Identification and Functional Characterization of Endophytic Microbes from Mulberry Tree（Morus alba L.）

doi:10.15933/j.cnki.1004-3268.2025.03.009

Abstract

Abstract: This study used methods such as microbial culture，target gene sequence amplification，and homology identification to isolate and identify endophytic microbes in Morus alba L.，and explored the strain types，distribution characteristics，and functional characteristics of endophytic bacteria and fungi in the roots，stems，and leaves of Yunsang No.6 mulberry.The results showed that a total of 43 endophytic bacteria belonging to 9 genera and 22 endophytic fungi belonging to 14 genera were isolated from mulberry trees. Among them，21 bacteria and 11 fungi were isolated from roots，13 bacteria and 5 fungi were isolated from stems，and 9 bacteria and 6 fungi were isolated from leaves.There were significant differences in the distribution of endophytic microbes in the roots，stems，and leaves of mulberry trees，there were Falsibacillus pallidus，Bacillus velezensis and Pseudomonas koreensis shared in the roots and stems of mulberry trees，Pantoea agglomerans，Bacillus subtilis and Pseudomonas koreensis shared in the stems and leaves，and Pseudomonas koreensis shared in the roots，stems，and leaves；Endophytic fungi Colletotrichum siamense was shared in mulberry roots and leaves，but no common fungi were found to exist in the stems and leaves，stems and roots.Further screening of functional microbes showed that 8 strains of nitrogen fixing microbes（7 bacteria，1 fungus），4 strains of phosphorus solubilizing microbes（2 bacteria，2 fungi），and 8 strains of potassium solubilizing microbes（6 bacteria，2 fungi）were obtained from 65 endophytic microbes，respectively.In summary，endophytic bacteria and fungi in mulberry trees exhibit diversity and distribution differences in roots，stems，and leaves.Among endophytic bacteria and fungi in mulberry trees，there are functional strains that can dissolve phosphorus，nitrogen，and potassium.

Key words: Morus alba L., Endophytic fungi, Endophytic bacteria, Isolation and identification, Functional microbes

摘要： 采用微生物培养、目标基因序列扩增与同源性鉴定等方法对桑树（Morus alba L.）内生菌进行分离与鉴定，探讨云桑6号桑树根、茎、叶中的内生细菌和内生真菌的菌株种类、分布特性及菌株功能特性。结果表明，从桑树中共分离得到9属43株内生细菌及14属22株内生真菌，其中从根部分离得到21株细菌和11株真菌，从茎中分离得到13株细菌和5株真菌，从叶中分离得到9株细菌和6株真菌。桑树根、茎、叶中的内生菌分布差异大，内生细菌在桑树根与茎中共有白色假芽孢杆菌（Falsibacillus pallidus）、贝莱斯芽孢杆菌（Bacillus velezensis）、韩国假单胞菌（Pseudomonas koreensis），茎与叶中共有成团泛菌（Pantoea agglomerans）、枯草芽孢杆菌（Bacillus subtilis）、韩国假单胞菌（Pseudomonas koreensis），根、茎、叶中共有韩国假单胞菌（Pseudomonas koreensis）；内生真菌在桑树根与叶中共有炭疽病菌（Colletotrichum siamense），茎与叶、根与茎中没有检测到共有真菌存在。进一步的功能菌筛选结果表明，从65株内生菌中分别得到8株固氮功能菌（7株细菌、1株真菌）、4株溶磷菌（2株细菌、2株真菌）和8株解钾功能菌（6株细菌、2株真菌）。综上，桑树内生细菌、内生真菌在根、茎、叶中具有多样性和分布差异性，其中有溶磷、固氮、解钾等功能菌存在。

关键词: 桑树, 内生真菌, 内生细菌, 分离鉴定, 功能菌

CLC Number:

S888.7

LI Sha, YANG Jiahu, WANG Yanhui, HUANG Xinqiu, YANG Wen. Identification and Functional Characterization of Endophytic Microbes from Mulberry Tree（Morus alba L.）[J]. Journal of Henan Agricultural Sciences, 2025, 54(3): 77-86.

