Metabolic Profiling on Alternaria Toxins and Components of Xinjiang

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Metabolic Profiling on Alternaria Toxins and Components of Xinjiang Jujubes Incubated with Pathogenic Alternaria alternata and Alternaria tenuissima via Orbitrap High Resolution Mass Spectrometry Dongqiang Hu, Yingying Fan, Yanglan Tan, Ye Tian, Na Liu, Lan Wang, Duoyong Zhao, Cheng Wang, and Aibo Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03243 • Publication Date (Web): 07 Sep 2017 Downloaded from http://pubs.acs.org on September 10, 2017

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Journal of Agricultural and Food Chemistry

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Metabolic Profiling on Alternaria Toxins and Components of Xinjiang Jujubes

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Incubated with Pathogenic Alternaria alternata and Alternaria tenuissima via

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Orbitrap High Resolution Mass Spectrometry

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Dongqiang Hu †, Yingying Fan ‡, Yanglan Tan †, Ye Tian †, Na Liu †, Lan Wang †,

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Duoyong Zhao ‡, Cheng Wang *‡, Aibo Wu *†.

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† SIBS-UGENT-SJTU Joint Laboratory of Mycotoxin Research, Key Laboratory of

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Food Safety Research, Shanghai Institutes for Biological Sciences, Chinese Academy

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of Sciences, University of Chinese Academy of Sciences, Shanghai, P.R.China,

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‡ Institute of Quality Standards & Testing Technology for Agro-Products, Key

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Laboratory of agro-products quality and safety of Xinjiang, Laboratory of Quality and

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Safety Risk Assessment for Agro-Products (Urumqi), Ministry of Agriculture,

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Xinjiang Academy of Agricultural Sciences, Urumqi 830091, Xinjiang Province,

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P.R.China

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Corresponding Author

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*E-mail: [email protected]. Phone: +86-021-54920716. Fax: +86-021-54920716

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ORCID

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Aibo Wu: 0000-0002-7161-1592

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Cheng Wang:

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Notes

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The authors declare no competing financial interest.

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ACS Paragon Plus Environment


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Abstract:

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Xinjiang jujubes (zizyphus rhamnaceae) are important agro-economically foods

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with the highest planting area and yields in China, however, black spot disease and

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contaminated Alternaria toxins have unfortunately caused decline or loss of jujubes

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nutritional quality in recent years. In this study, we used ultra-high performance liquid

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chromatography coupled to Orbitrap high-resolution mass spectrometry (UHPLC-

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Orbitrap HRMS) to profile both Alternaria toxins and components in three

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representative Xinjiang jujubes, Hami Huang (HM), Hetian Jun (HT) and Ruoqiang

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Hui (RQ). Before LC-MS analysis, jujubes were inoculated with two main pathogens

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of Alternaria alternata (Aa) and Alternaria tenuissima (At). Different combination of

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jujube varieties with pathogenic isolates display different metabolic profiles as

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expected. Moreover, four major Alternaria toxins, alternariol (AOH), alternariol

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monomethyl ether (AME), altenuene (ALT) and tenuazonic acid (TeA) were detected

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in all samples. The inoculation of both pathogens significantly decreased the levels of

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nutrients and metabolites in jujube, including 4 saponins, 3 organic acids and 3

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alkaloids, whereas it increased the level of several glycerol phosphates. The flavonoid

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profiles are diverse. Lastly, inoculation of Aa changes more metabolites in jujubes

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than At. Our data provides insights to better understand the detrimental contamination

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of Alternaria pathogens in Xinjiang jujubes and improve food safety of jujubes.

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Keyword:

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zizyphus rhamnaceae; Alternaria toxins; nutritients and metabolites; Orbitrap

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Introduction

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Jujube (zizyphus rhamnaceae) is a popular nutritional food fruit and a main

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source of carbohydrates in both China and worldwide. The natural distribution of

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jujubes is mainly in Central Asia, northern India and China. China is the largest

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producer and consumer of jujubes in the world. Xinjiang province of China produces

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30% of jujubes in China, more than 7 million tons per year, ranking No. 1 in China.

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Jujube production is the pillar industry in Xinjiang. Unfortunately, fungal

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contamination in jujube fruits can cause visible plant diseases, including black spot

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diseases, corky core, rust disease and anthracnose, etc. Particularly in the recent

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decade, black spot disease frequently occurred during rainy season in Xinjiang

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province, which has an arid climate. The main cause of black spot disease is

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Alternaria spp., which infect jujubes during their growth, processing and storage.

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In general, Alternaria black spot disease has been found in many garden and

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flowering plants. The lesions are found on branches, leaves(1), fruits(2), and the

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roots(3). Although Alternaria spp. are regarded as asexual fungi, sexual reproduction

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also exists (4) to generate new Alternaria strains. These new strains may produce new

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types of metabolites and toxins, thus leading to more challenges to control Alternaria.

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Alternaria toxins can be classified in two types, nonspecific toxins (NHSTs) and host-

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selective toxins (HSTs) (5). NHSTs aggravate plant diseases, including TeA, AOH,

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AME, ALT, and TEN, whereas HSTs bind to specific toxin receptors in specific

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hosts, for example, Alternaria citri toxin (ACT) is found in infected citrus(6),

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Alternaria fries toxin (AF) in strawberries(7), Alternaria kikuchiana toxin (AK) in



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pears(8), Alternaria lycopersici toxin (AL) in tomatoes(9), and so on.

