Role of Antagonistic Microorganisms and Organic Amendment in Stimulating the Defense System of Okra Against Root Rotting Fungi


1 Agricultural Biotechnology and Phytopathology Laboratory, Department of Botany,University of Karachi, Karachi, Pakistan
2 Biotechnology and Drug Development Laboratory, Department of Biochemistry, University of Karachi, Karachi, Pakistan
3 Postharvest and Food Biochemistry Laboratory, Department of Food Science and Technology, University of Karachi, Karachi, Pakistan
4 California Department of Food and Agriculture, Sacramento, CA, USA

* Corresponding author: M. Athar, California Department of Food and Agriculture, Sacramento, CA, USA; e-mail:

Submitted 7 May 2014, accepted 27 February 2015


Without application of chemical pesticides control of soilborne diseases is a great challenge. Stimulation of natural plant’s defense is considered as one of the most promising alternative strategy for crop protection. Organic amendment of soil besides direct suppressing the pathogen, has been reported to have an influence on phytochemicals in plants. In the present study, Pseudomonas aeruginosa, a plant growth promoting rhizobacterium and Paecilomyces lilacinus, an egg parasite of root knot and cysts nematodes were examined individually and in combination in soil amended with cotton cake for suppressing the root rotting fungi and stimulating the synthesis of polyphenols and improving the antioxidant status in okra. Application of P. aeruginosa and P. lilacinus in soil amended with cotton cake significantly (P < 0.05) suppressed Macrophomina phaseolina, Fusarium oxysporum, and Fusarium solani with complete reduction of Rhizoctonia solani. Combine use of biocontrol agents in cotton cake amended soil showed maximum positive impact on plant growth, polyphenol concentra­tion and antioxidant activity in okra.

Key words: antagonistic microorganisms, okra, organic amendment, root rotting fungi


Vegetable crops are vulnerable to a range of patho­genic organisms that reduce yield by killing the plant or damage the product and make it unmarketable. Plant diseases on average are responsible for up to 26% yield loss to global agriculture and sometimes there may be complete crop failure leading to 100% yield loss in a locality or a field (Khan et al., 2009). Okra [Abelmoschus esculentus (L.) Moench] is an important vegetable crop and is grown worldwide including Pakistan (Athar and Bokhari, 2006). Okra is a warm, rainy season crop and requires high temperature. However, diseases are the limiting factor in okra production. In Pakistan okra crop is attacked by various soil borne plant pathogenic fungi like Macrophomina phaseolina, Rhizoctonia solani, Fusarium spp. and the root knot nematodes Meloido­gyne spp. (Afzal et al., 2013; Ehteshamul-Haque et al., 1996). Without application of chemical pesticides con­trol of soilborne diseases is a great challenge. Among the new biological approaches, the stimulation ofnatural plant’s defense is considered as one of the most promising alternative strategies for crop protection (Anderson et al., 2006; Walters and Fountaine, 2009; Walters et al., 2005). This original biological approach does not exert direct effects on the pathogen (Walters and Fountaine, 2009) but stimulates natural defenses in plants, leading to a systemic acquired resistance (Vallad and Goodman, 2004).

Plants produce a wide range of secondary metabo­lites in response to biotic stress that are toxic to path­ogens and herbivores. Phenolic phytochemicals are secondary metabolites that are common constituents of fruits and vegetables that function in the defense against insect and animal herbivory (Stevenson et al., 1993). These phenolic metabolites protect the plants against biological and environmental stresses and there­fore are synthesized in response to pathogenic attack such as fungal or bacterial infection or high energy radiation exposure such as prolonged UV exposure (Briskin, 2000). Phenolic phytochemicals, because of their important protective biological functions, are ubiquitous in all plants and therefore find their place in almost all food groups. In resistant plants, phenolic based defense responses are characterized by the early and rapid accumulation of phenolics at the infection site resulting in the effective isolation of the pathogen (Chérif et al., 1991).

Farmers and agricultural scientists have long under-stood that organic amendments applied to field soils improve soil functions such as infiltration, water holding capacity, nutrient retention and release, and resistance to wind and water erosion and can suppress soilborne diseases (Bonanomi et al., 2007; Stone et al., 2003). Beside a wide variety of organic matters that have been tested as organic amendments for manag­ing plant pathogens, oil seed cakes can decrease the population of soil borne pathogens (Ehteshamul-Haque et al., 1995; Sharma et al., 1995). It has been observed that several antimicrobial by-products (e.g. organic acids, hydrogen sulfide, phenols, tannins and nitrog­enous compounds) are released during the decom­position of organic amendments or synthesized by microorganisms involved in such degradation (Rodriguez–Kabana et al., 1995). Furthermore organic fertilizers enhance the antioxidant content in plants and conse­quently improve plant defense against pests and dis­eases (Dumas et al., 2003).

