J. Hort. Sci. 
Vol. 1 (1): 44-47, 2006 

Management of bacterial wilt of tomato caused by Ralstonia solanacearum (Smith) 
Yabuuchi et al using biological control agents 

A. Manoj Kumar and Girija Ganesan' 
Department of Crop Physiology 

University of Agricultural Sciences 
G K. V. K., Bangalore-560 065, Kamataka, India. 

E-mail: manojarthik@rediffmail.com 

ABSTRACT 

Biological control agents, Glomus mosseae IIHR, Bacillus subtilis IIHR-1, Pseudomonas fluorescens IIHR*3, 
Trichoderma harzianum IIHR Pj and T. viride IIHR P̂ ^ were evaluated against tomato wilt caused by Ralstonia 
solanacearum in pathogen-infested plots during 2003 -2004. Microbial preparations were applied either as 
transplant root dips or root dip plus soil drench 30 days after transplanting. Per cent survivability increased with 
the use of all biological control agents tested. However, G mosseae treated plants resulted in better survival 
(25.75 and 28.79% in root dip alone and 60 and 66.67% in root dip plus drench against untreated control 0 and 
1.5% during 2003 and 2004 respectively), compared to the rest of the treatments, suggesting G mosseae amendment 
to pathogen-infested soil would result in substantially higher plant survival against the untreated controls. 

Key words: Biocontrol microorganisms, bacterial wilt, survivability, Ralstonia solanacearum, tomato 

INTRODUCTION 

Tomato (Lycopersicon esculentum Mill. Nom. 
Cons.) is one of the widely distributed vegetable crops 
grown worldwide. Tomatoes are a key food crop throughout 
the tropics and subtropics for low-income group of farmers 
and are also a valuable cash crop. In India, tomato occupies 
4180 ha with annual production of 6.4 million tonnes (Anon, 
1998). Bacterial wilt caused by Ralstonia (formerly 
Pseudomonas) solanacearum (Smith) Yabuuchi et al, 
(Yabuuchi et al, 1995) is a soil borne plant disease of great 
economic importance. It is endemic in most tropical, 
subtropical and warm temperate regions of the world 
(Hayward, 1991), proving a major limitation to tomato 
production in Karnataka, Andhra Pradesh, Kerala, 
Maharashtra, Assam, Orissa, Jharkhand and North Eastern 
states. 

The pathogen's host range is rapidly expanding and 
means of control of the disease seems limited. However, 
due to the oligogenic nature of plant resistance and because 
of the variation in aggressiveness of bacterial isolates from 
different locations around the world, alternative control 
measures such as biological control have been receiving 
increased interest. None of the chemicals is effective in 
achieving desired levels of disease control. Hence, 
biological control is gaining more importance as an eco-

friendly means of disease management. Bacteria, which are 
antagonistic towards P. solanacearum have been reported 
by many workers earlier (Anuratha and Gnanamanickam, 
1990; Ciampi Panno et al, 1989; Fucikovsky et al, 1989 
and Gallardo et al, 1989). In most cases, field experiments 
were limited and the degree of protection was in-sufficient 
for commercial use (Tanaka et al, 1990). In many cases 
protection failed because root colonization of the biological 
control agent was poor (Chen and Echandi, 1984). Hence, 
in the present study different biological control agents viz., 
Glomus mosseae IIHR, Trichoderma harzianum IIHR-P ,̂ 
T. viride IIHR-P^ ,̂ Bacillus subtilis IIHR-1 and 
Pseudomonas fluorescens IIHR'̂ S were collected from 
Indian Institute of Horticultural Research, Bangalore and 
tested against wilt causing bacteria of tomato under field 
conditions and results obtained are presented. 