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Xinjiang jujubes are not only edible drupes, also have great values for traditional

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Chinese medicine, because they are rich in soluble sugars including fructose, glucose,

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rhamnose, sorbitol and sucrose. Jujubes also contain abundant of other nutrients

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including potassium, phosphorus, calcium, manganese(10), vitamin C, phenolics,

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flavonoids, triterpenic acids, and polysaccharides (11). Previous studies used the 2,2-

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diphenyl-1-picrylhydrazyl and ferric-reducing antioxidant power assays to reveal that

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jujubes have antioxidant capacity(12). The results in mice showed that extracts of

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jujube fruits significantly decreased the activities of alanine aminotransferase and

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aspartate aminotransferase, and attenuated tissue damages that were caused by

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oxidative stress(13). The studies on animals also discovered that flavonoids and

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saponins in jujube extracts have both sedative and hypnotic functions (14), and jujube

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juice can prolong the hexobarbital-induced sleeping time in mice(15). In Korea,

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jujube fruit extracts from three different breeds were analyzed, the results showed that

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these three breeds contain different bioactive contents, particularly, the flavonoids

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varied widely (16). In China, jujube cultivars have different antioxidant capacity, and

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alpha-tocopherol and β-carotenes contents were found relevant in some of the jujube

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cultivars(17).

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Recently, Orbitrap HRMS has been widely used since its accuracy and precision

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are higher than that of other mass spectrometers. Researchers evaluated the glycation

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level of bovine serum albumin by using Orbitrap mass spectrometry, creating a new

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method to detect modified proteins(18). In another study, Orbitrap MS can detect


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significant increase levels of eleven exogenous metabolites in plasma of female rats,

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which have been fed with cranberry procyanidins(19). Orbitrap was also used for the

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environmental science to monitor the degradation of cytostatic etoposide, several

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etoposide by-products were found(20). In forensic investigations, Orbitrap allows to

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detect trace content of anabolic steroids and their esters in human hair, thus providing

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supporting evidence to identify suspects (21). Taken together, Orbitrap is a powerful

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analytical tool and widely used to perform comparison analysis, confirm new

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substances, detect trace components, and so on.

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Black spot disease not only causes yield loss to the pillar industry of Xinjiang,

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but also huge nutrient loss and quality decline for those harvested products. The

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normal color of jujube fruit is from cyan to dark red during growth phase, while

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disease area of jujube fruit colored from light red to black, which mislead people to

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ingest the infected fruit. The infected part of fruit feels hard, mildew, decaying and

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tastes astringent, differing from the soft and sweet taste of natural mature jujube fruits.

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Because of the saccharides and amino acids consumption by pathogens, there are no

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major nutrients in infected jujube fruits for food supply, on the contrary, potential

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Alternaria toxins can be dangerous to human.

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Most studies used postharvest jujube fruits to find the solutions to extend jujube

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storage time, however, it is largely unknown how various Alternaria isolates infect

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different jujube varieties to the black spot disease. Here, we used two Alternaria

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isolates, which infected Xinjiang jujube and caused black spot disease, to examine

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their pathogenic effects on three jujube fruits from Xinjiang province. These three



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jujube fruits include HM with ellipsoidal shape, HT with spheroidal shape, and RQ

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with the smallest size and higher glycogen contents. In vitro inoculation studies were

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carried out to examine the invasion of two pathogens in different Xinjiang jujubes.

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LTQ-Obitrap high-resolution mass spectrometric was used to identify the profiles of

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Alternaria toxins and metabolites in jujubes after inoculation.

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Overall, the primary purpose of this study is to analyze the profiles of Alternaria

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toxins and components of jujube fruits which were inoculated with Alternaria

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pathogens. The results will provide new insights into metabolic changes before and

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after the invasion of Alternaria spp. in jujubes.

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Materials and Methods

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Reagents and Medium

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To make 1 liter of potato dextrose agar medium (PDA), 20 g glucose, 15 g agar

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and 200 g boiled extracts of potatoes were mixed. 200 g jujube powders and 15 g agar

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were mixed to make 1 liter of jujube medium. Jujube fruits were first washed using

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distilled water, then washed using 75% ethanol and distilled water before

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denucleation, lyophilization and milling to obtain jujube powder. The powders were

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evenly spread out and sterilized using ultraviolet for 30 min. During sterilization, the

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powder was re-mixed every 10 min. All the medium contains 50 mg ampicillin per

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liter.

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Magnesium sulphate (MgSO4) and sodium chloride (NaCl) were purchased from

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Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Methanol, acetonitrile and

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ammonium acetate were chromatographic grade and purchased from Tedia Co., Ltd

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(Shanghai, China). Standards of TeA, AOH and TEN were purchased from Romar

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Supply, Inc (Tulln, Austria).

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Jujube Breeds and Pathogens

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Jujubes were collected from several orchards of Xinjiang Production &

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Construction Corp in the late autumn. The sampling location was at the Tarim river

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coast in southern Xinjiang province, along the river coast, RQ jujubes were from

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Ruoqiang county, HT jujubes were from Hetian county, HM jujubes were from Hami

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county. The jujubes from every breed include both healthy and sick samples, the sick



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samples were conformed to have the black spot disease. It can be clearly found lump

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and decay inside the flesh of jujubes with black spot disease-positive when isolating

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pathogens.

