Crop Protection 30 (2011) 1269e1273

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Crop Protection
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Actinomycetes mediated biochemical responses in tomato (Solanum lycopersicum)

enhances bioprotection against Rhizoctonia solani
Hemant J. Patil a, b, Alok K. Srivastava a, Dhananjaya P. Singh a, Bhushan L. Chaudhari b, Dilip K. Arora a, *
a
National Bureau of Agriculturally Important Microorganisms, Mau 275101, India
b
Department of Microbiology, School of Life Sciences, North Maharashtra University, Jalgaon 425 001, India

a r t i c l e i n f o a b s t r a c t

Article history: Six actinomycetes isolates, namely Streptomyces toxytricini vh6, Streptomyces flavotricini vh8, S. toxytricini
Received 19 July 2010 vh22, Streptomyces avidinii vh32, Streptomyces tricolor vh85 and vh41, an isolate of an unknown species
Received in revised form of Actinomycetales, were tested for their efficacy in protecting tomato (Solanum lycopersicum) against
10 April 2011
Rhizoctonia solani under green house conditions. Actinomycetes treated plants showed better growth in
Accepted 23 April 2011
terms of high chlorophyll content, higher phenylalanine ammonia lyase (PAL) activity and high total
phenolic content. Qualitative and quantitative estimation of phenolic compounds from tomato leaves
Keywords:
showed significant accumulation of six phenolic acids, gallic (29.02 mg g
1 fresh leaf wt), ferulic
Actinomycetes
Phenolics
(11.44 mg g
1 fresh wt), cinnamic (56.84 mg g
1 fresh wt), gentisic (24.19 mg g
1 fresh wt), chlorogenic
Phenylalanine ammonia lyase acid (1.72 mg g
1 fresh wt) and salicylic (0.39 mg g
1 fresh wt) acid, in actinomycetes treated plants.
Rhizoctonia solani Biochemical profiling, when correlated with plant mortality in actinomycetes treated and untreated
Streptomyces plants, indicated that isolates vh6 and vh8 offered 44.55% and 40.14% disease reductions, respectively
Tomato compared to the control. These results established that these organisms have the potential to act as
biocontrol agents.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction biocontrol of plant diseases, e.g. Mycostop, a formulation devel-

oped from Streptomyces griseoviridis and Actino-iron (S. lydicus, iron
Actinomycetes, filamentous saprophytic bacteria, are of special and humic acid) (Sabaratnam and Traquair, 2002; Minuto et al.,
interest in the rhizosphere since they are able to colonize plant 2006), these organisms are still less explored for their involve-
tissues and produce resistant spores that can survive long in agri- ment in development of host resistance through induction of
cultural soils (Hamdali et al., 2008a,b). These organisms have been biochemicals and biological control in comparison with plant
studied for their plant growth promoting as well as biocontrol growth promoting rhizobacteria.
abilities leading to increased agricultural yields (Doumbou et al., Phenolic compounds are well known natural constituents in
2002; Shahrokhi et al., 2005; Hamdali et al., 2008b). In soil, plants participating in constitutive and/or inducible defense
production of antibiotic metabolites (Hyang et al., 2005) and anti- mechanisms associated with non-host resistance (Stoessl, 1983;
microbial compounds (Sabaratnam and Traquair, 2002; Berdy, Elgersma and Liem, 1989; Nicholson and Hammerschmidt, 1992).
2005; Lehman et al., 2005) make actinomycetes able to restrict Many of these compounds are formed in response to pathogenic
the attack by pathogenic organisms in the habitat (Beom et al., stress and hence, are considered as a part of active defense
1999; El-Tarabily et al., 2000). There are reports on the agricul- response in plants (Nicholson and Hammerschmidt, 1992).
tural implications of these organisms in biological control of plant Phenylalanine ammonia lyase (PAL), a key enzyme in phenyl
pathogens (Cao and Forrer, 2001; Bressan, 2003; Ghorbani et al., propanoid pathway constituting the biosynthesis of phenolic
2007) and triggering of signal transduction in host plants to compounds, has also been demonstrated to act as an indicator of an
initiate defense responses to cope with the stresses at cell, tissue active defense response in plant (Havir, 1987). Enhanced defense
and organ level following inoculation of these organisms response against plant pathogen attack due to accumulation of
(Hasegawa et al., 2006). Although there are a number of reports on phenolic compounds following colonization with non-host bacteria
the developments of commercial formulations of actinomycetes for and fungi is well documented (Cherif et al., 2007), but the role of
actinomycetes in this respect is only scarce. Therefore, this study
* Corresponding author. Tel.: þ91 547 2530388; fax: þ91 547 2530358. was conducted to determine the effect of root colonization of
E-mail address: [email protected] (D.K. Arora). actinomycetes in tomato plants in terms of plant growth