The root colonizing bacteria that have a beneficial effect on plants are termed as plant growth promot­ing rhizobacteria (PGPR) and have been reported to improve plant growth either through direct stimulation of the plant by producing growth regulators or by sup­pression of pathogens (Inam-ul-Haq et al., 2012; Weller et al., 2002). Of the various rhizospheric bacteria, the bacteria belonging to the fluorescent Pseudomonas which colonize roots of a wide range of crop plants are reported to be antagonistic to soil-borne plant patho­gens (Siddiqui and Ehteshamul-Haque, 2001). PGPR may induce plant growth promotion by direct or indi­rect modes of action (Kloepper, 1993). Direct mecha­nisms include the production of stimulatory bacterial volatiles and phytohormones, lowering of the ethyl­ene level in plant, improvement of the plant nutrientstatus (liberation of phosphates and micronutrients from insoluble sources; non-symbiotic nitrogen fixa­tion) and stimulation of disease-resistance mecha­nisms (Antoun and Prévost, 2005). The present report describes the role of soil amendment and application of PGPR on the suppression of okra root diseases and polyphenol content and antioxidant activity in okra alone or with Paecilomyces lilacinus, an egg parasite of root knot and cyst nematodes.

Materials and Methods

Biological antagonist. Cultures of P. aeruginosaand P. lilacinus, used in this study were obtained from Karachi University Culture Collection (KUCC).

Experimental design / Screen house experiment. Dry powder of cotton cake was mixed in sandy loam soil, pH 8.0, @ 1.0% w/w. The soil had natural infes­tation of 5–11 sclerotia of M. phaseolina g–1 of soil, as determined by wet sieving and dilution technique (Shiekh and Ghaffar, 1975), 4–13% colonization of sor­ghum seeds was used as bait for R. solani (Wilhelm, 1955), and 3000 cfu.g–1 of soil of a mixed population of Fusarium solani and F. oxysporum as determined by a soil dilution technique (Nash and Synder, 1962). One kg of amended soil was transferred to 12 cm diam­eter clay pots. The pots were watered daily to allow the decomposition of the organic substrate. After two weeks, aqueous suspensions of P. aeruginosa (108 cfu/ml) grown on KB broth and P. lilacinus (107 cfu/ml) grown on potato dextrose broth were drenched onto each pot at 25 ml per pot. Pots without amendment/antagonists or fungicides served as control. Aqueous suspension (100 ppm) of a fungicide, carbondazim at 25 ml per pot served as positive control. Six seeds of okra were sown in each pot and pots were kept ran­domized on a screen house bench of Department of Botany at 50% water holding capacity with four repli­cates of each treatment. After germination, only four seedlings were kept and excess were removed.

Determination of fungal infection and growth parameter. To assess the efficacy of P. aeruginosa and P. lilacinus in suppression of root disease, plants were uprooted after 45 days of growth. To determine the incidence of fungi, roots were washed with running tap water then surface disinfested with 1% Ca(OCl)2 and 1 cm long root pieces from tap roots, (5 from each plant) were plated onto potato dextrose agar plates supplemented with penicillin (100,000 units/litre) and streptomycin (0.2 g/litre). After incubation for 5 days at 28°C, the incidence of root infecting fungi was recorded. Infection percentage for each pathogen was calculated using the formula:

Infection % of a pathogen= (Number of plants infected by a pathogen/Total number of plants) ×100

Plant growth parameters, such as plant height and fresh weight of shoot, root length and root weight were also recorded.

Determination of polyphenol. Okra leaves were oven-dried at 80°C for 24 hours. Dried leaves were ground into fine powder using a clean pestle mortar and finally crushed samples were suspended in ethanol. Samples were collected in screw capped centrifuge tubes. The extracts were centrifuged for 20 minutes at 3,000 rpm. The supernatants were collected and used for analyzing phenolic content and antioxidant activity.

The estimation of polyphenol was done by Folin-Ciocalteu phenol reagent as describe by Chandini et al., (2008). For estimation 100 μl aliquots of ethanolic leaves extract were mixed with 2 ml of 2% Na2CO3 and allowed to stand for 2 minutes at room temperature. After incubation 100 μl Folin-Ciocalteu phenol rea­gent was added and mixture was mixed thoroughly and allowed to stand for 30 minutes at room tempera­ture in dark. Absorbance of samples was recorded at 720 nm using spectrophotometer and phenolic content was expressed as gallic acid equivalents.

