Pest Management in Horticultural Ecosystems,
Vol. 18, No. 1 pp 60-65 (2012)
Lactic acid bacteria (LAB) as plant growth promoting bacteria (PGPB)
for the control of wilt of tomato caused by Ralstonia solanacearum
K. NARASIMHA MURTHY, M. MALINI, J. SAVITHA and C. SRINIVAS
Department of Studies in Microbiology and Biotechnology, Bangalore University, Jnanabharathi Campus,
Bangalore – 560056, Karnataka, India.
E-mail: [email protected]
ABSTRACT: Bacterial wilt caused by Ralstonia solanacerum is one of the production constraints of tomato (Lycopersicon
esculentum Mill). A study was conducted to evaluate the potential of Lactic acid Bacteria (LAB) to suppress the development
of wilt pathogen and their role as Plant Growth Promoting Bacteria (PGPB) in tomato. R. solanacerum was isolated from
infected tomato plants and their soil samples on triphenyl tetrazolium chloride (TZC) medium. In the invitro screening, two
LAB strains showed maximum inhibition against wilt pathogen. Seed treatment with LAB strains significantly improved the
quality of seed germination and seedling vigour. Efficacy of both the strains were evaluated under green house conditions
in suppressing incidence of bacterial wilt disease and promoting plant growth in susceptible tomato plant varieties. The
disease incidence was significantly reduced by about 63% in plants raised from seed treatment and soil drench method. The
present work demonstrates the ability of LAB to act as plant growth promoting bacteria and biocontrol agent against R.
solanacearum in vivo.
Key words: Lactobacillus paracasei subsp. tolerans, Lactobacillus paracasei subsp. paracasei, Ralstonia solanacearum,
tomato wilt, PGPB.
INTRODUCTION
Bacterial wilt caused by Ralstonia solanacearum is
deemed to be one of the most important plant diseases
in tropical agriculture (Hayward, 1991, Milling et al.,
2011). It has a large host range of more than 200
species in 50 families (Aliye et al., 2008). The disease
also affects other economically important crops such as
potato, eggplant, chilly and non solanaceous crops such
as banana and groundnut in India (Anuratha et al., 1990).
In India, a study showed 10 to 100% incidence of
bacterial wilt during the summer (Kishun, 1985). Infested
soil and water are the primary sources of inoculum. The
pathogen infects roots of susceptible plants, usually
through wounds and Colonizes within the xylem
preventing the water movement into upper portion of the
plant tissue (Pradhanang et al., 2005) R. solanacearum
is a Gram negative, rod shaped, strictly aerobic
bacterium, 0.5-0.7 µm x 1.5-2.0 µm in size, with a single
polar flagellum. Individual bacterial colonies are usually
visible after 36 to 48 hrs of growth at 30 ± 2 0C and
colonies of the normal or virulent type are white or cream
coloured with pink centered (Fig. 1), irregularly shaped,
highly fluidal and opaque. Occasionally colonies of the
mutant or non virulent type appear uniformly round,
smaller and butyrous or dry. A Kelman's selective nutrient
Tetrazolium chloride (TZC) medium (Kelman, 1954) can
differentiate the two colony types on this medium.
Fig 1. R. solanacearum on TZC agar plate
Management of bacterial wilt in tomato caused by
R. solanacearum has been difficult and it still threatens
commercial tomato production. Chemicals may be an
effective method in controlling many bacterial diseases,
but these chemicals potentially cause negative impact on
plant growth or yield. Lactic Acid Bacteria (LAB) are
known to improve human and animal health (probiotics)
and recognized as safe (Stiles, 1996). Several members
of the lactic acid bacteria are known to produce
antibacterial substances. The antibacterial effect has been
ascribed to the production of antibiotics or antibiotic like
60
Lactic acid bacteria as plant growth promoter
substances such as acidophilin, lactocidin, lactolin, nisin
etc and various inhibitory substances such as lactic,
acetic, probionic acids, bacteriocins (Ouwehand, 1998).
Many of the antimicrobial compounds affect the
physiological activities of a pathogen such as cell division,
biosynthesis of DNA, RNA, protein, lipid metabolism and
cell synthesis (Chaurasia et al., 2005). However,
information on the occurrence of lactobacilli on living
plants is scarce, and no information is available on the
interactions of plant-associated lactic acid bacteria with
phytopathogenic bacteria.
centrifuged at 12 000 g for 10 min at 10°C. The pellet
was resuspended in distilled water and was
adjusted spectrophotometrically to 108 CFU per ml (Ran
et al. 2005).
