Physiological Effects of Pseudomonas fluorescens on Tomato  

N. Shiva1 , G. Gomathi1 , S. Karthika1 , S. Ramya1 , B. Senathipathi1 , P. Senthil2 , K. Krishna Surendar2 , S. Ramesh Kumar2
1. Vanavarayar Institute of Agriculture, Pollachi-642103, India
2. Assistant Professors, Vanavarayar Institute of Agriculture, Pollachi-642103, India
Author    Correspondence author
International Journal of Horticulture, 2013, Vol. 3, No. 18   doi: 10.5376/ijh.2013.03.0018
Received: 07 May, 2013    Accepted: 20 May, 2013    Published: 24 May, 2013
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Shiva et al., 2013, Physiological Effects of Pseudomonas Fluorescens on Tomato, International Journal of Horticulture, 2013, Vol.3, No.18 104-108 (doi: 10.5376/ijh.2013.03.0018)


Tomato plants were grown in green house conditions and were studied under biological control of Fusarium oxysporum f.sp. lycopersici and Pythium aphanidermatum causing wilt and damping off in order to evaluate the relative water content, soluble protein content, total phenolic content and yield changes. Application of P.fluorescens+T.viridi +soil application had higher soluble protein content and total phenolic content in tomato. The highest increase in soluble protein and total phenolic content of 20 and 18 per cent over control leads to very lesser reduction in yield by the application of P.fluorescens + T.viridi +soil application during the presence of wilt and damping off diseases.

Tomato, soluble protein; Total phenolics; Pseudomonas fluorescens; Trichoderma viridi; Fusarium oxysporum<; Pythium aphanidermatum and yield

The country being blessed with the unique gift of nature of diverse climates and distinct seasons, it makes it possible to grow an array of vegetables (Mathura Raj et al., 2001). In vegetables production, India is next only to China with an annual production of 87.53 million tonnes from 5.86 million hectares having a share of 14.4 per cent to the world production. Vegetables form the most important component of a balanced diet for human beings . Present production of 1.5 million tonnes of vegetable supply only 145 g per capita per day against recommended requirements of 300 g. Tomato (Lycopersicon esculentum Mill) is important and remunerative vegetable crop in India. Tomato fruits provides a rich source of minerals, vitamins and organic acids and contains 3%~4% total sugar, 4%~7% total solids, 15~30 mg/100g of ascorbic aeid, 7.5~10 mg/100 mL titratable acidity and 20~50 mg/100g fruit weight of lycopene. Uttar Pradesh, Maharashtra, Karnataka, Binar and Orissa are major tomato growing states in India. The area under its cultivation is 865000 ha with a production of 16826000 tonnes,productivity 19.5 tonnes per ha contributing 7.95% of total area and production. Tomato is infected by various fungal, bacterial and viral diseases (Doolittle, 1948). Pre-hectare productivity of tomato is quite low because of sevaral factors in which diseases is one of the important factors. In nursery fungi like Fusarium, Pythium , Phytophthora and Sclerotium cause soil borne diseases. Among them Fusarium oxysporum f.sp. lycopersici is one of the problematic diseases currently threatening tomato production (Sokhi et al., 1991). The disease was first reported in India from Pusa Bihar as Fusarium oxysporum f.sp. lycopersici (Sacc). Fusarial wilt caused by Fusarium oxysporum schlechtent.f.sp. lycopersici (Sacc.) W.C Synder and N.H. Hans is a destructive disease of tomato crops world wide (Shen and Wang, 2006). The losses due to this disease ranging from 10 to 80 per cent have been reported from different parts of India (De Boer et al., 2003). Fusarium species are present in soil both in temperate and tropica! regions and are most frequently isolated. The species are involved in diseases of plants and animals and produce toxins which contaminate human and animal food. However, the fusarium species are most important as plant pathogens. Because of their reputation for complexity and their capacity for rapid change, many plant pathologists try to avoid them altogether (Haware, 1993).