李莎, 杨加虎, 王艳辉, 黄新球, 杨文. 桑树内生菌的分离鉴定及功能菌筛选[J]. 河南农业科学, 2025, 54(3): 77-86.

Figures/Tables 8

References

［1］何宁佳，赵爱春，秦俭，等. 桑树基因组计划与桑树产业［J］. 蚕业科学，2012，38（1）：140⁃145.
HE N J，ZHAO A C，QIN J，et al. Mulberry genome project and mulberry industry［J］. Science of Sericulture，2012，38（1）：140⁃145.

［2］ KATSUBE T，IMAWAKA N，KAWANO Y，et al.Antioxidant flavonol glycosides in mulberry（Morus alba L.）leaves isolated based on LDL antioxidant activity［J］. Food Chemistry，2006，97（1）：25⁃31.

［3］PARK E，LEE S M，LEE J E，et al. Anti⁃inflammatory activity of mulberry leaf extract through inhibition of NF⁃κB［J］. Journal of Functional Foods，2013，5（1）：178⁃186.

［4］陈佳佳，刘凡，廖森泰，等. 桑叶提取物抑菌活性及抑菌稳定性研究［J］. 食品工业科技，2012，33（9）：88⁃91.

CHEN J J，LIU F，LIAO S T，et al. Study on antibacterial activity and stability of extracts from mulberry Morus

alba L.）leaves［J］.Science and Technology of Food Industry，2012，33（9）：88⁃91.

［5］YANG N C，JHOU K Y，TSENG C Y.Antihypertensive effect of mulberry leaf aqueous extract containing γ⁃aminobutyric acid in spontaneously hypertensive rats［J］. Food Chemistry，2012，132（4）：1796⁃1801.

［6］CHAN K C，YANG M Y，LIN M C，et al. Mulberry leaf extract inhibits the development of atherosclerosis in cholesterol⁃fed rabbits and in cultured aortic vascular smooth muscle cells［J］. Journal of Agricultural and Food Chemistry，2013，61（11）：2780⁃2788.

［7］RICHE D M，RICHE K D，EAST H E，et al.Impact of mulberry leaf extract on type 2 diabetes（Mul⁃DM）：A randomized， placebo⁃controlled pilot study［J］.Complementary Therapies in Medicine，2017，32：105⁃108.

［8］杨晶，刘嘉琪，王宝昌，等. 桑不同药用部位中黄酮类成分的定量检测［J］.中国药房，2014，25（27）：2550⁃2552.

YANG J，LIU J Q，WANG B C，et al.Quantitative analysis of flavonoids in different medicinal parts of Morus alba［J］.China Pharmacy，2014，25（27）：2550⁃2552.

［9］HALLMANN J，QUADT⁃HALLMANN A，MAHAFFEE W F，et al. Bacterial endophytes in agricultural crops［J］.Canadian Journal of Microbiology，1997，43（10）：895⁃914.

［10］GLICK B R. Plant growth⁃promoting bacteria：Mechanisms and applications［J］. Scientifica，2012，2012：963401.

［11］翟彩霞，张彦才，刘灵娣，等. 氮、磷、钾肥对金银花产量及绿原酸、木犀草苷含量的影响［J］. 华北农学报，2012，27（S1）：328⁃332.

ZHAI C X，ZHANG Y C，LIU L D，et al.Effects of nitrogen，phosphorus and potassium fertilizer on the yield and contents of chlorogenic acid and luteolin of Lonicera japonica Thunb［J］. Acta Agriculturae Boreali⁃Sinica，2012，27（S1）：328⁃332.

［12］李金龙，熊俊芬，张海涛，等. 氮、磷、钾对滇重楼产量及皂苷含量的影响［J］.云南农业大学学报（自然科学），2016，31（5）：895⁃901.

LI J L，XIONG J F，ZHANG H T，et al. Effects of NPK fertilizer on yield and total saponin contents in Paris polyphilla var.yunnanensis［J］.Journal of Yunnan Agricultural University（Natural Science），2016，31（5）：895⁃901.

［13］张建海，冯彬彬，徐晓玉. 氮磷钾配合施用对野菊花内在品质的影响［J］.河南农业科学，2013，42（8）：92⁃97.