Seed bacterization
Ten mL of LAB inoculum containing 3 x 108 cfu
ML was added to Petri plates. Surface sterilized tomato
seeds (Guo et al., 2004) were soaked in a culture
suspension for 6h, amended with (0.2%) caboxymethyl
cellulose (CMC). After incubation, the seeds were air
dried overnight aseptically. Seeds soaked in distilled water
amended with CMC (0.2%) served as control.
-1
MATERIALS AND METHODS
Isolation and identification of R. solanacearum
The infected plant material and soil samples were
collected from the different agro climatic zones of
Karnataka and other parts of India. The collected
rhizosphere soil and surface sterilized plant samples were
plated onto SMSA (Elphinstone et al. 1998; Wenneker
et al. 1999) and TZC (Kelman 1954) media by serial
dilution and direct plating methods, respectively. The
plates were incubated at 28 ± 20C for 24-48 h. Isolation
was also done from a freshly wilted tomato plant by
streaking the bacterial ooze onto TZC agar plates (Danks
et al., 2000).
Effect of LAB on seed germination and seedling
vigor of tomato under Lab conditions
The effect of R. solanacearum and LAB strains on
seed germination and vigour of seedlings was evaluated
under laboratory conditions. Three different wilt
susceptible tomato cultivars viz., Arka Meghali, Arka
Sourabh, Arka Vikas were procured from IIHR Bangalore,
India. The germination tests were carried out according
to the paper towel method (ISTA 2005). Seeds treated
with distilled water served as negative controls. One
hundred seeds were placed at equidistant on the
germination paper presoaked in distilled water and
covered with another presoaked paper towel and
wrapped with polythene to prevent drying of towels. The
rolled towels were incubated for ten days at 24 ± 20C.
After incubation, paper towels were unrolled and
germinated seeds were counted and represented in
percentage. The vigour index was calculated by using
the formula VI = (mean root length + mean shoot length)
x Germination percentage (Abdul Baki and Anderson
1973). The experiment was conducted with four
replicates of hundred seeds each and the entire
experiment was repeated thrice.
Identification of Ralstonia solanacearum
The suspected colonies were observed for colony
characteristics, biochemical, physiological, hypersensitive
and pathogenicity tests for con?rmation of the identity
of the pathogen (Vanitha et al., 2009 and Narasimha
Murthy et al., 2012). Biovar characterization was carried
out according to Hayward (Hayward, 1964).
Isolation and identification of Lactic Acid Bacteria
(LAB)
Lactic Acid Bacteria were isolated from soil, dairy
products and environmental waste samples by standard
spread plate technique on MRS agar (Brashear et al.,
2003). Plates were incubated at 35 0 C for 24h.
Identification was done by studying the morphological
and biochemical characterization. The isolates were
further confirmed by VITEK 2 system (Biomeriux, USA)
and 16S r DNA.
In vivo experimental design
The efficacy of LAB to control bacterial wilt of
tomato in the greenhouse was tested. Seeds soaked
overnight in LAB culture broth and non soaked seeds
were sown in seedling trays with 98 eyes with sterilized
soil and coconut pith compost for seed treatment method
and soil drench method respectively. Twenty days old
seedlings were transplanted as five per pot with sterilized
Potting soil (soil, sand and coconut pith compost).
In vitro assays
The isolated LAB was screened for the antagonistic
activity against R. solanacearum by agar overlay and well
diffusion method. Plates were incubated at 300C for 22h
and zone of inhibition was measured (C V Jacobsen
et al,. 1999).
For seed treatment method, seedlings from LAB
treated seeds were transplanted into pots infested with
and without R. solanacearum, without any additional
supplementation of LAB. For soil drench method,
seedlings raised from non soaked seeds were transplanted
Preparation of bacterial inoculum
Inoculum of the pathogen was prepared by culturing
it in CPG broth (Kleman, 1954). Cultures were
Pest Management in Horticultural Ecosystems,
Vol. 18, No. 1 pp 60-65 (2012)
61
Narasimha Murthy et al.
into pots infested with and without R. solanacearum with
additional supplementation of LAB at a rate of 50 ml
culture broth per pot. Seedlings were maintained in a
glasshouse at 24-280C and 75-90% relative humidity,
uninoculated plants served as the control (Neelu Singh
et al., 2012). At the end of the experiment (2 months
after transplanting), plants including the roots were
harvested from the pots and fresh weight, mean shoot
length, mean root length and disease incidence were
recorded (Lim and Kim, 1997).