0261-2194/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cropro.2011.04.008
1270 H.J. Patil et al. / Crop Protection 30 (2011) 1269e1273

promotion, induced accumulation of phenolics and protection 2.5. Phenylalanine ammonia lyase activity
against soilborne disease caused by Rhizoctonia solani J.G. Kühn.
Phenylalanine ammonia lyase activity was determined using the
direct spectrophotometric method described by Havir (1987) with
2. Materials and methods slight modifications. Fresh tomato leaves were harvested at regular
time intervals. Leaf tissues (0.2 g) were extracted in 5 ml borate-HCl
2.1. Actinomycetes buffer (25 mM; pH 8.8) containing 5 mM mercaptoethanol and the
supernatant obtained after centrifugation (12,000  g for 20 min at
Actinomycetes (Streptomyces toxytricini vh6, S. flavotricini vh8, 4  C) served as enzyme extract. The reaction mixture contained
S. toxytricini vh22, S. avidinii vh32, A. bacterium vh41, S. tricolor 0.2 ml of enzyme extract, 0.5 ml of borate buffer and 1.3 ml of water.
vh85 and vh41, an isolates of an unknown species of Actino- The reaction was initiated by adding 1 ml of L-phenylalanine
mycetales) were isolated from non-rhizospheric and rhizospheric solution followed by incubation at 32  C for 45 min after which
soil of Indo-gangetic Plains (IGP) region of India. Selective isolation 0.5 ml trichloroacetic acid (1 M) was added to stop the reaction.
of the organisms and their morphological, chemotaxonomic, Quantification of enzyme was done using calibration curve of cin-
physiological and molecular characterization has been described namic acid and the enzyme activity was expressed in terms of mM
earlier (Patil et al., 2010). cinnamic acid produced per hour per gram fresh tomato leaves.

2.2. Inoculum preparation 2.6. Extraction of phenolic acids from tomato leaves

The pure cultures of actinomycetes isolates under study were Fresh leaves from the lower nodes of tomato plants from all
inoculated in starch casein broth at 32  C for one week. After incu- replications in each treatment were collected periodically and
bation, the broth was centrifuged at 8,000  g min
1 for 10 min at mixed together. Two hundred milligram of leaves was macerated in
10  C and supernatant was discarded. The pellet was washed with precooled mortar pestle with 5 ml of methanol: water (1:1 v/v). The
sterile distilled water (SDW) twice and re-suspended in SDW to reach suspension was collected in screw capped tubes and centrifuged at
a cfu 106 ml
1 approximately (called bioagent suspension hereafter). 10 000  g for 20 min at 4  C. The greenish supernatant was then
R. solani (Accession No. NAIMCC F-01971) was obtained from the subjected to charcoal treatment to remove pigments and after
National Agriculturally Important Microbial Culture Collection, filtration through Whatman filter paper No. 1 it was transferred to
India and used as a test pathogen. The pathogenic fungus was disk glass tube followed by evaporation at room temperature. Sample
inoculated in wheat bran (700 g in 2 l capacity flask; moisture level was dried, dissolved in one ml HPLC grade methanol and stored at
50%) and incubated for two weeks in the dark with proper mixing 4  C for further analysis.
at regular intervals for uniform distribution of fungal mycelia.