ZHANG J H，FENG B B，XU X Y. Effect of combined fertilization of nitrogen，phosphorus and potassium on intrinsic quality of chrysanthemi indici Flos［J］.Journal of Henan Agricultural Sciences，2013，42（8）：92⁃97.

［14］王宪菊，许艳萍，字淑慧，等. 大麻（Cannabis sativa L.）内生细菌的分离与鉴定［J］. 分子植物育种，2023，21（12）：3946⁃3956.

WANG X J，XU Y P，ZI S H，et al. Isolation and identification of endophytic bacteria in Cannabis sativa L.［J］. Molecular Plant Breeding，2023，21（12）：3946⁃3956.

［15］STIERLE A A，STIERLE D B，BUGNI T. Sequoiatones C⁃f，constituents of the redwood endophyte Aspergillus parasiticus［J］. Journal of Natural Products，2001，64 （10）：1350⁃1353.

［16］张飞官. 桑疫病原菌拮抗性桑树内生菌的筛选鉴定及其抑菌活性物质研究［D］. 重庆：西南大学，2014.

ZHANG F G. Screening， identification and antimicrobial activity of antagonistic mulberry endophytes against mulberry pest pathogens［D］.Chongqing：Southwest University，2014.

［17］王建军，刘成琴，刘涛. 一种促进通关藤生长的内生菌筛选方法：CN113388519A［P］. 2021⁃09⁃14.

WANG J J，LIU C Q，LIU T. A method for screening endophytic bacteria that promote the growth of Tengguan vine：CN113388519A［P］. 2021⁃09⁃14.

［18］何锦芝. 桑树可培养内生细菌的分离鉴定与优势菌的促生研究［D］. 重庆：西南大学，2020.

HE J Z. Isolation and identification of culturableendophytic bacteria from mulberry andgrowth promotion of dominant bacteria ［D］. Chongqing：Southwest University，2020.

［19］RAI N，KUMARI KESHRI P，VERMA A，et al. Plant associated fungal endophytes as a source of natural bioactive compounds［J］. Mycology，2021，12（3）：139⁃159.

［20］何玲敏，蔡新，毛鑫鑫，等. 基于Biolog ECO板技术的小麦内生菌多样性研究［J］.安徽农业科学，2019，47（23）：157⁃160.

HE L M，CAI X，MAO X X，et al. Biodiversity research of wheat endophytic bacteria based on Biolog ECO plate method［J］. Journal of Anhui Agricultural Sciences，2019，47（23）：157⁃160.

［21］郭玉华，尹焕松，林妃，等. 小花睡莲内生菌分离鉴定及抗菌活性评价［J］.分子植物育种，2024，22（20）：6767⁃6775.

GUO Y H，YIN H S，LIN F，et al. Isolation and identification of endophytes derived from small⁃flowered water lily and evaluation of antibiotic effects［J］. Molecular Plant Breeding，2024，22（20）：6767⁃6775.

［22］王伟，张怡荣，祝一鸣，等. 不同桉树叶片、果实和枝条内生真菌群落组成及多样性分析［J］. 仲恺农业工程学院学报，2023，36（4）：32⁃39.

WANG W，ZHANG Y R，ZHU Y M，et al. Community composition and diversity of endophytic fungi in leaves，fruits and branches of different Eucalyptus spp［J］. Journal of Zhongkai University of Agriculture and Engineering，2023，36（4）：32⁃39.
［23］张猛.三峡库区消落带桑树内生和根际真菌群落结构及其提升桑树抗逆性能的研究［D］.重庆：西南大学，2020.

ZHANG M. Study on the community structure of endophytic and rhizosphere fungi in mulberry in water⁃level⁃fluctuating zone of three gorges reservoir area and its improvement of mulberry’s stress resistance［D］.Chongqing：Southwest University，2020.

［24］ZHANG Q X，STUMMER B E，GUO Q G，et al.Quantification of Pseudomonas protegens FD6 and Bacillus subtilis NCD⁃2 in soil and the wheat rhizosphere and suppression of root pathogenic Rhizoctonia solani AG⁃8［J］. Biological Control，2021，154：104504.

［25］路国兵，冀宪领，张瑶，等.桑树内生细菌的分离及生防益菌的筛选［J］. 蚕业科学，2007，33（3）：350⁃354.