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Two Alternaria genus, Aa and At, were isolated from the sick jujubes with

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confirmed black spot disease. The infected parts of sick jujubes were torn out and

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cultured in PDA medium. After three days, single fungal colony was picked and re-

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inoculated in the PDA medium, DNA of single colony was extracted and sequenced

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by ITS sequencing, the sequences were aligned and analyzed using BLAST. After

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incubation and collection of fungal spores, two main pathogens of jujube black spot

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disease were obtained.

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Sample Preparation

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Five microliter spores of each Alternaria isolate were inoculated in PDA, HM,

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HT and RQ mediums respectively. Five microliter distilled water was added on each

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medium as blank control. All conditions contain three replications. After 7 days of

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incubation, all the mediums were lyophilized at -20 °C for overnight, then milled to

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powders. Jujube powder from each sample was mixed to 80 % of methanol, MgSO4

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and NaCl in proportion of 4:1. After vortexing for 5 min, the extract was sonicated for

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20 min. The solution was centrifuged for 10 minutes and the supernatant was

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collected.

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Parameters of UHPLC-Orbitrap and LC-MS

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UHPLC and HRMS were used to obtain the metabolic profile of pathogens in

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jujubes. The Waters Acquity UPLC system was used. It includes an autosampler,


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binary pump, an ultraviolet detector and a thermostatted column compartment.

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Chromatographic separation was performed on an Agilent Extend—C18 column (100

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mm × 4.6 mm, 3.5 µm) at 30 °C. The mobile phase mixture consists of 5 mM

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ammonium acetate and acetonitrile, which were mobile phase A and B respectively.

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The gradient elution program was performed as followings: 0-2 min, 95% A; 2-

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13min, 95%–0% A; 13–15 min, 0% A; 15–18 min, 0%–95% A and 18-20 min, 95%

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A. The injection volume was 5 µL and the flow rate was 300 µL/min. The component

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alteration was analyzed using Thermo Q Exactive Quadrupole Orbitrap HRMS. This

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system has the scanning range of more than four quantitative series with femtogram

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grad sensitivity. The instrument was operated in a negative mode, and the operation

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parameters were as follows: capillary temperature, 320 °C; pray voltage, 3.00 KV;

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auxiliary gas heater temperature, 300 °C; scan modes, full MS and MS/MS. The mass

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range was from 70 to 1050 dalton, thus covering most metabolic macromolecules.

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Thermo Finnigan TSQ VANTAGE triple quadrupole LC-MS was used to

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confirm the newly identified mycotoxin from incubated samples. Medium extracts

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and the standards of TeA, AOH and TEN, were mixed and injected to the

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chromatographic column. The standards were used for intercomparison. The LC

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parameters were as follows: mobile phase A was 5 mM ammonium acetate, mobile

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phase B was acetonitrile, and gradient elution program was 0-5 min, 80%-20% A; 5-

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7min, 20% A; 7–8 min, 20%-80% A and 8–10 min, 80% A. The MS scan mode was

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SRM, and the ions for quantitative analysis included TeA: m/z 196.1 [M-H], AOH:

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m/z 259.1 [M+H] and TEN: m/z 413.2 [M+H].



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Data Analysis

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The raw HRMS data were uploaded onto the website of XCMS online

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(https://xcmsonline.scripps.edu). The primary manipulation was creating a single job,

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uploading all the raw files, feature detection, correction and alignment of retention

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time, statistical analysis and data visualization. At last, the mass spectra for each

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sample were transformed to a two-dimensional matrix(22). Three jujube cultures and

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PDA samples were chosen to create a multi-group job, and the cultures with two

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pathogens were chosen to create a pairwise job. The results of XCMS were imported

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to the statistical software, SIMCA-P 11.5, for principal component analysis (PCA).

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METLLIN database was used for the analysis, each peak of chromatograms

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represented a metabolic feature, which can be exactly identified with accurate

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molecular weight. The seven-step protocol was used to characterize metabolites (23).

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Amount changed metabolites and their fold changes were listed to generate a heatmap

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to present metabolite profile.

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To confirm the jujube plates contained the Alternaria toxins, multiple reaction

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monitoring (MRM) was performed using the triple quadrupole LC-MS system. Once

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its retention time matched the exact mass of quantitative ion, the substance was well-

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determined.

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Results and Discussion

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Total Extracted Ion Chromatogram (TIC) and General Description

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In this study, the extracts of culture mediums were analyzed using UHPLC-

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Orbitrap, and the metabolic profile of Aa and At were investigated. The representative

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UHPLC-HRMS TIC of extracts from two incubated mediums and uncultured medium

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were shown (Figure 1). From the TIC, as the process of gradient elution program, the

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extracts from all samples have a chromatographic peak with high intensity at the 2nd

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minute. As the ratio of acetonitrile amount increased, more peaks appeared in the

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incubated samples than in the original jujubes, even though the intensities of some

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peaks attenuated. The metabolite profiles of the Aa inoculated samples are largely

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different from that of the At inoculated samples. Aa produced more metabolites than

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At. On the other hand, At produced the metabolites that Aa did not. The components

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of three jujube breeds also varied. HM and HT have the components that can’t be

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detected in RQ, thus showing a closer relationship between HM and HT (Figure 1 in

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the Supporting Information). However, chromatographic peak changes before and

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after incubation were similar among three breeds for jujubes in both pathogens. In

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summary, two pathogens can significantly change the metabolic profiles of three

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jujube breeds.

