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"

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