LU G B，JI X L，ZHANG Y，et al. Isolation of endophytic bacteria from mulberry and screening of beneficial bacteria［J］. Acta Sericologica Sinica，2007，33（3）：350⁃354.

［26］RATHI M，YOGALAKSHMI K N. Brevundimonas diminuta MYS6 associated Helianthus annuus L. for enhanced copper phytoremediation［J］.Chemosphere，2021，263：128195.

［27］XU T C，LU Y H，WANG J F，et al. Bioactive secondary metabolites of the genus Diaporthe and anamorph Phomopsis from terrestrial and marine habitats and endophytes： 2010⁃2019 ［J］.Microorganisms，2021，9（2）：217.

［28］窦学娥，牟志美，韩景瑞，等. 桑树内生真菌的分离、鉴定及其多样性分析［J］. 蚕业科学，2008，34（1）：6⁃10.

DOU X E，MU Z M，HAN J R，et al. Isolation and identification of endophytic fungi in mulberry and its diversity analysis［J］. Science of Sericulture，2008，34 （1）：6⁃10.

［29］张雪彬，孟磊，曹明，等. 氮肥配施硝化抑制剂对海南鲜食玉米生长及N2O排放的影响［J］. 南方农业学报，2024，55（11）：3265⁃3274.

ZHANG X B，MENG L，CAO M，et al. Effects of nitrogen fertilizer combined with nitrification inhibitor on growth and N₂O emission of fresh maize in Hainan［J］. Journal of Southern Agriculture，2024，55（11）：3265⁃3274.

［30］季美彤，刘宝勇，于皓，等. 污泥协同泥炭施加对矿区废弃地土壤养分的影响［J/OL］.山西农业科学，2024：1⁃9（2024⁃11⁃20）［2025⁃01⁃15］. https：//kns.cnki.net/kcms/detail/14.1113.S.20241118.2016.004.html.

JI M T，LIU B Y，YU H，et al. Effect of sludge combined with peat application on soil nutrients in mining wasteland［J/OL］.Journal of Shanxi Agricultural Sciences， 2024： 1⁃9 （2024⁃11⁃20）［2025⁃01⁃15］.https：//kns. cnki. net/kcms/detail/14.1113.S.20241118.2016.004.html.

［31］陈伟，王巍凝.氮肥与木醋液协同作用对甘薯生长发育的影响［J］.山西农业科学，2024，52（5）：98⁃106.

CHEN W，WANG W N. Effects of synergistic effect of nitrogen fertilizer and wood vinegar on growth and development of sweet potato［J］. Journal of Shanxi Agricultural Sciences，2024，52（5）：98⁃106.

［32］陈大江，杨青云，朱宏强，等. 不同肥料运筹模式对烤烟生长及产质量的影响［J］. 山西农业科学，2024，52（5）：107⁃113.

CHEN D J，YANG Q Y，ZHU H Q，et al. Effects of different fertilizer operation modes on growth and yield quality of flue⁃cured tobacco［J］. Journal of Shanxi Agricultural Sciences，2024，52（5）：107⁃113.

［33］马金丰，董晓杰，李志江，等. 氮肥施用对谷子产量及小米米色的影响［J］. 山西农业科学，2024，52（5）：91⁃97.

MA J F，DONG X J，LI Z J，et al. Effects of nitrogen fertilizer application on millet yield and millet color［J］. Journal of Shanxi Agricultural Sciences，2024，52 （5）：91⁃97.

［34］张银杰，苗玉红，盛开，等. 配施有机肥调控农田土壤磷素有效性研究进展［J/OL］. 河南农业大学学报，2025：1⁃15（2024⁃08⁃01）［2025⁃01⁃15］. https：//doi. org/10. 16445/j. cnki. 1000⁃2340. 20240801. 001.

ZHANG Y J，MIAO Y H，SHENG K，et al. Research progress on organic fertilizers application regulates

phosphorus availability in farmland soi［l J/OL］. Journal of Henan Agricultural University， 2025： 1⁃15（2024⁃08⁃01）［2025⁃01⁃15］. https：//doi. org/10. 16445/j. cnki. 1000⁃2340. 20240801. 001.

