Rajamangala University of Technology Tawan-ok International Conference, Thailand, 29-31 May, 2013
INDUCTION OF PLANT DEFENSE RELATED GENE EXPRESSION IN CHILI BY
PLANT ELICITORS AND ACTINOMYCETES CULTURE FILTRATE
Nisakorn Suwan1,2*, Sarunya Na-Lampang2,3
1
Department of Plant Science, Faculty of Ago-industrial Technology, Rajamangala
University of Technology Tawan-Ok, Chantaburi, 22210, Thailand.
2
Center of Excellence on Agricultural Biotechnology (Ag-BIO/PERDO-CHE), Bangkok
10900, Thailand
3
Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai
University, Chiang Mai 50200, Thailand
*E-mail: liu.nisakorn@gmail.com, Fax: (6639)307268
Abstract: The inducible defenses are subjected to prevent pathogen invasion infection and
colonization in chili seedlings. There are a various mechanism to activate such as used of
chemical elicitors, abiotic stress-wounding and antagonist bacteria to induce defense genes.
All defense related gene expressions were detected with differed pattern. The recognition
event leads to triggers a large range of inducible defense mechanisms and activates
expression of some plant defenses related genes including CHS (chalcone synthase) and LOX
(lipoxygenase) were investigated. To activate defense genes in chili plants, culture nonfiltrates of the Streptomyces NSP-1 (NF-NSP-1), abiotic stress-wounding and pathogen
inoculated were introduced to four-week-old chili plants. The transcription level of each gene
was analyzed by qRT-PCR. The results implied that CHS mRNA levels were markedly
increased within 24 h when plants were treated with NF-Streptomyces NSP-1. According to
treatment of MeJA was highly induction of LOXs expressed, after treated and Streptomyces
NSP1-NF treated was greatly induced within 12 h after treated. These results indicated that
Streptomyces NSP-1 (NF-NSP-1) could be involved in the action of defense-eliciting
molecules in plant defense mechanism.
Keywords:
bacteria.
Colletotrichum, Streptomyces, Capsicum, defenses mechanism, antagonist
Introduction: The anthracnose of chili is a problem arising throughout chili growing area
such as Thailand, Pakistan, Turkey and Mexico (Than et al., 2008). Colletotrichum
gloeosporioides and C. capsici is an important pathogenic fungi causing chili anthracnose
disease (Than et al., 2008), affecting chili pepper (Capsicum annuum), one of the importance
economically crop in Thailand, with severely devastation of chili fruits and stem as well. The
disease control of this disease has commonly relied on chemicals fungicide. Conversely, no
effective fungicides are available and chemical resistant of fungicides were concerned. In
present time, attempts to finding for the biological control agent of anthracnose disease by
using bacterial antagonists to induce defense mechanisms in plant were challenged (Howell,
2003). Streptomyces spp. are important soil bacteria which ubiquitous in soil and well known
as producers of antibiotics, extracellular enzymes (Crawford et al. 1993) and secondary
metabolites production (Trejo-Estrada, et al., 1998). The induction of plant defense
mechanism mediated by soil bacteria, Streptomyces spp. on foliar diseases has not been
documented previously. In the present study, the time-course expression levels of several
defense genes of chili seedling involved in exogenous application of methyl salicylic acid
(MeSA) methyl jasmonic acid (MeJA), oligomer-chitosan, wounding, Streptomyces sp.
Rajamangala University of Technology Tawan-ok International Conference, Thailand, 29-31 May, 2013
culture filtrate and challenge with virulent C. gloeosporioides were studied for induced
expression of defense gene group in chili plants.
