Determination of compatibility of Pseudomonas fluorescens and Trichoderma harizianum grown on deoiled cakes of neem and jatropha for mass multiplication of P . fluorescens and T . harizianum in vitro

Tomato plants grown in sterilized soil applied with Pseudomonas fluorescens grown on Jatropha cake resulted in highest recovery of P. flourescens from soil. Combined application of two bio-agents in the rhizosphere of tomato in sterilized soil resulted in comparatively lower recovery of P. fluorescens and Trichoderma harzianum but was found better for root growth, shoot growth and fruit yield enhancement than their single application. Combined application of bio-agents grown on any of the cake to the sterilized soil resulted in “three time” increase in root length, 1.5 times increase in shoot length and “five time” increase in fruit yields of tomato crop.


INTRODUCTION
Some naturally occurring soil bacteria and fungi have demonstrated great potential to antagonize disadvantageous microorganisms responsible for causing diseases in several crops, hence, use of such plant beneficial microorganisms for plant protection is being considered as an alternative to reduce the use of chemical pesticides (Compant et al., 2005).In certain soils, such indigenous plant-beneficial microorganisms are able to suppress the activities of several pathogenic microorganisms and these soils are referred as suppressive soils' (Weller et al., 2002).Thus, suppressive soils are regarded as sources of antagonistic microorganisms.Bio-control of plant diseases can be ensured by either augmentation of naturally occurring micro-biota or by introducing a new one to the soil/ rhizosphere.
Many agro-industrial bio-products such as deoiled cakes of tree born oils seeds (TBOs) like Neem and Jatropha which are either going waste or being used as a less profitable and usable products since quite long time.
The oils extracted from Neem and Jatropha, are either directly used as bio-fuel or as raw material for industrial inputs in various manufacturing industries like cosmetics, agrochemicals and pharmaceuticals.Deoiled cakes of these trees remain either unexploited or poorly exploited.These deoiled cakes contains lot of carbohydrates, proteins, fatty acids, minerals and many more biochemical constituents which may serve as source of nutrition for beneficial micro-organisms (growth promoting and bio-control agents) in crop cultivation, hence may be exploited as substrate for mass multiplication of bacterial bio-control agents such as Pseudomonas flourescens.
Mass multiplication of Trichoderma has been successfully done on Neem and Jatropha.
In addition, application of Bio-control agent (BCAs) grown with deoiled cakes which are rich in many nutrients may reduce soil-borne diseases by releasing allelochemicals generated during product storage or by subsequent microbial decomposition.
Tomato is susceptible to several pests and diseases causing considerable decreases in its productivity.In spite of the introduction of several high yielding varieties, its yield potential could not be exploited up to expectation due to pests and diseases.Tomato is found to suffer from a variety of diseases caused by fungi, bacteria, viruses and nematodes.The important diseases are damping off, early blight, late blight, Fusarium wilt, Verticillium wilt, bacterial wilt and tomato mosaic virus.

Collection of soil samples and isolation of bio-control agent
To isolate the biocontrol agent that is, P. fluorescens from tomato crop rhizosphere, soil samples were collected from crop research centre (CRC) of SVPUA&T University Modipuram Meerut Uttar Pradesh 250110 (India).For isolation, one gram of soil sample was placed in a 250 ml conical flask containing 100 ml of sterilized distilled water (SDW) and mixed thoroughly.Different dilutions of working samples were prepared by serially diluting the stock solution (10 -8 ). 1 ml of last serial dilution that is, 10 -8 was spread on P. fluorescens selective king's B Medium (King's et al., 1954) for isolation of P. fluorescens.The plates were incubated for 2 days at 28±2°C and after incubation, pure culture was grown; colour of bacterial colony was initially yellow but turned yellow green as pigmentation were produced (Bonds et al., 1957).Composition of culture media as given of P. fluorescens (Selective) King's B Medium (King et al., 1954).

Maintenance of the culture
The bacteria initially isolated in a pure culture on King's B media and subcultured on PDA slants were allowed to grow at 28±2°C temperature.The culture thus obtained was stored in refrigerator at 5°C for further studies and was sub cultured periodically.