Fig 3. Inhibition Zone of R. solanacearum by LAB strains
Statistical analysis
Data were analyzed using SPSS for windows
(SPSS Inc.) by means of a univariate and multivariate
ANOVA and subsequently differences between treatments
were determined using least significant differences
(LSD at  0.05).
RESULTS AND DISCUSSION
Isolation and Identification of R. solanacearum and
Lactic Acid Bacteria (LAB).
Around 100 strains of R. solanacearum (Rs) and 35
strains of LAB were isolated. The isolated pathogen
strains were identified as R. solanacearum (Hayward,
1964) and LAB strains as L. paracasei subsp. tolerans
(LAB I) (Yousef et al., 2008) and L. paracasei subsp.
paracasei (LAB II) (Ljungh et al., 2002).
In vitro efficacy of LAB against R.solanacearum:
LAB I and LAB II were screened against
R. solanacearum strains, of which Rs1, Rs 2, Rs 3, Rs
4 and Rs5 showed inhibition that ranged from an average
of 22-24 and 23-25 mm radius zone respectively.
(Fig 2 and Fig. 3).
Fig 4. Seed germination of tomato seeds A, B and C: LAB
I, LAB II, and control respectively D: Pathogen, treatments.
Effect of LAB strains on seed germination and
seedling vigor of tomato under Lab conditions
There was an improvement in seed germination and
seedling vigour upon LAB I and LAB II seed treatment
whereas seed germination of tomato seeds upon R.
solanacearum inoculation showed reduction (Fig. 4). The
LAB strains treated, enhanced the vigour index when
compared to control. The maximum germination was
recorded in LAB I and LAB II treated seeds, as tabulated
in (Table 1.)
Green house studies
The efficacy of LAB for the control of R.
solanacearum wilt in tomato plants was evaluated under
Fig 2. Inhibition Zone of R. solanacearum (RS) by
LAB I and LAB II
Pest Management in Horticultural Ecosystems,
Vol. 18, No. 1 pp 60-65 (2012)
62
Lactic acid bacteria as plant growth promoter
Table 1. Effect of seed treatment with pathogen and LAB strains on seed germination and seedling vigour of tomato under
laboratory conditions.
Organisms
Germination (%)
MSL
MRL
VI
Control
76.10b
3.50b
5.70b
700.08b
LABI
78.13b
5.76c
8.47c
1112.76c
LAB II
79.76b
5.80c
8.83d
1130.20c
RS 1
36.00a
1.80a
3.86a
204.20a
RS 2
35.00a
2.10a
3.66a
201.76a
RS 3
35.00a
1.73a
3.90a
196.80a
RS 4
36.33a
2.20a
3.76a
216.83a
RS 5
34.66a
1.80a
3.90a
197.33a
RS - Ralstonia solanacearum, LAB - Lactic Acid Bacteria, MRL - Mean root length; MSL - Mean shoot
length; VI - Vigour index.
mechanism of inhibition, assay for lytic enzymes, defense
enzymes and determination of inhibitor substances other
than antibiotics for application of LAB in biocontrol as a
viable alternative method to manage plant diseases.
greenhouse conditions. Pot trials with LAB I and LAB
II applied as seed treatment and soil drench in absence
of R. solanacearum, revealed their ability to enhance plant
growth as compared with control (Fig. 5). However, the
plant growth characteristics significantly differed in
response to LAB I and LAB II. The Fresh weight, shoot
length, root length and disease incidence were tabulated
as in (Table 2) for LAB treatment by seed treatment and
soil drench methods. These results reveal the capability
of LAB as PGPB. Application of PGPB has been
hampered by inconsistent performance in field tests,
usually attributed to their poor rhizosphere competence
(Thomashow, 1996). Rhizosphere competence of
biological control agents comprises effective root
colonization combined with the ability to survive and
proliferate along growing plant roots, (Stephane et al.,
2005). Our results confirm efficacy of LAB as probable
PGPB.
