factor activation and

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regulation of gene expression culminating in the production of defense products.13 (ⅲ)

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bio-conversion affecting plant chemical profiles. As shown for the endophytic

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Paraconiothyrium variabile which specifically bio-transforms glycosylated flavonoids to

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aglycone or other chemicals, which in turn alters the host Cephalotaxus harringtonia

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metabolomic composition.14

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Host plants on the other hand present selective pressure on endophytic fungal community

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and shape the assembly of fungal endophytes on any given host spp.15 A plant of certain

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genotype and age located in an environment/ecosystem harbors its own special endophyte

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composition that may govern aspects of bi-directional SM accumulation and cross-talk.16 For

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example, it is reported that comparison of Fusarium-infected spring wheat cultivars and

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winter wheat cultivars showed altered accumulation of SMs (benzoxazinoids, phenolic acids, 3



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carotenoids, and flavonoids), whose levels exhibited varying selective pressures on the fungal

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pathogen Fusarium at five time points. Furthermore, in addition to certain other wheat SMs

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the flavonoids, homoorientin and orientin were identified as potential inhibitors of the

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trichothecene mycotoxin deoxynivalenol (DON) produced under Fusarium infection.17

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The interaction between endophytic fungi and host plant is complex. The many challenges

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arise from: (ⅰ) the enormous number of endophytic fungi in association with the plant

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ecosystem; (ⅱ) complex interactions of host plants with numerous individual fungal species at

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the same time; (ⅲ) the interactions occurring among endophytes themselves; and (ⅳ) the

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elucidation of interactions that are authentic i.e., reproducibility under standard laboratory in

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vitro conditions.9 However, objectively elucidating the correlation between the special

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chemical composition and the corresponding endophytic fungus is a key first step in scientific

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exploration and application of fungal endophytes in providing the much needed and sort after

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resources of drugs in medicinal use against various forms of diseases.8,17,18 Recently, it is

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reported that Humicola fuscoatra and Aspergillus niger significantly (p≤0.05) relate to host

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development under salinity stress.19 The other notable example is that of the health hazard

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caused by Lolium perenne an important pasture and forage plant to livestock`s which was

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linked to the accumulation of alkaloids, lolitrem B and peramine which were found to

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significantly positively correlate with endophytic wild type E (WT) strain Neotyphodium lolii

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infection.20 Based on this find, N. lolii strains like AR1 (EAR1) and AR37 (EAR37) were

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modified and inoculated on L. perenne, leading to the reduction of hazardous effects and

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improved grass productivity.21

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As an annual, herbaceous and succulent plant, C. songaricum must be collected before or 4


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just at the developmental stage of sprouting to above ground emergence, which possesses the

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highest pharmacological activity or else the plant would become hollow with low

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pharmacological activity according to traditional experience and Chinese pharmacopoeia.22

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We aimed at addressing the key question of, do endophytic fungi participate in the

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physiological and developmental process of C. songaricum? To answer this question, we

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asked: (1) what is the composition of endophytic fungi in C. songaricum? (2) how do

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endophytic fungal communities evolve across its different developmental stages? (3) how do

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the accumulations of main active ingredients change under endophytic associations? (4) are

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there any correlations between active ingredients and the endophytic fungi? (5) If so, what are

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these correlations and what do they imply? In part all or some of these cruces form the main

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objective of this study. Our study shows a clear correlation between active ingredient

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accumulation and endophyte assembly and presents new opportunities to better understand

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plant fungal symbiosis illustrating the influential factors for improving medicinal quality of C.

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songaricum.

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MATERIALS AND METHODS

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Plant Material Collection and Sample Preparation. C. songaricum developmental growth

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was divided into five stages: Tubercle (T), Sprouting (S), Unearthing (U), Maturing (M) and

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Atrophy (A) (Cui et al., 2018). The plants located around area of Xilin-gaole town (39°05′ 45′′

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N, 105°23′27′′ E, ≈1133.98 m), Alashan League, Inner Mongolia of China, were collected in

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November, March, May, June and September, respectively during the calendar year 2015 to

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2016. The fresh rhizomes were collected and stored at 4℃, and transported to the laboratory

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in < 8 hours. Collected sample materials were cleaned and surface-sterilized following 5



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methods described earlier,18 then they were divided into two parts: one part of the collected

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material was dried in the shade, and used for chemical analyses; while the other part was used

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for DNA extraction and high-throughput sequencing of endophytic fungi.

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Extraction and Analysis of Active Ingredients. The dried samples of C. songaricum were

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powdered and passed through a 100 micron mesh sieve. A total of seven active ingredients in

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C. songaricum were evaluated, and all standards and/or reference chemicals including

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D-(+)-glucose (No.110833), rutin (No.100080), gallic acid (No. 110831), protocatechuic acid

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(No.110809), catechins (No.110877) and ursolic acid (No.110742) were purchased from

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National Institutes for Food and Drug Control, China. Total sugars, flavonoid and tannin

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content were analyzed using UV spectrophotometry UV2450 (Shimadu, Japan), and the other

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four ingredients (protocatechuic acid, catechins, gallic acid and ursolic acid) were determined

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by HPLC 1200 LC system (Agilent Technologies, USA) (Supplement Figure S1). The

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equations for linear regression, calibration coefficient, linear range, wavelength and retention

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time (tR, in HPLC) are shown in Table 1. All the experimental treatments were done in

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triplicate measurements.