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Alternaria Toxins Produced in Xinjiang Jujubes Incubated with Alternaria

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Isolates

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The ion chromatogram for each Alternaria toxin was obtained in the ms2 mode of

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the full scan chromatogram. The accurate mass of TeA, AOH, AME and ALT are



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197.11, 258.05, 272.07 and 292.09, respectively, in the negative mode, the ionized

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form of Alternaria toxin is [M-H]-, and the accurate mass of these ionized Alternaria

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toxins are 196.10, 257.05, 271.06 and 291.09. From the scan filter, each mycotoxin

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was detected, along with the peak time: TeA, 6.6min; AOH, 12.1min; AME 13.5min;

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and ALT, 11.2min (Figure 2). The intensity of extracted ion chromatographic peak for

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each Alternaria toxin in Aa is higher than At, suggesting more mycotoxins were

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produced in Aa. Therefore, it is very likely that Aa grows faster than At in jujube

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mediums. Furthermore, we used the standards of three Alternaria toxins to obtain the

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MRM chromatograms of mycotoxins that two pathogens emerged, and two main

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quantitative fragment ions of each toxin were detected in the same peak time (Figure

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2 and 3 in the Supporting Information). The MRM results were consistent with the

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extracted chromatogram of full scan mode, indicating that Aa produced more

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Alternaria toxins than At.

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Besides NHSTs of TeA, AOH, AME and ALT, HSTs were also searched in the

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full scan chromatograms (Figure 4 in the Supporting Information). Only in the

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samples that incubated with At, AK-V toxin appeared at 7.7 min, whereas AF-V toxin

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emerged at 14.3 min in both At and Aa. Relative contents of these Alternaria toxins

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were calculated (Figure 5 in the Supporting Information), and compared to general

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medium, contents of each Alternaria toxin decreased in most jujube media for

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Alternaria isolates, especially for At. The jujubes incubated with Aa produced more

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AF_2 than those incubated with At. The HM jujubes incubated with At produced

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more AK_2 and AF_2 than the HT jujubes incubated with At. Interestingly, neither of


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two HSTs was found in the RQ jujubes, the possible reason is that these two

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Alternaria isolates may produce different HSTs rather than AK_2 and AF_2 in RQ

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jujubes. In this study, no other HSTs were detected in the incubated jujube samples.

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For a long time, Alternaria toxins had not not been much accounted in the field

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of mycotoxins. Although FDA lists TeA as a poisonous substance, no food safety

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agent made any standards in food supply for Alternaria toxins. Our study revealed

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that mycotoxins are produced in the jujubes, which have Alternaria black spot

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disease. The results clearly showed the same abundance of other NHSTs with that of

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TeA. In addition to NHSTs, HSTs were also detected. We are the first to confirm that

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the jujubes having Alternaria black spot disease produce both AK_2 and AF_2. All

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these results suggested that the jujube black spot disease was the consequence of the

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additive effect of HSTs and NHSTs. HSTs were pathogenic factors that determine the

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parasitic range and host specificity, whereas NHSTs were virulence factors that

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aggravate the jujube black spot disease.

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PCA Analysis of Different Incubated Samples

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The profile of metabolites and components changes before and after Alternaria

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incubation were demonstrated by PCA analysis, clustering experimental samples

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based on metabolite profile. After PCA analysis, the multicomponent data of each

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medium sample was transformed into a two-dimensional matrix with two principal

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variables that avoid ion information loss. This analysis has better interpretation of the

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MS data. For the pathogen Aa in three jujube mediums, after incubation, significantly

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changes appeared and all the incubated samples were clustered together (Figure 3B).



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However, in jujube mediums incubated with At, two group-specific clusters

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overlapped, with a clear approach between HT jujube samples incubated with At and

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RQ jujube samples (Figure 3C). For the samples incubated with At, the plots in each

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group cluster were not very closely accumulational, the RQ samples were particularly

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far from others, regardless of being incubated or uncultured. It suggested that jujube

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breed of RQ was very different from the other two jujubes, thus producing different

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metabolites after pathogen inoculations.

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Additionally, the distribution of different culture mediums was demonstrated,

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showing a clear separation between general PDA mediums and jujube mediums. Both

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Pathogens, Aa and At that were incubated in PDA, were on the left side of the plot,

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whereas those incubated in jujube mediums were on the right side (Figure 3A). Also,

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the distance was closer between the same pathogens than that between the same

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jujube breeds, indicating that metabolites difference was the larger contribution to the

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principal component distribution.

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Alteration of Components in Jujubes

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The HRMS raw data was processed using XCMS online, the results showed that

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more than 5000 ions were detected in each sample. After normalization, the pairwise

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analysis jobs were performed between incubated samples and uncultured samples.

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Each comparison group contained all three jujube breeds. The mean value was

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calculated for each peak, which represent a metabolite.

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All the metabolites were ranked by change rate from largest to smallest. The

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metabolites with significant change rates of extracted ions were picked, some of the


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changed metabolites were listed (Table 1, 2, 3 and 4 in the Supporting Information).

