Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (18): 3520-3530.doi: 10.3864/j.issn.0578-1752.2018.18.008

• PLANT PROTECTION • Previous Articles     Next Articles

Identification of Pathogens Causing Brown Spot and the Role of MeJA in Disease Resistance in Stevia rebaudiana

XiaoXia CUI(), HongMei SHU, Lu JIANG, XiaoLan HE, YuanYong GONG, WanChao NI, ShuQiao GUO()   

  1. Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014
  • Received:2018-04-18 Accepted:2018-05-18 Online:2018-09-16 Published:2018-09-16

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Abstract:

【Objective】The objective of this study is to identify the pathogenic fungus causing brown spot disease on Stevia rebaudiana, and analyze the role of MeJA in response to Alternaria to provide a basis for disease prevention and resistance breeding. 【Method】 The samples of diseased S. rebaudiana leaves were collected from the stevia production base of Fuan Town, Dongtai City, Jiangsu Province. After isolation and purification, the isolates were identified by using the methods of morphological characteristics analysis. Morphology parameters mainly included the colony and conidial morphology, conidial size. The isolates pathogenicity was tested on detached S. rebaudiana leaves. The internal transcribed spacer (ITS) rDNA region of the 7 pathogenic strains was amplified by using the fungal universal primers ITS1 and ITS4, and then the amplified product was recovered and sequenced. MEGA 7 was used to construct phylogenetic tree to confirm the pathogen species based on the rDNA-ITS sequence and the related Alternaria sequences in GenBank. The germination status of A. tenuissima conidial spores and the way they invaded the leaves after stained with trypan blue were analyzed by microscopy. The effect on the growth of A. tenuissima mycelium was analyzed by exogenous application of MeJA to potato dextrose agar (PDA) medium. To test the role of MeJA in the S. rebaudiana resistance to A. tenuissima, detached leaves were fed with MeJA and then used for infection. The qPCR method was performed to analyze the expression of JA pathway-related genes before or after inoculation with A. tenuissima in leaves of S. rebaudiana. 【Result】 Seven Alternaria strains were collected from the diseased S. rebaudiana leaves. All the strains grew on the PDA medium in a nearly circular and equal diameter radiation. The aerial hyphae were relatively developed, with white at the beginning and gradually changed to different degrees of grayish black at the later stage. The conidia are solitary or in a chain, mostly nearly spherical, inverted bar-shaped, or inverted pear-shaped, with a size of (20.5-45.5) × (6.5-16.0) μm. The isolated 7 strains were inoculated on the detached leaves of S. rebaudiana, and the pathogenicity was different. ST2, ST3 and ST7 strains led to the lesions spread rapidly with stronger pathogenicity. The rDNA-ITS sequence length of the ST2, ST3, and ST7 strains was 569, 570, and 570 bp, respectively. Phylogenetic tree analysis indicated that the similarity of ST2 and ST3 with strains KY814634.1 and DQ491089.1 (Alternaria alternata, Alternaria sp.) is between 99%-100%, and similarity between ST7 and strain HQ402558.1 (Alternaria tenuissima) is 99%. The results of trypan blue staining of S. rebaudiana leaves inoculated with A. tenuissima showed that conidia of ST7 could germinate from the head, lateral and caudal of the spores, and the A. tenuissima mycelia could invade into epidermal cells from the stomata or the epidermis intercellular. Exogenous application of MeJA could effectively inhibit the growth of A. tenuissima mycelia when the concentration of MeJA was higher than 200 μmol·L-1. After inoculated with A. tenuissima, the lesion area of detached leaves that fed with 100 μmol·L-1 MeJA was significantly smaller than that of the control, indicating that MeJA could enhance the S. rebaudiana resistance to A. tenuissima. JA pathway related genes were involved in the response of S. rebaudiana to A. tenuissima, the expression of LOX3 and JAR1 was up-regulated while the expression of JAZ1 and JAZ4 was down-regulated after S. rebaudiana inoculated with A. tenuissima.【Conclusion】The pathogenic fungus that caused S. rebaudiana leaf brown spot in the stevia production base of Fuan Town, Dongtai City, Jiangsu Province was identified as Alternaria. A. tenuissima mycelia can invade epidermal cells from the stomata or the epidermis intercellular space of the leaves. The resistance of S. rebaudiana leaves to A. tenuissima was enhanced after exogenous application of MeJA, which will become the potential candidates for the control of S. rebaudiana brown spot disease in the field.