MATERIAL AND METHODS 

A field experiment was conducted in the R. 
solanacearum infested plot during 2003 and 2004. Twenty 
five day old tomato seedlings of cv. PKM-1 were 
transplanted in pathogen-infested soil. Three replications 
were maintained for each treatment with 22 plants/ 
replication. Plants were spaced 75 cm x 50 cm in a plot 
size of 7.5 m .̂ Untreated plots served as control. Randomly, 
ten pathogen-infested soil samples were picked f-orm each 

'Division of Plant Pathology, Indian Institute of Horticultural Researcli, Hesseraghatta Lake Post, Bangalore-560 089, India 

mailto:manojarthik@rediffmail.com


Manoj Kumar and Girija Ganesan 

plot, and were pooled, i.e., 'a composite sample'. Similarly, 
33 composite samples were collected from 33 plots. The 
bacterial population of the pathogen-infested plots were 
determined for all 33 plots by serial dilution on TTZ (2, 3, 
5, triphenyl tetrazolium chloride) medium. The mean 
populations of R. solanacearum were 2.4±0.4xl0'cfu g ' 
and 2.9±0.35xl0' cfu g ' during the year 2003 and 2004, 
respectively. 

Fungal antagonists viz., T. harzianum and T. viride 
were multiplied on Potato Dextrose Broth at 27°C for 7 
days in B.O.D incubator and the mycelium was blended 
thoroughly. Spore count of VxlO'conidial spores/ml in T. 
harzianum and 7.4xl0'conidial spores/ml in T. viride was 
recorded by using heamocytometer. The bacterial 
antagonists viz., B. subtilis and P. fluorescens were 
multiplied on nutrient broth at 35°C for 72h at 120 rpm in 
the incubator shaker. Bacterial antagonist population was 
determined by serial dilution method on Nutrient Agar 
medium and were found to be 4 x 1 0 ' c.f.u/ ml {P. 
fluorescens) and 4.3x10' c.f.u/ ml {B. subtilis). However, 
Glomus mosseae was maintained on Eleusine coracana for 
six months showing 60-70% of root colonization with 20 
spores/g soil. 

At the time of transplanting, roots were dipped in 
broth containing biological control agents viz., T. 
harzianum, T. viride, B. subtilis and P. fluorescens for 30 
min. and transplanted to the sick plots, in randomized block 
design (RBD). In case of Glomus mosseae, the seedlings 
were grown on G. mosseae soils and transplanted. 

In another set of experiment, one soil drench of 
bio control agents (root dip-i-drench) in the root zone @ 20 
ml per plant was given at 30 days after transplanting (DAT) 
in addition to root dip. Assessment of treatment effects 
were recorded as per cent wilt incidence at 90 DAT and the 
wilted plants examined in laboratory for confirmation of 
the presence of/?, solanacearum. Plant height was recorded 
at 30 DAT. Data were normalized through angular 
transformation and subjected to analysis of variance 
(ANOVA) followed by mean separation by the Student-
Newman-Keuls' test (p=0.05). All the analysis was 
performed using the SAS (1996) package. 

RESULTS AND DISCUSSION 

Of the five biological control agents used in the 
investigation, the highest per cent wilt incidence of 94% 
and 84.9% in tomato in seedling root dip experiment was 
recorded in T. harzianum and minimum of 74.25% and 
71.21% in G. mosseae as against 100% and 98.5% in 

untreated control for the year 2003 and 2004, respectively, 
these results indicate that the protection offered by G 
mosseae was better in both the years as compared to other 
treatments (Table 1). 

Further, the incidence of bacterial wilt in root dip 
plus soil drench treated with G mosseae was much lower 
(40 & 33.33%) as compared to 100 and 98.5% in untreated 
control. However, the maximum wilt incidence of 86.7 and 
66.7% was observed in T. harzianum during the successive 
years 2003 and 2004. Decreasing trend of wilt incidence 
was observed in G mosseae and T. harzianum treated plots, 
in both the experiments, i.e., seedling root dip and seedling 
root dip plus soil drench. Bacillus subtilis and P. fluorescens 
treatments showed no change in wilt incidence for both the 
years, i.e., 80.31% in root dip and 66.67% in root dip plus 
soil drench. However, wilt incidence was increased in 2004 
in both the experiments in T. viride treated plots. 