2.3. Experimental set-up 2.7. Estimation of total phenolics

Tomato (var. Nav Ratan) seeds were sown in earthen pots (15 cm To estimate total phenolic compounds (TPC), 1 g of fresh leaves
dia., 6 seeds/pot) containing sterile soil. After 25 days of germina- was extracted with methanol: water (10 ml, 1:1 v/v) twice and the
tion, thinning was done to maintain four seedlings per pot. Pots supernatant after evaporation was dissolved in 3 ml methanol. To
were kept in ambient green house conditions with natural this, 0.5 ml of Folin-Ciocalteau’s reagent was added followed by
temperature (25  2  C), 12 h light/dark photoperiod and relative incubation at room temperature and 2 ml of 20% sodium carbonate
humidity of 85e90%. Wheat bran containing pathogen was mixed was again added with thorough mixing. The appeared colour was
at a rate of 1.5% (w/w) with the same soil with which the pots were analyzed using UVeVIS spectrophotometer (Shimadzu UV-1700) at
filled and then 10 g of the same soil (containing pathogen inoc- 650 nm as described by Sadasivam and Manickam (1996). Total
ulum) was spread over the surface of the pots at four leaf stage of phenolics were expressed in terms of gallic acid equivalents per
the plants. After acclimatization for 3e4 days, the plants were again gram of fresh leaves. Analytical grade reagents were used
inoculated with actinomycete suspension. Each actinomycete throughout the experiment.
isolate, with and without pathogen, under investigation was
considered as one treatment. The changes in accumulation of
phenolic constituents and growth parameters were determined
every 48 h after inoculation. Mortality caused by R. solani was
recorded after 45 days of inoculation of the pathogen. Plants grown
in sterile soil served as control. Each treatment was replicated five
times in a completely randomized experiment design.

2.4. Determination of total chlorophyll

The chlorophyll content in tomato leaves was quantified by

extracting the pigment from 0.5 g of freshly harvested leaf tissues
using methanol: water (9:1, v/v). A pinch of calcium carbonate was
added to prevent degradation of chlorophyll to pheophytin. After
removal of the cell debris through centrifugation at 15,000 xg for Fig. 1. Total chlorophyll content in tomato leaves on 10th day of interaction following
5 min, chlorophyll content in the supernatant was quantified by treatment with bioagent under pathogenic stress. The bar line indicates the standard
taking OD at A665nm and A645nm (Ferjani et al., 2003). deviation at P < 0.05 by one-way analysis of variance (ANOVA).
 H.J. Patil et al. / Crop Protection 30 (2011) 1269e1273 1271

Fig. 2. Phenylalanine ammonia lyase (PAL) activity in tomato plants. A: plants treated with bioagents under pathogenic stress and B: plants treated with bioagents only (non-
pathogenic stress).

2.8. HPLC analysis 2.10. Statistical analysis

High-performance liquid chromatography (HPLC) was performed The data was subjected to one way analysis of variance (ANOVA)
for each sample according to a protocol by Singh et al. (2003). The using the GraphPad InStatÒ (Version 3.01, GraphPad Software Inc.).
HPLC system (Waters, Massachusetts USA) was equipped with two The differences in the mean values in each treatment were
Waters 515 reciprocating pumps, a photo diode array detector compared and significance level was calculated at P < 0.05 level.
(Waters 2996) and a system controller with WatersÒEmpowerÔ
software for data integration and analysis. Reverse phase chro- 3. Results
matographic analysis was carried out under isocratic conditions (C-
18 100 A, 5 mm, 250  4.6 mm i.d.; solvent system - methanol: acetic The actinomycetes isolates S. toxytricini vh22, S. avidinii vh32,
acid 0.4% in water, 60:40 V/V; flow rate, 1.0 ml min
1) with 10 ml Actinomycetales bacterium vh41 and S. tricolor vh85 along with S.
injection volume and detection at 290 nm. Samples were filtered toxytricini vh6 and S. flavotricini vh8 showed prominent antagonistic
through membrane filter (0.45 mm) prior to injection. Gallic, gentisic, potential against R. solani and exhibited significant level of accu-
ferulic, cinnamic, chlorogenic and salicylic acids were used as mulation of phenolic compounds in tomato plants under pathogenic
internal and external standards. Phenolic compounds were identified stress. Quantitative estimation showed significant increase in total
by comparing the retention time of standard compounds and by co- chlorophyll content in plants under treatments compared to control.
injection. Quantitative analysis was carried out by comparing peak The treatments with isolates vh8 and vh6 resulted in significant
areas of reference compounds with those of the sample run under increases compared to all other treatments (Fig. 1). Isolate vh22
the same elution condition. resulted in less chlorophyll content. Phenylalanine ammonia lyase
activity was more pronounced in tomato leaves after 48 h of inter-
action with all bioagents under pathogenic as well as non-pathogenic
2.9. Disease severity index stress. The highest PAL activities with vh32 and vh8 were recorded
among all treatments under pathogenic stress (Fig. 2A). In case of the
Disease assessment for root rot symptoms was recorded 45 days treatment of bioagents alone, vh41 and vh85 provided considerable
after bioagent inoculation and severity index of tomato root rot was induction compared to all other isolates and control (Fig. 2B).
determined in linear scale from 0 to 4, as follows: 0 ¼ Healthy roots, The TPC was significantly higher in tomato leaves treated with
1 ¼ 1/3 secondary roots do not exist, 2 ¼ 3/4 secondary roots do not biocontrol agents in both R. solani- inoculated and non-inoculated
exist, 3 ¼ primary roots exist, secondary roots do not exist, and plants as compared to the control. Among all six isolates, vh32
4 ¼ Primary roots do not exist induced the highest accumulation of TPC under pathogenic stress