Isolation and maintenance of Trichoderma harzianum culture
To isolate the biocontrol agent that is, T. harzianum from rhizosphere, soil samples were collected from crop fields of crop research centre (CRC) of university.For isolation, 10 g of soil sample was placed in a 250 ml conical flask containing 100 ml of sterilized distilled water (SDW) and mixed thoroughly.Different dilutions of working samples were prepared by diluting the stock solution (10 -6 ). 1 ml of last serial dilution that is, 10 -6 was spread on Trichoderma Selective Medium (TSM) for isolation of T. harzianum.The plates were incubated for 7 days at 28±2°C and after incubation, pure culture was grown; it was initially hyaline but turned green as colony were produced (Rhode, 1959).

Preparation of culture media
Trichoderma selective medium (TSM): All the ingredients were mixed with distilled water, agar was added to it in a stainless steel pan and steered with glass rod for proper mixing.Now the medium was filtered through a muslin cloth by squeezing out whole liquid.200 ml dissolved medium was placed in each 500 ml capacity flasks.Flasks were tightly plugged with non-absorbent cotton and wrapped with butter paper and rubber band.Then medium was autoclaved at 1.1 kg/cm 2 pressure for 20 min at 121.6°C and cooled before pouring into Petriplates (Elad and Chet, 1983).

Determination of population dynamics and longevity of P. fluorescens on deoiled cakes of neem and jatropha
Deoiled cakes of two tree born oilseeds that is, neem and jatropha were used during the course of investigation.These cakes were collected from local agricultural product-processing units.Cakes were used after adding sufficient amount of sterilized distilled water to maintain three level of moisture that is, 15, 25 and 35% (w/v) and autoclaved at 121.6°C (1.1 kg/cm 2 ) for 20 min.The flasks were allowed to cool at room temperature prior to inoculation.Flasks containing substrates were inoculated with 3-4 days old actively growing culture of P. fluorescens (2-3 bits of 5 mm size from the culture grown on PDA in Petri plates under aseptic conditions in laminar flow.
Flasks inoculated with P. fluorescens were incubated at 28±2°C and shaken thoroughly once a day.For each treatment, three replicates of flasks were maintained and arranged in a completely randomized manner.Population dynamics of P. fluorescens were determined by following serial dilution plate technique at each 15 days interval till these cakes yielded CFUs of P. fluorescens.After 15 days of incubation substrates were used for mixing it to rhizosphere of tomato plants to determine longevity and viability of bioagent in the soil planted with tomato.
Determination of population dynamics and longevity of T. harzianum on deoiled cakes of neem and jatrofa: Deoiled cakes of neem and jatropha were used after adding sufficient amount of sterilized distilled water to maintain two level of moisture that is, 15 and 25% (w/v) and autoclaved at 121.6°C (1.1 kg/cm 2 ) for 20 min.The flasks were allowed to cool at room temperature prior to inoculation.Flasks containing substrates were inoculated with 3-4 days old actively growing culture of T. harzianum (2-3 bits of 5 mm size from the culture grown on PDA in Petri plates under aseptic conditions in laminar flow.
Flasks inoculated with T. harzianum were incubated at 28±2°C and shaken thoroughly once a day.For each treatment, three replicates of flasks were maintained and arranged in a completely randomized manner.Population dynamics of T. harzianum were determined by following serial dilution plate technique at each 15 days interval till these cakes yielded CFUs of T. harzianum.After 15 days of incubation substrates were used for mixing it to rhizosphere of tomato plants to determine its longevity and viability in the rhizospheric soil.

Determination of effect of substrate concentration and dilution of inoculums on population dynamics of P. fluorescens
Initialy the P. fluorescens cultures were grown in King's B broth medium at 28 ± 2°C for 2 days.After 2 days of incubation, a serial dilution of 10 -5 , 10 -6 , 10 -7 and 10 -8 were prepared by the method given at para Dilution Plate Method.

Monitoring population dynamics in deoiled cakes
Population of P. fluorescens and T. harzianum were monitored after each 15 days interval.For this purpose, 1 g of each cakes where P. flourescens or T. harzianum were inoculated were taken from each flasks maintained for different duration that is, 15 to 120 days and CFUs were counted using PDA through dilution plate technique.Determination of population dynamics and longevity of P. fluorescens and T. harzianum in sterilized and unsterilized soil planted with tomato.A pot culture experiment was conducted to determine the longevity of P. fluorescens and T. harzianum grown on neem and jatropha cakes, which was subsquently incorported to pot soil planted with tomato.For this purpose, 2 days old P. fluorescens and 15 days old T. harzianum culture were mixed to the sterilized neem cake and jatropha cakes and incubated for 15 days for proper colonization and growth of these two organisms on substrates, now these substrates were added to sterilized and unsterilized soil in clay pots and mixed thoroughly as per detail given below.