REFERENCES
Abdul Baki, A. A. and Anderson, J. D. 1973. Vigour
determination in soybean seed by multiple criteria. Crop
Science, 13:630–633.
Aliye, N., Fininsa, C. and Hiskias, Y. 2008. Evaluation of
rhizosphere bacterial antagonists for their potential
to bioprotect potato (Solanum tuberosum) against
bacterial wilt (Ralstonia solanacearum). Biological
Control, 47: 282-288.
Anuratha, C. S. and Gnanamanickam, S. S. 1990. Biological
control of bacterial wilt caused by Pseudomonas
solanacearum in India with antagonistic bacteria. Plant
and Soil 124:109-116.
In the present study, Lactobacillus paracasei subsp.
tolerans and Lactobacillus paracasei subsp. paracasei
strains have shown good antagonism against the R.
solanacearum, exhibited plant growth promotion and
reduced disease severity. Bioprotection of tomato plants
by LAB is a finding reinforced not only by their inhibition
capacity but also by their persistence in the soil under
hard conditions. LAB seems to be more resistant to
stress conditions such as fluctuation in temperature and
relative humidity. The use of chemicals and bactericides
in agriculture as well as the environmental pollution would
be avoided by LAB and hence is a promising PGPB and
biocontrol agent. Future research should confirm the
Pest Management in Horticultural Ecosystems,
Vol. 18, No. 1 pp 60-65 (2012)
Barea, J. M. and Jeffries, P.1995. Arbuscular Mycorrhizas in
Sustainable Soil Plant Systems. In: Mycorrhiza Structure,
Function, Molecular Biology and Biotechnology, Hock,
B. and A. Varma (Eds.). Springer, Heidelberg, pp: 521559.
Brashears, M. M., Jaroni, D., Trimble, J. 2003. Isolation,
selection and characterization of lactic acid bacteria for
a competitive exclusion product to reduce shedding of
Eschericia coli 0157:H7 in cattle. Journal of Food
Protection, 66(3): 355-363.
Chaurasia, B., Pandey, M. and Palni, M. 2005. Diddusible and
volatile compounds produced by an antagonistic
63
Pest Management in Horticultural Ecosystems,
Vol. 18, No. 1 pp 60-65 (2012)
Total fresh weight of pathogen inoculated control without treatments = 8.14c, Disease incidence of pathogen inoculated control= 100%. MSL: Mean Shoot Length,
MRL: Mean Root Length, TFW: Mean Total Fresh Weight, DI: Disease Incidence. Scheffe post hoc test: Means sharing different alphabetical (a, b, c) superscripts
in a column significantly different (P<0.05).
Table 2. Effect of LAB on bacterial wilt disease in three varieties of tomato
Narasimha Murthy et al.
64
Lactic acid bacteria as plant growth promoter
Bacillus subtilis strain couse structural deformations in
pathogenic fungi in vitro. Microbiology Research,
160:75-81.
Narasimha Murthy, K. and Srinivas, C. 2012. Invitro
screening of bioantagonistic agents and plant extracts
to control bacterial wilt (Ralstonia solanacearum) of
tomato (Lycopersicon esculentum) Journal of
Agricultural Technology, 8(3): 999 – 1015.
Danks, C. and Barker, I. 2000. On site detection of plant
pathogens using lateral flow devices. Bulletin OEPP/
EPPO Bulletin, 30: 421-426.
Neelu Singh and Siddiqui, A. 2012. Inoculation of Tomato
with Ralstonia solanacearum, Xanthomonas
campestris, and Meloidogyne javanica. International
Journal of Vegetable Science, 18: 78–86.
Elphinstone, J. G., Stanford, H. M. and Stead, D. E. 1998.
Detection of Ralstonia solanacearum in potato tubers,
Solanum dulcamara, and associated irrigation water. In:
Bacterial Wilt Disease: Molecular and Ecological
Aspects (Eds.): P. Prior, Allen C & Elphinstone J),
SpringerVerlag, Berlin (DE):133-139.
Ouwehand, A. 1998. Antimicrobial components from lactic
acid bacteria. In Lactic acid bacteria Microbiology and
Functional Aspects ed Salminen, S. Von Wright, A.
pp.139-159.New York: Marcel Dekker Inc.
Guo, J. H., Qi, H. Y., Guo, Y. H., Ge, H. L., Gong, L. Y., Zhang,
L. X. and Sun, P. H. 2004. Biocontrol of tomato wilt by
plant growth-promoting rhizobacteria. Biological
Control, 29: 66-72.