Glomus mosseae treated plots recorded higher plant 
growth of 46.17cm and 50.67cm followed by T. harzianum 
during both the years as against all other treatments (Table 
1). In the present investigation, some of the tested biological 
control agents had antagonistic effect on R. solanacearum. 
Among these, bacterial antagonists (B. subtilis & P. 
fluorescens) performed better than fungal antagonists. 
However, G mosseae amended infested soil recorded less 
wilt incidence compared to control and other treatments. 
The biological control agent must compete with complex 
biological and physical factors, including soil composition 
and structure, moisture and pH, which influence the 
structure of the microbial population (Nesmith and Jenkins, 
1985; Weller, 1988), in order to show its better performance 
under field conditions. Anuratha and Gnanamanickam 
(1990) reported that P. fluorescens str. Pfcp protected tomato 
plants from bacterial wilt up to 95% in greenhouse and 
36% in the field. Similarly, Bacillius sp. strain BA-46 
controlled the bacterial wilt disease by 21.13% in tomato 
under greenhouse conditions (Silveira et al, 1995). T. 
harzianum, isolated from Sikkim was found effective in 
controlling ginger wilt caused by R. solanacearum (Raj an 
et al, 2002). Arbuscular mycorrhiza forms a beneficial 
symbiotic association with roots that increased plant ability 
to absorb phosphorous, minor elements and water, thus 
enhancing the tolerance of the plant against the pathogen 
damage (Harley and Smith, 1983). Kobayashi, etal (1991) 
reported that VAM -i- charcoal compost proved effective in 
reducing the level of bacterial wilt of tomato in greenhouse 
tests. The extracts from mycorrhizal tomato roots infected 
with Glomus fasciculatum reduced populations of P. 

J. Hon. Sci. 
Vol. 1 (1): 44-47, 2006 

45 



Managing bacterial wilt of tomato with biological control agents 

Tablel. Effect of different microorganisms on survival and growth in tomato in R. solanacearum infected soil 

Treatment Wilt incidence (%) Plant height (cm) 
2003 2004 2003 2004 

Root dip alone Root dip + Drench Root dip alone Root dip + Drench 
Glomus mosseae 

T. harzianum 

T. viride 

B. subtilis 

P. fluorescens 

Control 

CD (p=0.05) 

74.25 
(60.04) 
94.00 

(75.98) 
75.76 

(61.59) 
77.28 

(62.47) 
80.31 

(67.45) 
100.0 

(88.19) 
NS 

40.00 
(38.85) 
86.67 

(75.71) 
46.67 

(43.07) 
53.33 

(46.92) 
66.67 

(54.99) 
100.0 

(88.19) 
31.63 

(22.44) 

71.21 
(57.56) 
84.85 

(63.68) 
83.33 

(62.61) 
78.79 

(67.31) 
80.31 

(66.25) 
98.50 

(84.71) 
7.35 

(7.32) 

33.33 
(35.01) 
66.67 

(54.99) 
60.00 

(50.77) 
53.33 

(47.29) 
66.67 

(54.99) 
98.50 

(79.93) 
21.69 

(15.11) 

46.17 

36.92 

32.08 

32.58 

36.83 

31.97 

~ 

50.67 

39.17 

33.83 

35.25 

38.00 

32.83 

" 

* DAT - Days after transplanting; NS- non significant; Figures in parentheses are angular transformed values 

solanacearum in nutrient broth, however, the active 
principle constituent of VAM fungi inhibiting P. 
solanacearum are yet to be studied (Suresh et al, 1991). It 
could be concluded from the present investigation that, the 
G. mosseae amended pathogen-infested soil resulted in 
substantially higher plant survival in R. solanacearum 
infected soil. In addition to the transplant root dip an 
additional drench of bioagents at 30 DAT was of an added 
advantage in protecting the plant from bacterial wilt 
pathogen. 

ACKNOWLEDGEMENTS 

The authors are grateful to the Director, I.I.H.R., 
Bangalore, for providing facilities and Mr. Puttamada 
Shetty, Technician, and Ms. N. Rama, Research Associate, 
for assistance rendered during the study. The authors are 
also thankful to Prof. M. Udaya Kumar, and Dr. Rohini 
Sreevathsa, GKVK, Bangalore for their support. 

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Manoj Kumar and Girija Ganesan 

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(MS Received 4 May, 2006 Revised 13 July, 2006) 

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Vol. 1(1): 44-47, 2006 

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