Treatments details
T 1 : Tomato plants grown in sterilized soil applied with P. fluorescens grown on neem cake.T 2 : Tomato plants grown in sterilized soil applied with P. fluorescens grown on jatropha cake T 3 : Tomato plants grown in sterilized soil applied with P. fluorescens grown on neem cake + T. harzianum grown on Neem cake.T 4 : Tomato plants grown in sterilized soil applied with P. fluorescens grown on jatrofa cake + T. harzianum grown on jatropha cake T 5 : Tomato plants grown in unsterilized soil applied with P. fluorescens grown on neem cake in unsterilized soil.T 6 : Tomato plants grown in unsterilized soil applied with P. fluorescens grown on jatropha cake in unsterilized soil.T 7 : Tomato plants grown in unsterilized soil applied with P. fluorescens grown on neem cake+ T. harzianum grown on Neem cake.T 8 : Tomato plants grown in unsterilized soil applied with P. fluorescens grown on neem cak + T. harzianum grown on jatropha cake.T 9 : Tomato plants grown in sterilized soil without any amendments.(Control-1).T 10 : Tomato plants grown in unsterilized soil without any amendments (Control-2).

Pot filling and planting with tomato seedlings
After sterilization, soil was left overnight for proper cooling.Now 15 pots were filled with sterilized soil at 3 kg per pot and in another set 15 pots were filled with unsterilized soil at 3 kg per pot.However, both the soil lots were from similar field to maintain similarity in physico chemical and biological properties of soil.In each pot 75 g mass culture of either P. fluorescens or T. harzianum grown on oil cakes of neem and jatropha, were added either separately or mixture of these two as per treatment needs .After pot filling and application of amendments same day pots were planted with 3 seedlings of tomato (cv.Arjun H-1) per pot.Two sets of check were maintained that is, in sterilized and unsterilized soil, without application of any amendments to have a comparison.Each treatment was replicated thrice.

Assessment of population dynamics of P. fluorescens and T. harzianum
After 15 days of planting, one gram soil sample from each replicate of every treatment was taken from 10 cm depth with sterilized disk cutter into test tube.It was put into another test tube containing 10 ml sterilized distilled water and shaken well, and diluted up to 10 -8 for P. fluorescens and 10 -6 for T. harzianum.1.0 ml of the suspension was put into sterilized Petri plate poured with king's B medium for P. fluorescens and spread throughout surface with gentle shaking.For monitoring of T. harzianum PDA was used.All the procedure of serial dilution was done under aseptic condition in Laminar Air Flow.The Petri dishes were then incubated for 5 days at 28±2°C for T. harzianum and for 2 days at 28±2°C for P. fluorescens.Within this period colonies were formed which were counted.Population dynamics of P. fluorescens was continuously determined till it yielded with CFUs of bioagents at each 15 days interval by following the procedure given above.

Assesement of root length, shoot length and fruit weight of tomato
Observations were also recorded to measure root and shoot length of tomato along with weight of total fruits in individual pots.Percent increase in root and shoot length and fruit yield were calculated using the following formula:

Statistical analysis
The data were subjected to analysis of variance, and treatment means were differentiated using Fischer's T test.The data taken into percentage were first transformed into angular value and then analyzed for test of significance (Gomez, and Maravall 1996;Chandel, 2002).
Length in treated pot -Length in control pots % length increase = x 100 Length in control pots Yield under protected -Yield under unprotected Percent yield increase = x 100 Yield under unprotected

RESULTS
Present studies entitled "longevity and survival of P. fluorescens on neem and jatropha cakes" were conducted under laboratory and pot conditions with the objectives to determine the suitability of deoiled cakes of neem and jatropha for mass multiplication of P. fluorescens in vitro and to determine the longevity of P. fluorescens grown on two deoiled cakes in the rhizosphere of tomato at the Department of Plant Pathology, S.V.P. University of Agriculture and Technology, Meerut.

Screening of deoil cakes of neem and jatrofa for mass multiplication of T. harziniaum
Two deoiled cakes which were earlier tested for suitability for survival and longevity of P. fluorescens, were also tested further for supporting population dynamics and longevity of T. harzianum at two different level of moisture that is, 15 to 25% upto a period of 120 days at each 15 days interval.Results obtained have been presented in Tables 2 and 3.