Fusarium oxysporum (Schlecht) is a cosmopolitan fungus that exists in many pathogenic forms (Armstrong and Armstrong, 1981). It survives in soil in the form of chlamydospores and mycelia. The mycellium is septate, hyaline, branched and intra-cellular.The pathogen is highly specific to tomato occurring world wide in distribution, wherever tomato is grown (Walker, 1969). It is primarily a soil inhabiting pathogen. Once introduced it remains at all stages of plant growth, starting from nursery upto flowering, it causes Clearing of veinlets and drooping of petioles of young plants lower leaves show yellowing and later the whole plant wilts and die prematurely. Browning of vascular system can be seen in a cross section of the lower stem. In wet weather condition fungal mycelial growth can be seen on dead plants in the form of pinkish mycelial layers (Singh, 1985). The pathogen being soil borne and soil inhabitant, persist for long periods in the soil. The most serious soil borne fungi causing damping off (Hesse,1874) of tomato seedlings in florida is Pythium aphanidermatum (Edson) Fitzp. Pythium species are essentially soil borne pathogenic fungi, it cause seed rot and seedling damping off of many crops, including tomato and chilli(shah-smith and burns,1996). Damping off of tomato seedlings is a destructive disease in nurseries causing heavy losses in transplantable population. Damping off of direct seeded and transplanted tomatoes is most serious during crop seasons when warm, humid weather with frequent rains fallows planting. The pathogen penetrates in soil through its oospores present in infected plant debris or most commonly through mycellium.The mycellium is colourless, slender, coenocytic, profusely branching and rapidly growing. Symptoms of this disease generally manifest at two stages, they are pre-emergence stage and post-emergence stage. In Pre-emergence stage the sown seeds generally fail to germinate, become softy and mushy, then turns brown, shrink and finally disgenerate. In Post-emergence stage the germinated seedlings are infected at young tissues at or below the ground level and the infected tissue become discoloured and water-soaked and soon collapse. The infectedd part of he seedling looks much thinner and softer trhan the healthy ones and it fails in supporting the seedling. This resuluts in toppling over of infed seedlings on the soil anytime after they emerge from the soil until the stem has hardened sufficiently to resist infection. More over, the pathogen continues to invade the seedling tissues after it has fallen on the ground,the seedling quickly withers and dies. Eradication of this soil borne pathogens is a difficult problem because of its polyphagous nature and its survival in the soil through its resting structure. Protection of crop plants from diseases by using Fungicides has been a widely adopted strategy and a regular practice followed for many years. However there have been many drawbacks in using chemical fungicides for managing the crop diseases. The pollution to soil, water and air caused by the accumulation of obnoxious chemical residues due to continuous use of fungicides and development of resistant strains of pathogens to these fungicides are forcing the scientists to look for methods which are eco-friendly, safe and more specific to pathogens. Biological control of soil borne plant pathogens by addition of antagonistic microorganisms to the soil is a potential non-chemical means (Harman, 1991) and is known to be a cheap and effective method for the management of soil borne diseases (Selvarajan, 1990; Sheela, 1996).
Biological approach is eco-friendly, does not leave any residual toxicity, besides being cost effective and can be successfully exploited in the framework of integrated disease management (Malarajan, 1996). Application of a single antagonist often results in inconsistent management of the disease and the antagonistic strain may not grow equally well in a variety of environmental conditions. One of the strategies to overcome this problem is to combine the disease suppressive activity of two or more beneficial antagonists. Till today no single method is found to be very effective and economical for the management of damping off and fusarium rot disease. Hence, an integrated approach would always ensure maximum disease suppression without any deleterious effect on the ecosystem. Application of organic amendments to soil is a traditional practice in Indian Agriculture. Besides providing nutrients to plants organic matter reduces the inoculums density of soil borne pathogens through changes in the general microbial balance of soil (Lukade, 1992). Hence, integration of organic amendments along with antagonists would ensure better consistency in controlling the pathogen. With this above background, the experiment aimed at evaluating the physiological effects of Pseudomonas fluorescens the progressive Fusarium and Pythium diseases, as well as to investigating the physiological and biochemical behavior in tomato submitted to biological control of Fusarium and Pythium.
Soluble protein
The data on soluble protein content were significantly differed between the treatments. Among the nine treatments, T7 (T.v+P.f+Dipping+Soil application) showed highest soluble protein content, which was followed by T1 and T8. The lowest soluble protein content were recorded in T9 (control) treatments (Table 1; Figure 1).