［35］刘新，蒋雨洲，张吉立，等. 秸秆还田条件下大豆-玉米轮作体系中磷肥用量对玉米产量和磷利用效率的影响［J］. 河南农业科学，2024，53（4）：65⁃75.

LIU X，JIANG Y Z，ZHANG J L，et al. Effects of phosphorus fertilizer application rates on maize yield

and phosphorus utilization efficiency in soybean⁃maize rotation system under straw returning conditions［J］.Journal of Henan Agricultural Sciences，2024，53（4）：65⁃75.

［36］王俊，李广鑫，李畅，等. 外源施用磷肥缓解小麦镉积累的效应［J］. 河南农业科学，2024，53（3）：78⁃86.

WANG J，LI G X，LI C，et al. Mitigation effect of exogenous application of phosphate fertilizer on cadmium accumulation in wheat［J］. Journal of Henan Agricultural Sciences，2024，53（3）：78⁃86.

［37］EGAMBERDIYEVA D. The effect of plant growth promoting bacteria on growth and nutrient uptake of maize in two different soils［J］. Applied Soil Ecology，2007，36（2/3）：184⁃189.

［38］刘晔，刘晓丹，张林利，等.花生根际多功能高效促生菌的筛选鉴定及其效应研究［J］.生物技术通报，2017，33（10）：125⁃134.
LIU Y，LIU X D，ZHANG L L，et al. Screening，identification of multifunctional peanut root⁃promoting rhizobacteria and its promoting effects on peanuts（Arachis hypogaea L. ）［J］. Biotechnology Bulletin，2017，33（10）：125⁃134.
［39］杨苗. 具有ACC脱氨酶活性促生细菌的筛选、鉴定及其接种效应研究［D］. 杨凌：西北农林科技大学，2018.
YANG M. The Study on Isolation and Identification of growth Promoting bacteria Pruducing ACC deaminase and Inoculation Effests［D］. Yangling：Northwest A & F University，2018.
［40］王继雯，谢宝恩，周伏忠，等. 高产吲哚乙酸及高泌氨巴西固氮螺菌的筛选与鉴定［J］.生物技术，2009，19（6）：14⁃17.
WANG J W，XIE B E，ZHOU F Z，et al. Isolation and identification of Azospirillum brasilense excreting high concentration of IAA and ammonium ［J］.Biotechnology，2009，19（6）：14⁃17.
［41］姜云，冉超，陈长卿，等.人参抗病、促生菌的筛选及田间效果评价［J］. 中国中药杂志，2018，43（11）：2230⁃2235.
JIANG Y，RAN C，CHEN C Q，et al. Evaluation of isolation and field effect of ginseng disease resistance and growth⁃promoting bacteria［J］.China Journal of Chinese Materia Medica，2018，43（11）：2230⁃2235.
［42］刘俊清，金晓雪，宋科稷，等. 一株溶磷真菌的筛选及相关促生特性初探［J］.安徽大学学报（自然科学版），2023，47（3）：90⁃97.
LIU J Q，JIN X X，SONG K J，et al. Screening and identification of a high⁃efficient phosphate⁃solubilizing fungus Penicillium decumbens and analysis of its phosphate⁃solubilizing characteristics［J］. Journal of Anhui University（Natural Science Edition），2023，47（3）：90⁃97.