Population dynamics of T. harzianum on neem cake
It is evident from Table 2 that neem cake containing 15% level of moisture resulted in 13.00×10⁶ level of CFUs of T. harzianum at 15 days of inoculation.At 30 days the population of T. harzianum increased to the level of 21.67×10⁶.After 45 days, population of T. hearzianum on neem cake further increased to 32.33×10⁶.At 60 days of inoculation, population of T. harzianum started declining and showed 30.67×10⁶ level of CFUs.At 75 days the population of T. harzianum was 27.33×10⁶ and at 90 days, it was only 23.00×10⁶.At 105 days of inoculation population of T. harzianum was 13.67×10⁶ and at 120 days the population level was 4.67×10⁶.The level of population of T. harzianum at each 15 days interval significantly different from each other.
Neem cake containing 25% moisture resulted in 15.00×10⁶ number of CFUs of T. harzianum after 15 days of inoculation, whereas at 30 days the population increased to the level of 25.00×10⁶.At 45 days this increased to the level of 39.00×10⁶ CFUs of T. harzianum.At 60 days of inoculation, population was 36.00×10⁶ and further decreased to the level of 33.00×10⁶ after 75 days of inoculation.At 90 days of inoculation the neem cake containing 25% moisture resulted in 27.33×10⁶ level of CFUs of T. harzianum.At 105 days and at 120 days the population of T. harzianum decreased to 15.33×10⁶ and 6.77 ×10⁶ respectively.Level of CFUs recorded after each 15 days interval and each level of moisture were significantly different from each other.

Effect of application of P. flourescens grown on neem and jatrofa cakes and T. harzianum grown on neem and jatropha cakes either alone or their mixture on recovery of P. fluorescens and T. harzianum population from soil planted with tomato
Data regarding this experiment have been presented in Table 1.At the time of application to the pot soil neem cake was having CFUs of P. fluorescens 139×10⁸, which was declined to the level of 114.00×10⁸ after 15 days of application in the pot soil planted with tomato (T-1) (Table 3), but with an initial decrease, population get increased from 30 days of application to upto 60 days of application to sterilized pot soil planted with tomato.The recovery of P. fluorecens population was 142.33×10⁸, which get increased further at 45 days (169.00×10⁸).Highest recovery of Pseudomonas population was on 60 days of application, which was 192.33×10⁸.Starting from 75 days onward, the population of P. fluorescens started decreasing as there was 136.33 ×10⁸ CFUs at 75 days, 93.00×10⁸ at 90 days, 43.00×10⁸ at 105 days, and only 9.00×10⁸ at 120 days of application.The population of Pseudomonas recovered from sterilized soil planted with tomato at each 15 days interval were siganificantly different from each other.Application of P. fluorescens grown on jatropha cake to sterilized soil planted with tomato (T-2) resulted in 120.33×10⁸ after 15 days of application which was significantly less than initial population at the time of application (148.00×10⁸),but when the population was monitored after 30 days of application, the population get increased to 151.00×10⁸, which further increased to 181.00×10⁸ and 200.33×10⁸ (highest among all), at 45 days and 60 days of application respectively, to the pot soil.After 75 days onwards the population of P. fluorescens started declining in the rhizosphere of tomato plants as there was 152.00×10⁸, 94.00×10⁸, and 53.00×10⁸ and 12.67×10⁸, number of CFUs of P. fluorescens, at 75, 90,105 and 120 days of application respectively to the sterilized soil planted with tomato.
The population of Pseudomonas recovered from tomato rhizosphere, after application with jatropha cake grown Pseudomonas were significantly different from each other, at each 15 days interval.It was also observed that population of P. fluorescens was comparatively higher in the rhizospheric soil applied with jatropha cake, than, those applied with neem cake.When mixture of neem cake grown P. fluorescens + neem cake grown T. harzianum were applied to the sterilized pot soil (T-3), there was recovery of 103.66×10⁸CFUs of P. fluorescens after 15 days of application, which get increased to122×10⁸,146.66 ×10⁸, and 171.33×10⁸ after 30, 45 and 60 days of application respectively.After 75 days onward population of P. fluorescens were found to be decreasing as recovery of CFUs were 120.00×10⁸, 72.66×10⁸, 21.66×10⁸, 6.66×10⁸ after 75, 90, 105 and 120 days respectively after inoculation.This treatment showed  When mixture of jatropha cake grown P. fluorescens + jatropha cake grown T. harzianum were applied to the sterilized pot soil (T-4), there was recovery of 110.33×10⁸, 139.33×10⁸, 169.00×10⁸and 187.33×10⁸P. fluoroscens after 15, 30, 45 and 60 days of application respectively.
Application of P. fluorescens grown on neem cake to unsterilized soil planted with tomato (T-5) with an initial population of 139×10 8 , for starting 15 days it was declined to the level of 104.00×10 8 after 15 days of application.However, the population of P. fluorescens started further increasing upto 60 days of application.The population of P. fluorescens was131.33×10 8, 157.67×10 8 and 181.00×10 8 number of CFUs of P. fluorescens after 30, 45 and 60 days of application respectively, to the unsterilized soil planted with tomato.From 75 days onwards the population of Pseudomonas started declining as number of CFUs recovered were 162.67×10 8 , 109.00 ×10 8 , 69.67×10 8 and 13.33 ×10 8 at 75, 90, 105 and 120 days of application respectively to the unsterilized soil planted with tomato.The number of CFUs recovered after each 15 days in unsterilized soil provided with Pseudomonas was grown on neem cake was significantly different from each other.Application of P. fluorescens grown on jatropha cake in unsterilized soil planted with tomato (T-6) with an initial 148.00×10 8 CFUs of P. fluorescens resulted in a decline in the population to the level of 114.00×10 8 after 15 days of application.Number of CFUs of P. fluorescens were increased upto 60 days in the rhizosphere of tomato plants in unsterilized soil, which were 142.67×10 8 , 174.33×10 8 and 192.00×10 8 after 30, 45 and 60 days of application respectively.After 75 days onwards the population of P. fluorescens population of P. fluorescens was found to be decreasing which were 173.33.×10 8 , 115.66.× 10 8 , 80.00×10 8 and 17.66×108 after 75, 90, 105 those where these bioagents were applied along with some of the cake.