Fig. 3. Quantification of total phenolic compounds (mg gallic acid equivalent g
1 fresh wt) in tomato leaves following induction by bioagent (actinomycetes isolates). A: plants
treated with bioagents under pathogenic stress and B: plants treated with bioagents only (non-pathogenic stress).
1272 H.J. Patil et al. / Crop Protection 30 (2011) 1269e1273

(Fig. 3A), followed by vh6 and vh22 in comparison to control (Fig. 3B).
HPLC analysis of tomato leaves following treatment of plants with the
six biocontrol agents and challenging with R. solani revealed differ-
ential expression of phenolic compounds. Of these, six peaks of
phenolic acids could be identified as gallic (Rt. 2.56 min), gentisic
(3.47), chlorogenic (3.56), ferulic (4.07), salicylic (6.68) and cinnamic
(9.81) acids. The highest accumulation of gallic acid (29.02 mg g
1
fresh leaf wt) was detected in treatment with vh8 at 48 h as compared
to non-treated control (2.21 mg g
1 fresh wt)(Table 1). The pattern
was observed to be consistent in all treatments up to 8 days after
treatment with gradual decrease in quantity.
The accumulation of other phenolic compounds was observed at
the highest level after 48 h after treatment under the pathogenic
condition. The highest accumulation of gentisic acid (24.19 mg g
1
fresh wt) was detected in leaves following the treatment with vh6.
Higher accumulation of ferulic acid (11.44 mg g
1 fresh wt) in vh8-
treated plants was detected 48 h after treatment than the control
(0.09 mg g
1 fresh wt). Similarly, cinnamic acid was found at the Fig. 4. Percent disease reduction of tomato treated with actinomycetes isolates under
pathogenic stress over control (LSD ¼ 2.23 at P < 0.05).
highest level (56.84 mg g
1 fresh wt) in the treatment with vh6. The
accumulation of chlorogenic acid was the highest (1.72 mg g
1 fresh
wt) in plants treated with vh41 (Table 1). Salicylic acid was detected
accumulation of antimicrobial phenolics and salicylic acid,
at the highest level (0.39 mg g
1 fresh wt) in plants treated with vh8
a signaling molecule with a proven role in systemic induced
48 h after treatment. The treatment of plants with all biocontrol
resistance. Thereby, we can speculate that these organisms
agents without inoculation with R. solani showed remarkable
behaved in a similar way to offer growth promotion and disease
decline in accumulation of all the phenolic compounds with
suppression in plants as has been previously defined for plant
maximum accumulation of gallic acid (3.41 mg g
1 fresh wt) in the
growth promoting rhizobacteria (PGPRs) like Pseudomonas and
treatment with vh6 (Table 1). There was a greater accumulation of
Bacillus and beneficial biocontrol fungi like Trichoderma (Meyer
almost all phenolic acids in tomato leaves treated with actinomy-
et al., 1998; Ramamoorthy et al., 2001; Compant et al., 2005;
cetes and further challenged with R. solani than those treated with
Cherif et al., 2007).
biocontrol agents only and the control (Table 1). Inoculation with R.
The effect of bioagent treatment in plants not inoculated with
solani produced the typical disease symptoms and resulted in final
a pathogen offers a practical way of immunizing plants against
plant death. All bioagents used in this study reduced disease
biotic stress (Jones and Dangl, 2006). In the present study, isolates
symptoms as well as mortality at different levels (23e41%) with
vh6 and vh8 were effective in inducing resistance in tomato plants
maximum reduction in the treatment with vh6 as compared to
in the presence of R. solani. Based on the HPLC analysis of phenolic
control. Isolates vh8 and vh85 also showed considerable reduction
acids particularly gallic, ferulic, cinnamic, genteisic, chlorogenic and
of disease severity.
salicylic acids revealed that isolates vh6 and vh8 which provided
significant protection of tomato plant against the pathogen (Fig. 4)
4. Discussion also showed the highest accumulation of these compounds. These
compounds have antibacterial and antifungal potential, which has
This study revealed the impact of the inoculation of actinomy- great importance in plant cell protection. Cinnamic acid is a key
cetes isolates on tomato in terms of biochemical changes in plants product of phenyl propanoid pathway, synthesized from precursor
inoculated with the fungal pathogen R. solani. Substantial differ- molecule phenylalanine through catalysis by the PAL activity. This
ences in the level of induction of phenolic compounds in tomato enzyme plays a key role in host resistance to biotic stress (Singh
leaves treated with actinomycetes biocontrol agents with and and Prithiviraj, 1997). The product of PAL and cinnamic acid is
without R. solani-inoculation were recorded (Fig. 3A). From these directly linked to cell lignifications process and the maximum PAL
results, it is evident that as a bioinoculant in the root rhizosphere, activity usually occur about 24e48 h after pathogen infection. In
the actinomycetes isolates improved plant growth as well as the experiments, the PAL activity was average in all treatments with
enhanced protection against the pathogen due to the induced little elevation in vh32 and vh8 against R. solani compared to plants