Key words: Stevia rebaudiana, brown spot disease, Alternaria, identification of pathogens, methyl jasmonate, signaling pathway

Table 1

List of qPCR primer sequences used in this study"

引物名称
Primer name		 核酸序列
Primer sequence (5′-3′)		 基因登录号
Accession number
SrJAR1-F
SrJAR1-R
SrLOX3-F
SrLOX3-R
SrJAZ1-F
SrJAZ1-R
SrJAZ4-F
SrJAZ4-R
26 S rRNA-F
26 S rRNA-R		 ACCGATGTCAAGCTCGGTGA
GGGTCGCGTTATGGAACCCA
TGCTTTGGCTCGAGAAGCAC
TCAGCTGGCAATGCTTCATGGT
GCCGTGCAGAAGCAGCATGA
CGTCTGGTTTCGATGGTTTCCG
GCACCACAACCAAGGACCCA
GAGACGGGTGAGCAGGGTTG
CAAAACGACTCTCGGCAACG
CACACACGAGACACTCCGAT		 BG523392.1

BG524650.1

BG525787.1

BG526796.1

AB457301.1

Fig. 1

Morphological characteristics of isolated strains Colony morphology of ST1-ST7 which cultured on potato dextrose agar at 25℃ for 10 days, from above and below"

Fig. 2

Conidial morphology of 7 isolated strains"

Fig. 3

Symptoms on S. rebaudiana leaves after inoculated with isolated fungi strains ST1-ST7, respectively The phenotype of S. rebaudiana leaves inoculated with ST1-ST7 strains, respectively;The control leaf inoculated with PDA medium"

Fig. 4

PCR results of 7 Alternaria rDNA-ITS"

Fig. 5

Phylogenetic tree of 7 strains from S. rebaudiana and related species of Alternaria based on sequences of rDNA-ITS region"

Fig. 6

Germination and invasion of A. tenuissima conidia on S. rebaudiana leaves The A. tenuissima conidia of ST7 germinated from the head (A), lateral (A, B) and caudal (C) of the spores on the S. rebaudiana leaves of cultivar Shoutian 3 at 6 h post inoculation; mycelium invaded into leaf epidermal cells from the leaf stomata (D, E, F) and epidermal cell gap (G, H) at 24 h post inoculation"

Fig. 7

Effect of MeJA on mycelial growth of A. tenuissima ST7 on PDA medium"

Fig. 8

Exogenous application of MeJA enhances the resistance of S. rebaudiana leaves to A. tenuissima The phenotype of S. rebaudiana leaves which inoculated with ST7 after feeding 100 μmol·L-1 MeJA for 10 h;II： The phenotype of S. rebaudiana leaves which inoculated with A. tenuissima ST7 after petiole feeding ddH2O;III： The control which leaves inoculated with PDA medium。 The time points for taking pictures and measuring the disease spots are 3, 4, 5 days after inoculation"

Fig. 9

Expression pattern of JA pathway related genes in response to A. tenuissima infection"