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Total sugars including oligosaccharides and polysaccharides were determined by modified

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phenol-sulfate method.23 Firstly, lipid and protein content were removed with Soxhlet

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circulation at 85.5℃ for 50 min and later the residue was dried and extracted with 80%

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ethanol using circulation reflux at 80℃ for 2 h. The residue was dissolved with 25 mL

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ultra-pure water by ultrasonication (350 w, 35 Hz) for 30 minutes. The mixture was

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centrifuged at 5000 rpm for 5 min, to the supernatant 100 mL of 95% ethanol was added,

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mixed well and keep static at 4 ℃ overnight, centrifuged at 5000 rpm for 5 min. The 6


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pellet/residue was washed with 5 mL of 80% ethanol, followed by pure diethyl ether and

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acetone, respectively dried at 50 ℃ under low pressure (0.09 MPa) using vacuum drying oven

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DZF-6020 (Ruijia Scientific co. LTD, China). Lastly, the residue was weighed and dissolved

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in 50 mL of ultra-pure water.

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Flavonoids were determined by colorimetric method.24 Briefly, 1 g powder was placed in a

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25 mL flask, sonicated (350 w, 35 Hz) with 25 mL of 50% ethanol for 35 min three times and

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the extraction combined into a 100 mL volumetric flask, then adjusted to final volume of 100

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mL with 50% ethanol and used as sample for determination of flavonoid content. The

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calibration curve was generated based on ten concentrations of reference rutin from 12.50

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μg/mL to 125.00 μg/mL in 50% ethanol, with increments of 12.50 μg/mL.

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Analysis of tannins was done by the method described in Chinese Pharmacopoeia.22 A

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powdered sample (1 g) of C. songaricum was placed in brown flask, ultra-pure water to a 25

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mL final volume was added and incubated overnight. Next day, ultrasonic extraction was

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done for 30 min, and then filtered through Whatman® qualitative filter paper, Grade 1. The

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above process was repeated three times, the extracts combined, and the volume made up to

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100 mL with ultra-pure water. Two mL of sample was mixed with 1 mL

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phosphomolybdotungstic acid and 10 mL H2O, and the total phenol content was determined

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by standard curve using UV spectrophotometry UV2450. For determination of unreacted

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phenol, casein (0.6 g) was dissolved in 25 mL of sample in a 100 mL conical flask, sealed and

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shaken at 30 ℃ for 1 h, then 2 mL of cold sample filtrate mixed with 1 mL

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phosphomolybdotungstic acid and 7 mL of H2O. Finally, 29% Na2CO3 was added and the

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final volume made up to 25 mL with ultra-pure water. The total tannin content (TC) was 7



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calculated using the formula: TC (mg) = total phenol (mg) – unreacted phenol (mg).

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The contents of protocatechuic acid, catechins and gallic acid were simultaneously

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determined by UV measurements at 280 nm by RP-HPLC-DAD.25 One gram of the sample

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was weighed accurately and placed in a 50 mL flask and ultrasonicated with 40 mL of 50%

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aqueous methanol (v/v) for 40 min. This was repeated twice, the combined extract volumes

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were adjusted to a final volume of 50 mL, the solution was then passed through 0.45 μm

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nylon membrane filter (Merck Millipore, USA). The samples were then analyzed using

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Agilent 1200 LC system equipped with diode array detector (DAD), a quaternary pump

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equipped with Eclipse XDB-C18 column (5.0 μm, 250×4.6 mm, Agilent, USA). The column

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oven temperature was fixed at 25℃. A mobile phase consisting of acetonitrile (A)

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(ThermoFisher Scientific, USA) and 0.1% glacial acetic acid:water buffer (B), was applied for

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the gradient elution: 8A/92B to 100A/0B in 25 min with a flow rate of 1.0 mL /min with an

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injection volume of 10 μL.

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Ursolic acid content was determined using HPLC method.26 One gram of C. songaricum

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sample powder was placed in a 50 mL volumetric flask, sonicated with 50 mL chloroform for

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40 min, filtered, and impregnated twice with petroleum ether after evaporation to complete

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dryness. After decanting the petroleum ether, the residue was taken up into a 10 mL

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volumetric flask and the final volume adjusted with chloroform. The dissolved sample was

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then filtered with 0.45 μm nylon membrane and used directly for HPLC analysis as described

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above. The mobile phase consisted of acetonitrile (A) and 1% glacial acetic acid:water buffer

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(B): 88A/12B in 15 min.