Table 1
Phenolic compounds in fresh tomato leaves treated with actinomycetes after 48 h of interaction.

Bioagent Concentration of phenolic compound (mg g
1 fresh leaf wt)a

GA Gent FA CA ChA

A B A B A B A B A B
Streptomyces sp. vh6 7.83 ± 0.57 3.41 ± 0.74 24.19 ± 1.07 0.62 ± 0.08 N/Db 0.15  0.04 56.84 ± 6.18 0.28  0.08 N/D 0.83 ± 0.19
Streptomyces sp. vh8 29.02 ± 3.01 0.29  0.08 10.12 ± 1.88 0.01  0.01 11.44 ± 1.52 0.02  0.01 23.41 ± 1.29 N/D 0.10  0.02 0.01  0.01
S. toxytricini vh22 12.61 ± 1.62 3.74 ± 1.23 0.37  0.11 0.02  0.01 2.99 ± 0.49 0.12  0.02 0.43  0.10 N/D 0.49  0.09 0.03  0.02
S. avidinii vh32 3.53 ± 1.10 3.13 ± 0.87 0.02  0.01 0.29  0.08 0.36  0.11 0.10  0.02 0.22  0.06 0.11  0.03 0.08  0.02 0.39  0.12
A. bacterium vh41 2.65  0.48 2.34  0.48 0.15  0.04 0.26  0.06 0.19  0.05 0.09  0.03 0.05  0.02 0.03  0.02 1.72 ± 0.48 0.35  0.11
S. tricolor vh85 4.16 ± 0.33 3.34 ± 0.43 0.64  0.08 0.55 ± 0.09 2.94 ± 0.33 0.22  0.06 N/D 0.23  0.06 0.02  0.01 0.74 ± 0.11
Non-treated (control) 2.21  0.25 2.21  0.25 0.28  0.08 0.28  0.08 0.09  0.03 0.09  0.03 1.48  0.29 1.48  0.29 0.37  0.11 0.37  0.11
Pathogen 3.39 ± 1.05 3.39 ± 1.05 0.11  0.95 0.11  0.95 0.06  0.65 0.06  0.65 0.92  0.32 0.92  0.32 0.15  0.27 0.15  0.27
a
GA: gallic acid, Gent: gentisic acid, FA: ferulic acid, CA: cinnamic acid, ChA: chlorogenic acid; A: Treated with bioagent under pathogenic stress, B: Treated with bioagent
only. Some means are significantly different at level 0.05 and marked as bold.
b
N/D: not detectable.
 H.J. Patil et al. / Crop Protection 30 (2011) 1269e1273 1273

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