[1] 刘琼, 潘芸芸, 吴卫. 甜叶菊化学成分及药理活性研究进展. 天然产物研究与开发, 2018, 30(6): 1085-1091.
LIU Q, PAN Y Y, WU W.Review on chemical compositions and pharmacological activities ofStevia rebaudiana(Bertoni) Hemsl. Natural Product Research and Development, 2018, 30(6): 1085-1091. (in Chinese)
[2] CHATURVEDULA V S, CLOS J F, RHEA J, MILANOWSKI D, MOCEK U, DUBOIS G E, PRAKASH I.Minor diterpene glycosides from the leaves ofStevia rebaudiana. Phytochemistry Letters, 2011, 4(3): 209-212.
[3] YADAV A K, SINGH S, DHYANI D, AHUJA P S.A review on the improvement of stevia [Stevia rebaudiana(Bertoni)]. Canadian Journal of Plant Science, 2011, 91(1): 1-27.
doi: 10.4141/cjps10086
[4] 吴则东, 张文彬, 吴玉梅, 刘乃新. 世界甜叶菊发展概况. 中国糖料, 2016, 38(4): 62-65.
doi: 10.13570/j.cnki.scc.2016.04.019
WU Z D, ZHANG W B, WU Y M, LIU N X.Review ofStevia development in the world. Sugar Crops of China, 2016, 38(4): 62-65. (in Chinese)
doi: 10.13570/j.cnki.scc.2016.04.019
[5] 徐鸿. 甜菊糖的特点及功能. 现代农业科技, 2013(2): 286-287.
doi: 10.3969/j.issn.1007-5739.2013.02.183
XU H.Characteristic and functions of stevioside. Modern Agricultural Sciences and Technology, 2013(2): 286-287. (in Chinese)
doi: 10.3969/j.issn.1007-5739.2013.02.183
[6] 张天宇. 中国真菌志. 第16卷. 链格孢属. 北京: 科学出版社, 2003.
ZHANG T Y. Flora Fungorum Sinicorum.Vol. 16. Alteranria .Beijing: Science Press, 2003. (in Chinese)
[7] FARR D F, BILLS G F, CHAMURIS G P, ROSSMAN A Y.Fungi on Plants and Plant Products in the United States. St. Paul (Minnesota): The American Phytopathological Society (APS) Press, 1989.
[8] 陈伟群. 链格孢及相似属代表种的分子系统学研究[D]. 杨凌: 西北农林科技大学, 1997.
CHEN W Q.Molecular phylogenetic study of Alternaria and similar genera[D]. Yangling: Northwest A&F University, 1997. (in Chinese)
[9] TISDALE W B, WADKINS R F.Brown spot of tobacco caused byAlternaria longipes(E. & E.), n. comb. Phytopathology, 1931, 21(6): 641-660.
[10] YU Q.Loss rate estimation of yield and output value of tobacco leaf infected by tobacco blown spot (Alternaria alternata). Plant Diseases and Pests, 2010, 1(6): 23-27, 63.
[11] 李金花, 柴兆祥, 王蒂, 李敏权. 甘肃马铃薯贮藏期真菌性病害病原菌的分离鉴定. 兰州大学学报 (自然科学版), 2007, 43(2): 39-42.
doi: 10.3321/j.issn:0455-2059.2007.02.009
LI J H, CHAI Z X, WANG D, LI M Q.Isolation and identification of the pathogens of potato fungus diseases during storage in Gansu Province. Journal of Lanzhou University (Natural Sciences), 2007, 43(2): 39-42. (in Chinese)
doi: 10.3321/j.issn:0455-2059.2007.02.009
[12] 邵玉琴, 吕佩珂. 番茄早疫病发生、流行与生态因子关系的研究. 内蒙古大学学报 (自然科学版), 1993, 24(2): 208-211.
SHAO Y Q, LÜ P K.A study about the relationship betweenAlternaria solani on the happening prevalence and ecological factors of tomatoes. Journal of Inner Mongolia University (Natural Science Edition), 1993, 24(2): 208-211. (in Chinese)
[13] ISHIBA C, YOKOYAMA T, TANI T.Black spot disease of stevia caused byAlternaria steviae. Annals of the Phytopathological Society of Japan, 1982, 48(1): 44-51.
[14] MAITI C K, SEN S, ACHARYA R, ACHARYA K.First report ofAlternaria alternata causing leaf spot on Stevia rebaudiana. Plant Pathology, 2007, 56(4): 723.
[15] ATGHIA O, JAVAN-NIKKHAH M, POURSAFAR A, KHOJASTEH M, ESLAMZADEH B.First report of Alternaria species as the causal agent of leaf spot on Stevia and Catalpa plants in Iran//2nd Iranian Mycological Congress . University of Tehran, Karaj,Iran, 2015: 130.