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DNA Extraction and Amplicon Preparation. To characterize the endophytic fungal 8


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community in C. songaricum in different developmental stages, genomic DNA was extracted

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from surface-sterilized rhizome of C. songaricum using E.Z.N.A. Plant DNA Mini Kit

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(Omega Bio-Tek, Doraville, GA, USA). PCR amplicons were then purified using PowerClean

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Pro Clean-Up DNA Kit (MO BIO Laboratories, Inc., USA). The internal transcribed spacer 2

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(ITS2) region was selected for PCR amplification with primer combinations of ITS3f

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(GCATCGATGAAGAACGCAGC) and ITS4 (TCCTCCGCTTATTGATATGC) complete

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with Illumina adapters and unique 7-bp barcode sequences that allowed for identification of

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individual samples in multiplexed runs.18 PCR was performed in a 25 μL reaction system

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containing of 1 μL of each primer (10 μM), 2 μL of template (50-100 ng/μL), 0.2 μL of

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TaqDNA Polymerase (5 U/μL), 1 μL of dNTP (10 mM), 2.5 μL of 10×buffer, and 17.3 μL of

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ddH2O. PCR amplifications were performed using: 3 min of initial denaturation at 94℃,

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followed by 40 cycles of: 94℃ for 30 s, 47℃ for 30 s, and 72℃ for 45 s; and a final

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extension of 7 min at 72℃. PCR products from triplicate runs were pooled to make one

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sample type to avoid PCR bias. After purification using PureLink Quick gel extraction Kit

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(Thermo Fisher Scientific, USA), DNA concentration was determined with Nanodrop

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ND-1000 with software ver.3.3 (Thermo Scientific, USA). Finally, all bar-coded samples

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were then pooled together in an equimolar ratio and were sent to high-throughput amplicon

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sequencing using the Illumina Miseq platform (USA) at Sangon Biotech (Shanghai) Co., Ltd.

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(Shanghai, China).

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Next-generation Sequencing and Bioinformatic Analysis. Following sequencing, forward

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sequence reads were de-multiplexed and sorted according to their barcodes. Similarly, the

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reverse primer barcode sequences and low-quality regions of each sequenced sample were 9



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removed through CUTADAPT and TRIMMOMATIC. Bioinformatics and statistical analyses

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of pair-end sequences were done as described recently in Cui et al.18 Sequences obtained from

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next-generation sequencing were deposited in the Sequence Read Archive (SRA) database in

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NCBI (https://submit.ncbi.nlm.nih.gov) (SRA Accession: SRP128486).

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The fungal OTU was picked with open picking method through the centroid based

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algorithm.27 The similarity between samples was tested to determine the OTU occurrences

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and statistical analysis on the diversity and evenness of the sample species was performed.

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The alpha-diversity indices including ACE and Chao-1 richness index predicting the total

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numbers of OTUs in each sample, Shannon and Simpson index were used to investigate the

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biodiversity based on the OTU richness. The similarity criteria that were used was: species

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98%, genus 94%, family 90%, order 85%, class 80%, and phylum 75%.28 The difference of

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endophytic fungal communities based on OTUs in different developmental stages of C.

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songaricum was evaluated by ANOVA statistics, multi-dimension and Venn Diagram

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package in R software. Furthermore, the difference among all developmental groups was

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analyzed through beta-diversity including non_multi_dimension_analysis (NMDS), Principal

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Co_ordinates (PCoA), Principal Component Analysis (PCA), Unweighted pair group method

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with arithmetic mean (UPGMA) and Unweighted unifrac. The phylogenetic relationship in

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representative OTUs was analyzed by Hierarchical Clustering dendrogram, Bray_Crutis_tree

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and “OTU_co_network”, based on R software. Test of the null hypotheses of no differences

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among the microbial community compositions were examined using permutation multivariate

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analysis of variance (PERMANOVA)18 and the p-value was recalculated based on the

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Bonferroni significance. 10


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Dynamic Analysis of Endophytic Fungi. The assembly and dynamics of endophytic fungal

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community was characterized in C. songaricum across T, S, U, M and A developmental

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stages corresponding to their individual sampling dates in November, March, May, June and

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September, respectively encompassing one developmental cycle. All samples analyzed in the

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five developmental stages were replicated three times.

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Analysis of Correlation. Unitary and multiple linear regression analyses was performed to

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evaluate the correlation between endophytic fungal richness and the accumulation of active

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components in C. songaricum across T, S, U, M and A developmental stages, which included

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simple correlation (Pearson correlation coefficient) and multi-correlation coefficient. As for

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samples not conforming to a normal distribution, non-parametric test of Wilcoxon Signed

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Rank was used to evaluate the variation in fungal richness and the contents of seven active

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components using IBM SPSS Statistics 19.0 (Chigago, USA) with the parameters of

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zero-order correlation coefficient (Pearson) and two-tailed test. In the matrices, correlation

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coefficients are marked in red and green for positive and negative correlations, respectively.

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While, light blue and dark blue colors are used to mark significant difference (p

Secondary Metabolite Accumulation Associates with Ecological

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