Table 1 Effects of Pseudomonas fluorescens on total phenolics, soluble protein & poly phenol oxidase enzyme activity in tomato


Figure 1 Green house field view

Poly phenol oxidase (PPO)
The result on poly phenol oxidase enzyme activity were significantly differed at all the nine treatments. Among the treatments, T7 (T.v+P.f+Dipping+Soil application) maintained its superiority in poly phenol oxidase enzyme activity, which was followed by T2 and T5. The lowest soluble protein content were recorded in T9 (control) treatments (Table 1; Figure 2).

Figure 2 Treatment effect

The present study revealed that tomato plants treated with consortium (seed+soil) showed higher induction of soluble protein and Poly phenol oxidase. These findings are in agreement with several workers The ISR stimuli were shown to be salicylic acid, a virulent pathogen and antagonistic biocontrol agent like T. viride (Wei et al., 1996).
Accumulation of peroxidase has been correlated with induced systemic resistance in several plants (Ramamoorthy and Samiyappan, 2001; Chen et al., 2000; Dolisay and Kuc, 1995 and Hammesschmidt et al.,1982). Similarly, enhanced PPO activity due to application of biocontrol agents have been reported by several workers. P. fluorescens induced PPO isozymes in rice against R.solani (Radjacommare, 2000). P. fluorescence plus chitin bio formulation was found to enhance the activity of polyphenol oxidase and suppressed the incidence of anthracnose in mango leaves (Vivekananthan, 2004). Early and increased synthesis of PPO enzyme was observed in the T. viride, P. fluorescens and Bacillus subtilis pre treated peppermint plants challenged with R. solani (Kamalakannan et al., 2003). Accumulation of PPO was higher in the combination of Pseudomonas strains treated banana plants (Harish, 2005) and in rice plants pretreated with PGPR isolates (Radjacommare, 2005).
From this study it has been concluded that tomato plants treated with consortium of bio-agents enhance induction of defense related enzymes such as soluble protein, PPO and total phenol content, which could have induced defense responses and enhanced the induced systemic resistance (ISR) of the tomato plants, which in turn would have reduced the disease incidence.
Materials and Methods
The experimental design was a completely randomized block design with three replications (Figure 1). The treatment details viz., T1=T.v+Dipping, T2=P.f+Dipping, T3=T.v+Soil application, T4=P.f+Soil Application, T5=T.v+Dipping+Soil application, T6=P.f+Dipping+Soil application, T7=T.v+P.f+Dipping+Soil application, T8=Chemical, T9=Control. The physiological mechanisms of soluble protein content and polyphenoloxidase enzyme activity were estimated during flowering stages and fruit formation stages. The procedure was given below.
Soluble protein content
The content of soluble protein was estimated from the leaf samples following the method of Bradford (1974) and expressed as mg/g fresh weight.
The leaf sample of0.5g was macerated with 10 mL of phosphate buffer (0.1M, pH 7.0) using a pestle and mortar. The extract was centrifuged at 10000 rpm @ 4℃ for 20 minutes. 0.1 mL of supernatant was taken and 5 ml of dye mixture was added. The solution was mixed well and kept aside for 15 minutes. The colour intensity was recorded at 595 nm optical density.
Polyphenol Oxidase (PPO)
The Poly Phenol Oxidase (PPO) activities of the leaf sample was estimated at all the stages of the crop by the method of Bray and Thrope (1954) and expressed as unit-1 min-1 mg of protein-1.
The leaf sample of 0.5g was macerated with 10 ml of sodium phosphate buffer (0.1M, pH 7.0) using a pestle and mortar. The extract was centrifuged at 10000 rpm at 4℃ for 20 minutes. The supernatant solution of 0.5 mL was taken in a test tube and 2 mL of sodium phosphate buffer (125µmol, pH 6.8), 0.5 mL of pyrogallol solution (50 µmoles) was added and kept in water bath for 5 minutes at 25 to 30℃ or at room temperature and 0.5 ml of H2SO4was added. The Optical Density was recorded at 420 nm against blank.
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