Methodology: To induce resistance, four-week-old chili plants each having two or four
expanded leaves were separately treated with six different methods as follow:- (1) Methyl
jasmonic acid (2) Methyl salicylic acid (3) Oligomer chitosan (4) wounding (5) culture nonfiltrate (NF) of Streptomyces sp. isolate NSP-1 (6) pathogen inoculation. The seedling leaves
were collected at 0, 12 and 24 h after induced resistance with different, quickly frozen in
liquid nitrogen, and stored at 80oC until required. The experiment was independently repeated
with three replications. The total mRNA of treated chilies was isolated. The expression
levels of Pathogen Related genes; Chalcone Synthase (CHS) and Lipoxygenase (LOXs),
respectively were analyzed by quantitative real-time PCR.
Results, Discussion and Conclusion: In order to examine the expression pattern of plant
defense genes group in response to MeSA, MeJA, oligomer-chitosan, wounding,
Streptomyces NSP1-NF and challenged inoculation of C. gloeosporioides to chili seedlings,
the total mRNA of chili were isolated and the corresponding primes were set according to the
sequence of plant defense genes isolated from chili. At 12 h after treated with MeSA the
expression of CHS (Campos et al., 2003) and LOX (Marmey et al., 2007) were induced at
high level. The CHS gene was up-regulated as consequent treated with pathogen inoculation
and NSP1-NF, 23.00 and 10.22 fold, respectively. The similar pattern of inducing CHS gene
by wounding and pathogen inoculations were demonstrated high induction after treated and
higher after 24 HAI accept for chitosan (1.39 fold) was exhibited higher after treated, The
treatments of MeJA (3.10 fold) and NSP1-NF were exhibited gradually increasing and higher
at 24 HAI (Figure 1). According to this experiment results was agreement with Compos et
al., (2003), the induction of enzymes CHS activity in common bean (cvs. Rio Tibagi and
Carioca) by salicylic acid was detected. Hence the induction of CHS in the production of
phytoalexin isoflavonoids was depended on plant cultivars and treatments (Tepper et al.,
1989) and the accumulation of phytoalexins (Hartleb, et al., 1997). Furthermore, the mRNA
accumulation would directly affect the biosynthesis of phenols and, possibly, CHS was
involved in the increase of these compounds during infection (Hartleb et al., 1997). The
relative expression levels of lipoxygenase (LOXs) was exhibited the highest increasing when
compared with mock treatment of MeJA and NSP1-NF treatments for 5.49 and 4.70 fold
simultaneously and after 12 HAI, respectively.
Rajamangala University of Technology Tawan-ok International Conference, Thailand, 29-31 May, 2013
Figure 1 Relative expression levels of CHS gene (A) Streptomyces NSP-1 metabolite, (B)
chitosan, (C) C. gloeosporioides spore suspension, (D) MeJA, (E) wounding and (F)
MeSA. Values are means SE and indicated with a bar.
The induction of LOXs gene was declined rapidly to the control level after reached their
highest level of induction. The expression levels of other treatments were increasing
gradually after 12 HAI and decreasing after 24 HAI (Figure 2).
Rajamangala University of Technology Tawan-ok International Conference, Thailand, 29-31 May, 2013
Figure 2 Relative expression levels of LOXs gene. (A) Streptomyces NSP-1 metabolite, (B)
chitosan, (C) C. gloeosporioides spore suspension, (D) MeJA, (E) wounding and (F) MeSA.
Values are means SE and indicated with a bar.
The chili seedlings were treated with MeJA revealed the induction of LOX simultaneously
although decreased gradually after 12 h. in agreement with Marmey et al. (2007), reported
that both salicylic acid (SA) and methyl-jasmonate (MeJA) were induced LOX activity and
GhLOX1 gene expression in cotton and lead to cell death in response to HR.
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Acknowledgements: This Research is partially supported by the Center of Excellence on
Agricultural Biotechnology, Science and Technology Postgraduate Education and Research
Development Office, Commission on Higher Education, Ministry of Education. (AGBIO/PERDO-CHE) Bangkok, Thailand, 10900 and Prof. Dr. Kazuya Akimitzu, Dept. of Life
Science, Faculty of Agriculture, Kagawa University, Japan.