[1]	ZHANG Chao, LÜ Jun. Isolation，Identification and Antibiotic Susceptibility Test of Aeromonas caviae from Common Carp（Cyprinus carpio） [J]. Journal of Henan Agricultural Sciences, 2025, 54(1): 153-159.
[2]	PENG Zhifeng, ZHANG Han, HUANG Huimin, SONG Xinghui, ZHU Yabo, LI Ziyi, QIAO Hongxing, BIAN Chuanzhou. Isolation，Identification and Susceptibility Analysis of One Morganella morganii Strain from Swine [J]. Journal of Henan Agricultural Sciences, 2024, 53(9): 126-131.
[3]	LA Guixiao, WANG Linlin, GUO Junqi, ZHAO Yulong, GUO Hongxia, JIA Hui, YANG Tiegang. The Complete Gene Sequencing of Bacilus subtilis Isolated from Rehmannia glutinosa and Study of Genes Related to Growth‑promoting Effects [J]. Journal of Henan Agricultural Sciences, 2024, 53(12): 75-85.
[4]	LI Shanmin, ZENG Xinhua, HUANG Weichang, FU Hao, HUANG Qingjun. Effects of Different Forest Type on the Growth of Bletilla striata and Diversity of Endophytic Fungi [J]. Journal of Henan Agricultural Sciences, 2023, 52(7): 70-80.
[5]	ZHAO Wenying, ZHANG Yunjing, BAI Xiaofei, SUN Yujie, GUO Linghua, HUANG Baicheng, TIAN Kegong. Isolation，Identification and Whole Genome Sequence Analysis of Porcine Parvovirus BJ2 Strain [J]. Journal of Henan Agricultural Sciences, 2023, 52(2): 136-144.
[6]	WU Jiayu, HU Wei, YANG Zhiyu, YANG Hongyi, LI Lili. Recent Advances in the Interaction between Mycorrhizal Fungi and Different Fungi in Plant Root Microecosystem [J]. Journal of Henan Agricultural Sciences, 2022, 51(2): 1-9.
[7]	SU Zhuowen, LU Caige, CAI Shulin, ZHANG Taotao, DONG Dan, ZHAO Juan, ZHANG Dianpeng. Screening and Identification of Antagonistic Endophytic Bacteria against Asparagus Stem Blight [J]. Journal of Henan Agricultural Sciences, 2022, 51(10): 106-113.
[8]	ZHANG Yuqi, REN Yueying, WEN Xiangsui, WANG Yumeng, FANG Ping, LI Baoquan, LENG Shihe. Screening and Control Effect of Endophytic Fungi for Brown Spot Disease of Schisandra chinensis [J]. Journal of Henan Agricultural Sciences, 2021, 50(8): 94-102.
[9]	JIN Qianyue, GUO Yonggang, LI Junpeng, QIN Baoliang, WANG Yinbiao, GUO Zhenhua, WANG Li, XING Guangxu, DENG Ruiguang, ZHANG Gaiping. Isolation,Identification and Genetic Characterization of Goose Astrovirus Isolate XX [J]. Journal of Henan Agricultural Sciences, 2021, 50(6): 134-141.
[10]	TENG Zheng, YANG Cuifeng, JIA Guikang, LAN Zhaoyun, GU Lili. Study on Screening and Biological Characteristics of Endogenous Manganese-tolerant Bacteria in Conyza canadensis [J]. Journal of Henan Agricultural Sciences, 2021, 50(4): 72-78.
[11]	JIA Yanyan, LI Qi, WANG Xiaoli, NIU Junhui, MAO Jing, LIAO Chengshui, ZHANG Haokai, ZHANG Mingliang. Isolation,Identification and Nuclease Activity of Extracellular Secretory Protein of Klebsiella pneumoniae in Mink [J]. Journal of Henan Agricultural Sciences, 2021, 50(3): 142-147.
[12]	DU Cuiying, LU Jiayi, HU Lang, YAO Guoxin, DAI Yujun, LI Jing, ZHENG Cao. Isolation and Identification of Soil Bacterial Strains Metabolizing Propionic Acid [J]. Journal of Henan Agricultural Sciences, 2021, 50(10): 69-75.
[13]	DENG Longjun. Isolation and Identification of Aeromonas veronii from Micropterus salmoides and Pathological Lesions of Its Infection [J]. Journal of Henan Agricultural Sciences, 2021, 50(1): 164-171.
[14]	TIAN Qizhen, TANG Chenchen, SHEN Danqing, HUANG Yinyun, CAI Bingyan. Isolation,Identification and Prebiotic Characteristics of Three Strains of Lactobacillus from Pastoral Dogs [J]. Journal of Henan Agricultural Sciences, 2020, 49(8): 149-155.
[15]	GAO Zhenfeng, LI Na, ZHANG Xiaoyu, HAN Jucai. Screening and Colonization Characteristics of Efficient Endophytic Antifungal Bacteria against Botrytis cinerea [J]. Journal of Henan Agricultural Sciences, 2020, 49(3): 88-100.