Pradhanang, P. M., Ji, P., Momol, M. T., Olson, S. M.,
Mayfield, J. L. and Jones, J. B. 2005. Application of
acibenzolar-S-methyl enhances host resistance in
tomato against Ralstonia solanacearum. Plant disease,
89: 989-993.
Hayward, A. C. 1991. Biology and epidemiology of bacterial
wilt caused by Pseudomonas solanacearum. Annual
Review of Phytopathology, 29: 65–87.
Ran, L. X., Liu, C. Y., Wu,G. J., Van Loon, L. C. and Bakker,
P. A. H. M. 2005. Suppression of bacterial wilt in
Eucalyptus urophylla by fluorescent Pseudomonas spp.
in China. Biological Control, 32:111–120.
International Seed Testing Association (ISTA), 2005.
Proceedings of the International Seed Testing
Association. International rules of seed testing. Seed
Science and Technology, 15 A, 1-9.
Schaad, N. W. 1992. Laboratory guide for identification of
plant pathogenic bacteria. 2 nd edition. American
phytopathological Society, pp 138.
Jacobsen, C. V., Rosenfeldt, V., Hayford, A. E., Moller, P. L.,
Michaelsen, and Paerregaard K. F. 1999. Screening of
probiotic activities of forty-seven strains of
Lactobacillus spp. by In vitro techniques and
evaluation of the colonization ability of five selected
strains in humans. Journal of Applied Microbiology,
11: 4949-56.
Stiles, M. E. 1996. Biopreservation by lactic acid bacteria.
Antonie van Leeuwenhoek, 70: 333-345.
Stephane, C., Brion, D., Jerzy, N., Christophe,C. and Essaid,
A. 2005. Use of plant growth-promoting bacteria for
biocontrol of plant diseases: principles, mechanism of
action, and future prospects. Applied and
Environmental Microbiology, 71: 4951-4959.
Johan, S. and Jesper, M. 2005. Antifungal lactic acid bacteria
as biopreservatives. Trends Food Science and
Technology, 1:70-78.
Thomashow, L. S. 1996. Biological control of plant root pathogens. Current Opinion in Biotechnology, 7: 343-347.
Kelman, A. 1954. The relationship of pathogenicity in
Pseudomonas solanacearum to colony appearance on
a tetrazolium medium. Phytopathology, 44: 693-695.
Vanitha, S. C., Niranjana, S. R. and Umesha, S. 2009. Role of
Phenylalanine Ammonia Lyase and Polyphenol Oxidase
in host resistance to Bacterial Wilt of Tomato. Journal
of Phytopathology, 157: 552-557.
Kishun, R. 1985. Effect of bacterial wilt on yield of tomato.
Indian Phytopathology, 38: 606.
Wenneker, M., Verdel, M. S. W., Van Beuningen, A. R., Derks,
J. H. J. and Janse, J. D. 1999. Ralstonia (Pseudomonas)
solanacearum race 3 (biovar 2) in surface water and
natural weed hosts: first report on stinging nettle
(Urticadioica). European Journal of Plant Pathology,
105: 307-315.
Lim, H. and Kim, S., 1997. Role of siderophores in biocontrol
of Fusarium solani and enhanced growth response of
bean by Pseudomonas fluorescens GL20. Journal of
Microbiology and Biotechnology, 7: 13–20.
Ljung A, Lan, J. and Yanagisawa N. 2002. Isolation, selection
and characteristics of Lactobacillus paracasei subsp.
paracasei F19. Microbial Ecology in Health Disease,
14 (3): 4-6.
Yousef, I. H., Lloyd, B. B. 2008. Antifungal activity of
Lactobacillus paracasei ssp. tolerans isolated from a
sourdough bread culture. International Journal of Food
Microbiology, 121: 112–115.
Milling, A., Babujee, L. and Allen, C. 2011. Ralstonia
solanacearum Extracellular Polysaccharide is a Specific
Elicitor of Defense Responses in Wilt Resistant Tomato
Plants. PLoS ONE 6(1): 158.
Pest Management in Horticultural Ecosystems,
Vol. 18, No. 1 pp 60-65 (2012)
MS Received : 19 May 2012
MS Accepted : 13 June 2012
65