[16] 魏景超. 真菌鉴定手册. 上海: 上海科学技术出版社, 1979.
WEI J C. Fungal Identification Manual.Shanghai: Shanghai Scientific and Technical Publishers, 1979. (in Chinese)
[17] SUN H H, WANG L, ZHANG B Q, MA J H, HETTENHAUSEN C, CAO G Y, SUN J L, WU J Q, WU J S.Scopoletin is a phytoalexin againstAlternaria alternata in wild tobacco dependent on jasmonate signalling. Journal of Experimental Botany, 2014, 65(15): 4305-4315.
doi: 10.1093/jxb/eru203 pmid: 4112635
[18] GEUNS J M C. Stevioside. Phytochemistry, 2003, 64(5): 913-921.
doi: 10.1016/S0031-9422(03)00426-6
[19] PHILIPPAERT K, PIRONET A, MESUERE M, Sones W, Vermeiren L, Kerselaers S, Pinto S, Segal A, Antoine N, Gysemans C, Laureys J, Lemaire K, Gilon P, Cuypers E, Tytgat J, Mathieu C, Schuit F, Rorsman P, Talavera K, Voets T, Vennekens R.Steviol glycosides enhance pancreatic beta-cell function and taste sensation by potentiation of TRPM5 channel activity. Nature Communications, 2017, 8: 14733.
doi: 10.1038/ncomms14733 pmid: 23850737
[20] GEUNS J.Stevia and Steviol Glycosides. Euprint, 2010.
[21] GEUNS J, STRUYF T.Radical scavenging activity of steviol glycosides and steviol glucuronide. Emergency Medicine Journal, 2010, 6(8): 2025-2028.
doi: 10.1136/emj.6.4.282
[22] HAJIHASHEMI S, GEUNS J M C. Free radical scavenging activity of steviol glycosides, steviol glucuronide, hydroxytyrosol, metformin, aspirin and leaf extract ofStevia rebaudiana. Free Radicals and Antioxidants, 2013, 3(44): S34-S41.
[23] 唐志发. 甜菊糖的崛起与发展战略. 中国食品工业, 1999, 6(2): 52.
TANG Z F.The rise and development strategy of stevioside. China Food Industry, 1999, 6(2): 52. (in Chinese)
[24] 朱东顺, 宋晴晴, 傅在秋, 臧义战. 山东省甜叶菊的主要病害及防治措施. 中国糖料, 2002(3): 27-29.
doi: 10.3969/j.issn.1007-2624.2002.03.008
ZHU D S, SONG Q Q, FU Z Q, ZANG Y Z.The diseases and control measures ofStevia rebaudiana in Shandong Province. Sugar Crops of China, 2002(3): 27-29. (in Chinese)
doi: 10.3969/j.issn.1007-2624.2002.03.008
[25] 柴再生, 蒋宏, 王亮. 河西走廊甜叶菊拱棚育苗三大病害发生原因及防治策略. 中国糖料, 2017, 39(4): 79-80.
doi: 10.13570/j.cnki.scc.2017.04.026
CHAI Z S, JIANG H, WANG L.Causes and prevention strategies of three major diseases in stevia nursery seedlings of Hexi Corridor. Sugar Crops of China, 2017, 39(4): 79-80. (in Chinese)
doi: 10.13570/j.cnki.scc.2017.04.026
[26] BLÉE E. Impact of phyto-oxylipins in plant defense. TRENDS in Plant Science, 2002, 7(7): 315-321.
doi: 10.1016/S1360-1385(02)02290-2 pmid: 12119169
[27] GUO Q, MAJOR I T, HOWE G A.Resolution of growth-defense conflict: mechanistic insights from jasmonate signaling. Current Opinion in Plant Biology, 2018, 44: 72-81.
doi: 10.1016/j.pbi.2018.02.009 pmid: 29555489
[28] GRIMES H D, KOETJE D S, FRANCESCHI V R.Expression, activity, and cellular accumulation of methyl jasmonate-responsive lipoxygenase in soybean seedlings. Plant Physiology, 1992, 100(1): 433-443.
doi: 10.1104/pp.100.1.433 pmid: 16652980
[29] CHINI A, BOTER M, SOLANO R.Plant oxylipins: COI1/JAZs/ MYC2 as the core jasmonic acid-signalling module.The FEBS Journal, 2009, 276(17): 4682-4692.
doi: 10.1111/j.1742-4658.2009.07194.x pmid: 19663905
[30] MOSBLECH A, THUROW C, GATZ C, FEUSSNER I, HEILMANN I.Jasmonic acid perception by COI1 involves inositol polyphosphates inArabidopsis thaliana. The Plant Journal, 2011, 65(6): 949-957.
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