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Among them, some components in jujubes were selected. Both pathogens, Aa and At,

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caused significant reduction of the components including organic acids, alkaloids and

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amino acids, however, At did not change the levels of flavonoids and saponins (Table

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1). Jervine and 2-methylbutanoic acid were the same decreased components between

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them, still, jujubes incubated with Aa showed a higher changed rate. Also, metabolites

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with increased level were also picked in two groups, compared to the uncultured

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jujubes, flavonoid and glycerol phosphate were included (Table 2). The flavonoids in

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increased components included naringenin, theaflavin, shoyuflavone B, etc., and

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naringenin was the common flavonoids for two pathogens (Table 3 and 4).

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Interestingly, isoflavone was a flavonoid with reduced level, however, the level of its

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derivative, licoisoflavone, increased, suggesting a transformation for the flavonoid

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forms after incubation with Aa.

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Early studies on plant soft-rot diseases showed a decline in plant amino acids

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during infection(24), whereas that several amino acid biosynthesis genes were

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overexpressed during fusarium crown rot(25), the amounts of triterpenes and fatty

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acids were significantly increased in grape berries with noble rot(26). Overall, all the

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detectable responses of metabolite alterations related to pathogen perception and

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defense signaling. Previous studies were focused on the pathology and plant

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responses, for example, in the leaf spot of withania somnifera plant, the levels of total

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alkaloids increased, whereas the levels of certain types of alkaloids decreased(27).

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Different from in vivo experiments, our in vitro studies revealed that the levels of most



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alkaloids decreased, particularly, it is the first time to observe a decrease of the

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amount of saponins. Two organic acids, jasmonic acid and salicylic acid, were both

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identified to increase plant resistance to Fusarium, but not Alternaria(28). Our results

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may suggest that Alternaria can severely damage the plant components, including

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organic acids.

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Profile of Metabolites Emersion and Components Alteration

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The results of the pairwise jobs were displayed in the volcano plots. Over 5000

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ions of each jujube sample were scattered with its fold change and statistically

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significant difference. The result of jujube mediums incubated with two pathogens

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were in agreement with the previous experiments, the fold change of Aa was larger

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than that of At, regardless of the component levels (Figure 4). The heatmap also

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confirmed that after Aa incubation, original component levels in jujubes significantly

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decreased and new metabolites appeared. Each variation was much more than that of

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the samples incubated with At. It suggested that the predominant pathogenic

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Alternaria spp. in the jujube black spot disease was Aa, which produced more

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Alternaria toxins. The metabolic profiles of three jujube breeds were displayed

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(Figure 5), all content values were equal to the average value. Compared to the

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average sample of three breed of uncultured jujube mediums, the samples incubated in

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HT showed a less decrease for those with high contents in origin.al samples, and the

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samples incubated in RQ showed a higher variation for all substances in original

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samples, which represents more metabolites emerged for both pathogens and more

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jujube components consumption in RQ jujube. And it confirmed again that RQ jujube


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was different with other two breed jujubes, substances in RQ could stimulate the

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growth of pathogens better than others, producing more secondary metabolites.

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It was a field study that jujube black spot disease occurred more frequently in HT

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rather than RQ jujubes. Interestingly, the simulation model of Alternaria jujube black

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spot disease in experimental conditions was not identical with that from the field

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investigation, on the contrary, more metabolites emerged and components altered in

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RQ jujubes. Possibly, the results of HSTs may provide an explanation that HSTs were

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only found in HT and HM, not in RQ, leading to the rare invasion of two isolates in

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RQ. We know that Alternaria black spot disease was a host-specific disease, different

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from other plant diseases. It was truly coincident that Alternaria black spot disease in

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Xinjiang jujubes had never been reported till recent decades, and it only infected some

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certain fruits. Therefore, it is obviously that Alternaria isolates have evolved to infect

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Xinjiang jujubes. We predicted that black spot disease in RQ can also be as severe as

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HT in the late few years and new Alternaria strains in favor of RQ may finally appear.

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In conclusion, this study was the first report about the Alternaria toxins and

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metabolic profile of pathogens in jujube black spot disease. Our study also makes a

369

comparison of components variation in three breeds of Xinjiang jujube, China.

370

Although it has not been reported, Alternaria jujube black spot disease has been

371

arisen for the past several years in Xinjiang. We performed a simulation study about

372

the pathogen invasion in jujube. From the metabolic profile to the secondary

373

metabolites particularly Alternaria toxins, after incubation, two main Alternaria spp.

374

have different pathogenic effects on Xinjiang jujubes, generating different Alternaria



375

toxins and metabolic profiles. Additionally, as the predominant jujube breeds in

376

Chinese market, HM, HT, and RQ showed potentials with predisposition to the

377

Alternaria spp. Although RQ can provide more nutrients, black spot disease has rare

378

incidence in RQ, probably due to the host specificities of two Alternaria isolates.

379

Jujube fruits are good sources of nutrients, this study demonstrated the metabolic

380

profiles of jujube Alternaria black spot disease, opening an avenue to investigate the

381

unknown pathogenic mechanisms of Alternaria jujube black spot disease.