DPPH radical scavenging activity. Antioxidant activity in okra was determined using DPPH (2, 2-Di-phenyl-1-picrylhydrazyl) assay (Zubia et al., 2007) with some modification. An aliquot of 200 μl of ethanolic leaves extract (0.2 mg/ml of ethanol) was mixed with 800 μl of 100 mM Tris-HCl buffer (PH 7.4). The mix­ture was added to 30 μM DPPH (dissolved in DMSO) and vortex, then left to stand at room temperature in the dark. The absorbance was measured at 517 nm after 1 minute and 30 minute of incubation, using UV-visible spectrophotometer against ethanol, used as blank. One ml ethanol with 1 ml of DPPH was used as control. Synthetic BHT was used as positive control. The ability to scavenge the DPPH radical was calculated using the follow equation:

% of inhibition = [(Acontrol – Asample)/ Acontrol] × 100

Where the Acontrol is the absorbance of the control (DPPH solution without sample), the Asample is the absorbance of the test sample (DPPH solution plus test sample).

Statistical analysis. The experiment was conducted twice and data were subjected to analysis of variance (ANOVA) and means were separated using the least significant difference (LSD) according to (Gomez and Gomez, 1984).


Influence of cotton cake and antagonistic micro­organism on plant growth and development of root rot infection. Application of cotton cake 1% alone or with PGPR and P. lilacinus showed positive impact on plant growth by improving plant height, fresh shoot weight and root length. PGPR and P. lilacinus with cot­ton cake 1% w/w significantly (P < 0.05) increased plant growth and caused maximum reduction in diseases severity. Greater plant height was observed in mixed treatment of cotton cake with PGPR and P. lilacinus fol­lowed by P. lilacinus together with cotton cake. A signifi­cant increase in fresh shoot weight was found where cot­ton cake 1% w/w was used followed by cotton cake with carbendazim or cotton cake with P. lilacinus (Table I).Highest root length was observed by the application of carbendazim used in amended soil, whereas greater fresh root weight was recorded in cotton cake with P. lilacinus treatment (Table I). Application of PGPR and P. lilacinus in soil amended with cotton cake 1% significantly sup­pressed infection of M. phaseolina, Fusarium oxysporum, F. solani with complete inhibition of R. solani (Table II). Use of carbendazim with cotton cake also significantly inhibited root rotting fungi as compare to the control. Soil application of cotton cake alone also showed reduc­tion of M. phaseolina, R. solani, and F. solani. P. lilacinus in cotton cake amended soil also showed suppressive effect on root rotting fungi (Table II).

Table I
Effect of P. aeruginosa and P. lilacinus on growth of okra plants in soil amended with cotton cake (1% w/w)
Treatments Shoot length


Fresh shoot weight (g) Root length (cm) Root weight (g)


Cotton cake (1% w/w)

P. aeruginosa

P. lilacinus

P. aeruginos+P. lilacinus

Cotton cake+ carbendazim

Cotton cake + P. aeruginosa

Cotton cake + P. lilacinus

Cotton cake+ P. aeruginosa + P. lilacinus











































5.781 1.541 2.911 Ns
1 Mean values in column showing differences greater than LSD values are significantly different at p < 0.05
Table II
Effect of P. aeruginosa and P. lilacinus on the infection of M. phaseolina, R. solani, F. solani and F. oxysporum in soil amended with cotton cake (1% w/w)
Treatments F.oxysporum, F. solani M. phaseolina R. solani
Infection %  


Cotton cake (1% w/w)

P. aeruginosa

P. lilacinus

P. aeruginos+P. lilacinus

Cotton cake+ carbendazim

Cotton cake + P. aeruginosa

Cotton cake + P. lilacinus

Cotton cake+ P. aeruginosa + P. lilacinus










































LSD0.05 = Treatments= 18.091, Pathogens= 11.442
1 Mean values in column showing differences greater than LSD values are significantly different at p < 0.05
2 Mean values in rows showing differences greater than LSD values are significantly different at p < 0.05

Polyphenols content. In this study, phenolic con-tent was measured in terms of mg% gallic acid equiva­lent (mg% GAE) using the Folin Ciocalteu reagent (Table III). Cotton cake 1% alone or with PGPR and P. lilacinus showed phenolic content ranged from (45.25 mg% GAE to 57.75 mg% GAE). Combined soil application with cotton cake + PGPR + P. lilaci­nus showed significantly (P < 0.05) higher polyphe­nols (57.75 mg% GAE) as compared to the control and chemical fungicide, carbendazim treatments i.e., 37.25 mg% and 34.25 mg% GAE respectively. Appli­cation of PGPR alone also showed higher phenolic content (50 mg% GAE) as compared to the P. lilacinus (39.15 mg% GAE) used alone but with cotton cake showed (48.25 mg% GAE).