Effect on root length
Application of mixture of P. flourescens grown on jatrofa cake and T. harzianum grown on jatrofa cake in sterilized soil (T-4) planted with tomato resulted in highest root length of tomato (42.50 cm) (Table 2).Root length in T-4 was almost three times higher than control (14.50 cm).Second highest root length (40.50) were observed from the tomato plants which were planted in sterilized soil applied with mixture of P. flourescens grown on neem cake and T. harzianum grown on neem cake (T-3).Root length from T-3 and T-4 were significantly different from each other.Tomato root length noticed from the plants provided with P. flourescens grown on jatrofa cake in sterilized soil (37.83cm) (T-2), P. flourescens grown on neem cake in sterilized soil (37.50cm) (T-1), mixture of P. flourescens grown on jatropha cake and T. harzianum grown on jatropha cake in unsterilized soil (37.50 cm)(T-8) and mixture of P. flourescens grown on neem cake and T. harzianum grown on neem cake in unsterilized soil (36.50 cm)(T-7) were at par to each other but significantly less than T-3 and T-4.Application of either P. flourescens grown on jatrofa cake (T-6) or P. flourescens grown on neem cake (T-5) resulted in a root length of 34.50 and 33.50 cm respectively were significantly quite less than T-3 and T-4.However root lenth in sterilized control pots (T9) were significantly less (14.50 cm) than root length noticed in unsterilized control pots (17.50 cm) (T-10).