382 383 384 385 386 387 388 389 390 391 392 393 394 395 396


Page 18 of 36

Page 19 of 36


397 398

Abbreviations Used

399

UHPLC-Orbitrap HRMS, ultra-high performance liquid chromatography coupled to

400

Orbitrap high-resolution mass spectrometry; HM, jujube of Hami Huang; HT, jujube

401

of Hetian Jun; RQ, jujube of Ruoqiang Hui; Aa, Alternaria alternata; At, Alternaria

402

tenuissima; NHST, nonspecific toxin; HST, host-selective toxin; TeA, tenuazonic

403

acid; AOH, alternariol; AME, alternariol monomethyl ether; ALT, altenuene; TEN,

404

tentoxin; ACT, Alternaria citri toxin; AF, Alternaria fries toxin; AK, Alternaria

405

kikuchiana toxin; AL, Alternaria lycopersici toxin; LC-MS, liquid chromatography-

406

mass spectrometry; PDA, potato dextrose agar medium; PCA, principal component

407

analysis; MRM, multiple reaction monitoring; TIC, total extracted ion chromatogram.

408 409 410 411 412 413 414 415 416 417 418



419 420 421 422

Acknowledgments We thank Yuanhong Shan in the Core Facility Centre of the Institute of Plant Physiology and Ecology for LTQ-Orbitrap mass spectrometry assistance.

423 424

Funding Sources

425

1. The authors highly appreciate the financial support from National Natural Science

426

Foundation of China (31471661)

427

2. Project supported by the National Science Foundation for Young Scholars of China

428

(31601575)

429

3. Study on Occurrence and Prevention of Alternaria Toxins in Xinjiang Jujube,

430

P161210005, 2016.07-2018.06, the Urumqi Talent Project Plan

431

4. The Evaluation and Detection of Mycotoxin Contamination in Xinjiang Jujube,

432

2017.01-2018.12, Basic Scientific Research of Public Welfare Research Institutes in

433

the Autonomous Region

434 435

Supporting Information Description

436

Brief descriptions in nonsentence format listing the contents of the files supplied as

437

Supporting Information. TIC of jujube powder culture mediums, confirmation of two

438

NHSTs, Orbitrap HRMS chromatograms of two HSTs, production of Alternaria toxins

439

produced by two pathogens in different culture mediums; Lists of metabolites with

440

increased or decreased level after incubation with pathogens.

441


Page 20 of 36

Page 21 of 36


442 443

References

444

1.

445

Spot Caused by Alternaria alternate on Aronia melanocarpa in Korea. Plant Dis. 2016, 100,

446

1011-1011.

447

2.

448

phylogenetic lineage of Alternaria alternata causing citrus brown spot in China. Fungal Biol

449

2015, 119, 320-30.

450

3.

451

Identification and community dynamics of fungi associated with root, crown, and foot rot of

452

field pea in western Canada. Eur. J. Plant Pathol. 2017, 147, 489-500.

453

4.

454

Peever, T. L., Signatures of Recombination in Clonal Lineages of the Citrus Brown Spot

455

Pathogen, Alternaria alternata sensu lato. Phytopathology 2013, 103, 741-749.

456

5.

457

Yamamoto, M.; Otani, H., Host-selective toxins produced by the plant pathogenic fungus

458

Alternaria alternata. Fems Microbiol Rev 2013, 37, 44-66.

459

6.

460

pathogenicity and toxin production in Tangerine pathotype of Alternaria alternata the causal

461

agent of citrus brown spot in Iran. Phytopathology 2010, 100, S6-S6.

462

7.

463

of fruit rot of strawberry caused by an Alternaria sp in Taiwan. Plant Dis. 2008, 92, 1248-

Kwon, J. H.; Kang, D. W.; Lee, S. Y.; Choi, O.; Kim, J., First Report of Brown Leaf

Huang, F.; Fu, Y.; Nie, D.; Stewart, J. E.; Peever, T. L.; Li, H., Identification of a novel

Taheri, A. E.; Chatterton, S.; Foroud, N. A.; Gossen, B. D.; McLaren, D. L.,

Stewart, J. E.; Thomas, K. A.; Lawrence, C. B.; Dang, H.; Pryor, B. M.; Timmer, L. M.;

Tsuge, T.; Harimoto, Y.; Akimitsu, K.; Ohtani, K.; Kodama, M.; Akagi, Y.; Egusa, M.;

Amini, N.; Kakvan, N.; Zamanizadeh, H.; Hajmansoor, S., Relationship between

Ko, Y.; Chen, C. Y.; Yao, K. S.; Liu, C. W.; Lin, C. H.; Maruthasalam, S., First report



464

1248.

465

8.

466

Alternaria alternata apple pathotype (A-mali) causes black spot of European pear. Eur. J.

467

Plant Pathol. 2016, 145, 787-795.

468

9.

469

Comparative Evaluation of Biochemical Changes in Tomato (Lycopersicon esculentum Mill.)

470

Infected by Alternaria alternata and Its Toxic Metabolites (TeA, AOH, and AME). Frontiers in

471

plant science 2016, 7, 1408.

472

10.

473

of chinese jujube. Food chemistry 2007, 103, 454-460.

474

11.

475

of Current Knowledge of Fruit Composition and Health Benefits. Journal of agricultural and

476

food chemistry 2013, 61, 3351-3363.

477

12.