DPPH radical scavenging activity. DPPH Radi­cal Scavenging Assay-1, 1-diphenyl-2-picryl hydrazyl (DPPH) was used in this study to determine the free-radical scavenging activity of the plant samples. This is a stable free radical whose color changes from violet to yellow when it is reduced by hydrogen donation. Butylated hydroxytoluene (BHT) was used as a stand­ard. The activity of leaves extract at two different time intervals i.e. at 1 minute and 30 minute were observed. The activity of extracts increased with the time of incu­bation as compare to BHT as shown in (Table III). The antioxidant activity initially was weaker than standard but increased with time. The activity was significantly (P < 0.05) higher (57.66%) in PGPR + P. lilacinus treated plants in amended soil. Okra plants with chemical fun­gicide and without any treatment showed lowest anti­oxidant activity (31.5%, 31.05%). It was also observed that extracts which show highest polyphenols, showed highest antioxidant activity (Table III). Furthermore antioxidant activity was reached more than 50% in plants grown in cotton cake amended soil alone or with carbendazim or with PGPR + P. lilacinus (Table III).

Table III
Effect of P. aeruginosa and P. lilacinus on antioxidant activity and polyphenol contents in okra in soil amended with cotton cake (1% w/w)
Treatments Antioxidant activity

(% inhibition)

Phenolic contents

(mg% gallic acid)

1 minute 30 minutes  











Standard (BHT)



Cotton cake (1% w/w)

P. aeruginosa

P. lilacinus

P. aeruginos+P. lilacinus

Cotton cake+ carbendazim

Cotton cake + P. aeruginosa

Cotton cake + P. lilacinus

Cotton cake+ P. aeruginosa + P. lilacinus























12.641 16.131 14.091
Mean values in column bearing same superscript letters are not significantly (P < 0.05) different according to Duncan’s multiple range test
1 Mean values in column showing differences greater than LSD values are significantly different at (P < 0.05)


The biological control of soil-borne pathogens with mixture of biocontrol agents, organic amendments and micronutrients is a new approach in crop protection to reduce the disease damage level in economically important crops (Bharathi et al., 2004). In this study soil amendment with cotton cake caused significant con­trol of root rotting fungi and improved growth of okra. Organic amendments are generally used for improv­ing crops, increasing agricultural productivity and sup­pressing soil borne diseases (Stone et al., 2003; Sultana et al., 2011). Among the wide variety of organic mat­ters tested as organic amendments for managing plant pathogens oil seed cakes significantly suppressed the soilborne pathogens (Ehteshamul-Haque et al., 1995; Sharma et al., 1995).

In this study application of PGPR or P. lilacinus in amended soil showed promising results by reduc­ing the soilborne pathogens, producing the healthier plants and improving the antioxidant status of the okraplants. There are reports that microbiota, e.g. rhizobac-teria, Trichoderma, and Pseudomonas spp., present indecomposing organic matter may enhance growth andyield of crops (Sylvia, 2004) by producing plant growth hormones and chemical compounds (e.g. siderophores, tannins, phenols) which are antagonistic to various soil-borne pathogens (Antonio et al., 2008). In this study plants grown in cotton cake amended soil and received both PGPR and P. lilacinus showed maximum amount of polyphenols as compared to other treatments. Organicfertilization has been reported to have larger impact on the phyto-nutritional quality of crops. Phenolic com­pounds are important plant secondary metabolites that can help plants tide over oxidative stress working as antioxidants (Grassmann et al., 2000; Urquiaga and Leighton, 2000). Toor et al. (2006) reported that organic fertilizers increased the content of ascorbic acid and total phenolics in tomato. Similarly Dumas et al. (2003) reported that inorganic fertilizers reduce the antioxi­dants while organic fertilizers were proved to enhance the antioxidant content in plants. It is also known that phenolic compounds are potential antioxidants and free radical- scavengers. Kumar et al. (2008) reported that there should be a close relation between the content of phenolic compounds and antioxidant activity.

Fertilizer and pesticide affect the human health and cause damage to the environment. Application of organic amendment and biocontrol agents are envi­ronmental friendly and an alternative strategy to the prevalent use of synthetic pesticides. Combination of introduced biocontrol agents with oil cake was more consistent against disease suppression. The results of the present study show that mixed application of oil cake with PGPR and P. lilacinus enhance plant growth and suppress the infection of soil borne root rotting fungi via increasing the polyphenols and antioxidant activity in okra plants.


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