478

capacities of the ethanolic extract of different tissues of jujube (Ziziphus jujuba Mill.) from

479

China. Food Chem. Toxicol. 2010, 48, 1461-1465.

480

13.

481

Zizyphus jujube fruit on carbon tetrachloride-induced hepatic injury in mice by anti-oxidative

482

activities. Journal of ethnopharmacology 2009, 122, 555-560.

483

14.

484

C.; Chen, Y. F., Possible mechanism involved in the sedative activity of jujubasaponins I in

485

mice. CNS neuroscience & therapeutics 2013, 19, 282-4.

Tanahashi, M.; Nakano, T.; Akamatsu, H.; Kodama, M.; Otani, H.; Osaki-Oka, K.,

Meena, M.; Zehra, A.; Dubey, M. K.; Aamir, M.; Gupta, V. K.; Upadhyay, R. S.,

Li, J.-W.; Fan, L.-P.; Ding, S.-D.; Ding, X.-L., Nutritional composition of five cultivars

Gao, Q.-H.; Wu, C.-S.; Wang, M., The Jujube (Ziziphus Jujuba Mill.) Fruit: A Review

Zhang, H.; Jiang, L.; Ye, S.; Ye, Y.; Ren, F., Systematic evaluation of antioxidant

Shen, X.; Tang, Y.; Yang, R.; Yu, L.; Fang, T.; Duan, J.-a., The protective effect of

Shi, G. B.; Wu, Q.; Zhang, B.; Sun, X. H.; Zong, W. T.; Zhao, X. R.; Xin, Y.; Zhao, Q.


Page 22 of 36

Page 23 of 36


486

15.

487

Ziziphus jujuba in mouse models of anxiety. Journal of ethnopharmacology 2000, 72, 435-

488

441.

489

16.

490

Amino Acids, Flavonoids, Total Phenolics, and Antioxidative Activities of Jujube (Ziziphus

491

jujuba) Fruits and Seeds Harvested from Plants Grown in Korea. Journal of agricultural and

492

food chemistry 2011, 59, 6594-6604.

493

17.

494

( Ziziphus jujuba Mill.) on the contents of sugars, organic acids, alpha-tocopherol, beta-

495

carotene, and phenolic compounds. Journal of agricultural and food chemistry 2012, 60,

496

9642-8.

497

18.

498

after ultrasonic pretreatment revealed by high-performance liquid chromatography-linear ion

499

trap/Orbitrap high-resolution mass spectrometry. Journal of agricultural and food chemistry

500

2014, 62, 2522-30.

501

19.

502

by cranberry procyanidins in plasma of female rats using (1) H NMR and UHPLC-Q-Orbitrap-

503

HRMS global metabolomics approaches. Mol Nutr Food Res 2015, 59, 2107-18.

504

20.

505

in chlorinated water by liquid chromatography coupled to quadrupole-Orbitrap mass

506

spectrometry: Identification and quantification of by-products in real water samples. The

507

Science of the total environment 2015, 506–507, 36-45.

Peng, W. H.; Hsieh, M. T.; Lee, Y. S.; Lin, Y. C.; Liao, J., Anxiolytic effect of seed of

Choi, S.-H.; Ahn, J.-B.; Kozukue, N.; Levin, C. E.; Friedman, M., Distribution of Free

Gao, Q. H.; Wu, C. S.; Wang, M.; Xu, B. N.; Du, L. J., Effect of drying of jujubes

Zhang, Q.; Tu, Z.; Wang, H.; Huang, X.; Shi, Y.; Sha, X.; Xiao, H., Improved glycation

Liu, H.; Garrett, T. J.; Tayyari, F.; Gu, L., Profiling the metabolome changes caused

Negreira, N.; López de Alda, M.; Barceló, D., Degradation of the cytostatic etoposide



Page 24 of 36

508

21.

509

A.; Pascali, V. L., Screening for exogenous androgen anabolic steroids in human hair by

510

liquid chromatography/orbitrap-high resolution mass spectrometry. Anal Chim Acta 2013, 793,

511

61-71.

512

22.

513

Platform to Process Untargeted Metabolomic Data. Anal. Chem. 2012, 84, 5035-5039.

514

23.

515

Siuzdak,

516

characterization of metabolites guided by the METLIN database. Nat. Protocols 2013, 8, 451-

517

460.

518

24.

519

Massive production of butanediol during plant infection by phytopathogenic bacteria of the

520

genera Dickeya and Pectobacterium. Molecular microbiology 2011, 82, 988-97.

521

25.

522

Colgrave, M. L.; Gardiner, D. M.; Manners, J. M.; Henry, R. J.; Kazan, K., The Fusarium

523

crown rot pathogen Fusarium pseudograminearum triggers a suite of transcriptional and

524

metabolic changes in bread wheat (Triticum aestivum L.). Annals of botany 2017, 119, 853-

525

867.

526

26.

527

Morales-Cruz, A.; Doyle, C. L.; Ye, Z.; Allen, G.; Heymann, H.; Ebeler, S. E.; Cantu, D.,

528

Developmental and Metabolic Plasticity of White-Skinned Grape Berries in Response to

529

Botrytis cinerea during Noble Rot. Plant Physiol. 2015, 169, 2422-2443.

Strano-Rossi, S.; Castrignano, E.; Anzillotti, L.; Odoardi, S.; De-Giorgio, F.; Bermejo,

Tautenhahn, R.; Patti, G. J.; Rinehart, D.; Siuzdak, G., XCMS Online: A Web-Based

Zhu, Z.-J.; Schultz, A. W.; Wang, J.; Johnson, C. H.; Yannone, S. M.; Patti, G. J.; G.,

Liquid

chromatography

quadrupole

time-of-flight

mass

spectrometry

Effantin, G.; Rivasseau, C.; Gromova, M.; Bligny, R.; Hugouvieux-Cotte-Pattat, N.,

Powell, J. J.; Carere, J.; Fitzgerald, T. L.; Stiller, J.; Covarelli, L.; Xu, Q.; Gubler, F.;

Blanco-Ulate, B.; Amrine, K. C. H.; Collins, T. S.; Rivero, R. M.; Vicente, A. R.;


Page 25 of 36


530

27.

531

leaf spot disease of Withania somnifera and its impact on secondary metabolites. Indian

532

journal of microbiology 2008, 48, 432-437.

533

28.

534

signalling mediates resistance of the wild tobacco Nicotiana attenuata to its native Fusarium,

535

but not Alternaria, fungal pathogens. Plant Cell and Environment 2015, 38, 572-584.

Pati, P. K.; Sharma, M.; Salar, R. K.; Sharma, A.; Gupta, A. P.; Singh, B., Studies on

Van Thi, L.; Schuck, S.; Kim, S.-G.; Weinhold, A.; Baldwin, I. T., Jasmonic acid

536 537 538

Figure Captions

539

Figure 1

540

TIC of jujube powder culture mediums before and after inoculation obtained

541

from LTQ-Obitrap HRMS. (A) Chromatograms of jujube powder extract. (B) After

542

incubation with Aa, a high intensity chromatographic peak appeared at the 12th

543

minute, some peaks attenuated. (C) After incubation with At, few new

544

chromatographic peak appeared with low intensity compared to Aa.

545 546

Figure 2

547

Orbitrap HRMS chromatograms of four NHSTs produced by two predominant

548

isolates in the jujube medium, TeA, AOH, AME and ALT. (A) NHSTs produced by

549

Aa. (B) NHSTs produced by At.

550 551

Figure 3



552

PCA score plots based on different experiments. (A) HT, HM and PDA were

553

incubated with two pathogens. Two isolates incubated in PDA showed a similar

554

cluster, whereas samples of HM and HT showed an obviously different distribution.

555

(B) PCA plots of samples before and after incubating with Aa, which showed

556

apparently clusters for both samples. (C) PCA plots of samples before and after

557

incubating with At, which showed an overlap in samples with RQ jujube.

558 559 560

Figure 4

561

Metabolite profile of incubated samples before and after incubation with two

562

isolates. All values equal to arithmetic mean. (A) Volcano plot of changed metabolites

563

in jujubes samples incubated with Aa. (B) Volcano plot of changed metabolites in

564

jujubes samples incubated with At. (C) Heatmap of metabolites profile before and

565

after incubation. Samples incubated with Aa (Jujube_aa) showed much more changed

566

metabolites in both increase and decrease compared to At (Jujube_at).

567 568

Figure 5

569

Metabolite profile of three jujubes after incubation with pathogens. All values

570

equal to arithmetic mean. (A) HM jujube; (B) HT jujube; (C) RQ jujube. Each

571

displays an average level, and the change rate was ordered: HM < HT < RQ. (D)

572

Heatmap of changed profile in three jujube breeds after incubation. HM and HT

573

jujubes were more closely similar in reply to the pathogens inoculation, whereas RQ


Page 26 of 36

Page 27 of 36


574

jujube showed greater amount and change rate of metabolites.

575 576 577 578 579 580 581 582

Tables

583

Table 1

584

Major components with decreased level after incubation with Aa. metabolites

name isoflavone

flavonoid

saponin

organic acid

alkaloid

amino acid

Others

fold change [log2(jujube/jujube_aa)] 3.64

dihydroxy-6,7,4'-trimethoxyflavone 8-glucoside 7,4'-dihydroxyflavone 7-rutinoside

7.68** 5.79

p-value 0.292 0.004 0.280

oenanthoside A

4.00**

0.005

isopentyl gentiobioside

3.98

0.177

perilloside E

3.88

0.175

2-methylbutanoic acid

9.34

0.184

N-palmitoyl-l-serine phosphoric acid

7.45

0.140

jervine

11.64

0.196

colchicine salicylate

3.66

0.169

Caranine

7.67

0.168

Ala Lys Thr Trp

7.19*

0.018

Lys Ser Trp Trp

3.91

0.054

Asp Ile Val Tyr

7.73

0.056

His Arg Val Val

7.52*

0.016

Phe Pro Pro Pro

5.32*

0.016

dihydrojasmonic acid, methyl ester

5.49**

0.003

corycavamine 4-benzoyl-1,3,5,7-tetraphenylheptane-1,7-dione

5.27*


4.76**

0.042 0.001


1,1,1,3,3,3-hexafluoro-2-(iodomethyl)propane

Page 28 of 36

3.75*

0.013

585

The fold changes were calculated using the formula log2(jujube/jujube_aa), *p

Metabolic Profiling on Alternaria Toxins and Components of Xinjiang

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