Effect on shoot length
Tomato plants grown in control sterilized soil pots (T-9) exhibited a shoot length of 37.50 cm.Tomato plants grown in sterilized soil, Applied with mixture of P. flourescens grown on jatrofa cake and T. harzianum grown on jatrofa cake (T-4) resulted in highest shoot length (59.50 cm) (Table 3).Second highest shoot length (58.30) were observed from the tomato plants which were planted in sterilized soil applied with mixture of P. flourescens grown on neem cake and T. harzianum grown on neem cake (T-3).Shoot length from T-3 and T-4 were at par with each other.Shoot length of tomato plant (55.83 cm) in sterilized soil applied with P. flourescens grown on jatrofa cake (T-2) and in unsterilized soil applied with P. flourescens grown on jatrofa cake (55.50 cm)(T-6) were at par with each other but significantly less than T-4 and T3.In the same way, shoot length of tomato plants (53.50 cm) in unsterilized soil applied with mixture of P. flourescens grown on neem cake and T. harzianum grown on neem cake (T-7) and in sterilized soil applied with P. flourescens grown on neem cake (53.17 cm) (T-1) were also at par with each other but significantly less than T-4,T-3, T-2 and T-6 respectively.Tomato grown in unsterilized soil applied with mixture of P. flourescens grown on jatrofa cake and T. harzianum grown on jatrofa cake (T-8) exhibited a shoot length of 51.50 cm.Tomato grown in unsterilized soil applied with P. flourescens grown on neem cake (T-5) exhibited shortest shoot length (48.50 cm).In unsterilized control pot (T-10) shoot length was only 42.50 cm, whereas in sterilized control pots (T-9) it was only 37.50 cm.Shoot length of tomato in T-9 was significantly quite less than shoot length noticed in T-10.

Effect on fruit weight
Fruit weight of tomato in sterilized (T-9) and unsterilized control pots (T-10) were 88.90 and 143.33 g respectively, which were significantly different from each other.Tomato plants which were planted in sterilized soil applied with mixture of P. flourescens grown on neem cake and T. harzianum grown on neem cake (T-3) produced highest fruit yield (480.00 g) which was at par with the fruit yield (430.00 g) and (398.33 g) produced by the tomato plants grown in sterilized soil applied with P. flourescens grown on jatrofa cake (T-2) and in sterilized soil applied with P. flourescens grown on neem cake (T-1) respectively.Tomato plants grown in sterilized soil, applied with mixture of P. flourescens grown on jatrofa cake and T. harzianum grown on jatrofa cake (T-4) resulted in a fruit weight of 381.67 cm which was significantly less than the fruit weight observed in T-3 but at par with the fruit weight noticed in T-2 and T-1 respectively.It was interesting to note that tomato grown in T-5, T-6,T-7 and T-8 were significantly quite less than those treatments which were planted in sterilized soil that is, T-1,T-2,T-3 and T-4 respectively.

DISCUSSION
Deoiled cakes of two trees born oilseeds (TBOs) viz., neem and jatropha, were tested for their suitability for mass multiplication of P. fluorescens and T. harzianum and also that for how long they are able to support the survival of P.fluorescens and T. harzianum with a considerable level of population dynamics.Different concentration of two cakes was also tested for their ability to enhance the population dynamics of P. fluorescens.In addition population dynamics and longevity of P. fluorescens and T. harzianum in sterilized and unsterilized soil planted with tomato were alsodetermined with a mandate to know that for how long different substrates are able to support them when applied to soil with some crop.
Effect of application of P. flourescens grown on neem and jatropha cakes and T. harzianum grown on neem and jatropha cakes either alone or their mixture on recovery of P. fluorescens and T. harzianum population from soil planted with tomato Highest recovery of Pseudomonas population was noticed on 60 days of application in sterilized soil where jatropha cake grown P. fluorescens was applied.Application of neem cake grown P. fluorescens was also resulted in quite higher level of recovery of CFUs of this bioagent but comparatively slightly less than jatropha cake.Mixing two bio-agent resulted in comparatively lower level of recovery of CFUs.Sterilized soil gave higher recovery of P. fluorescens than unsterilized soil.Up to 60 days, population of P. fluorescens tend to increased, whereas after 75 days onward population started decreasing.In case of T. harzianum, its highest population was recorded when mixture of T. harzianum grown on jatrofa cake + mixture of P. fluorescens grown on jatropha cake were applied to sterilized soil planted with tomato after 45 days of application.After 60 days onward population of T. harzianum started declining.Although population started declining after 75 days of application in pot soil but even then it was of quite higher level.
Higher population dynamics in the tomato rhizosphere as compared to laboratory condition may be attributed to the fact that different root exudates and lechates along with some other organic matter available in the soil might have helped P. fluorescens to multiply profusely to maintain comparatively higher level of population dynamics along with quite longer longevity by providing abundant food sources.However it is a unique type of investigation which throw some light on the role of different substrates in supporting the population dynamics of P. fluorescens and longevity after mixing to the soil which will help in checking out the strategy for biological control in a long term perspective in such a way that after how much interval reapplication of P. fluorescens should be done in the soil.
According to Thangavelu et al. (2004), when T. harzianum grown on five different organic substrates (rice bran, rice chaffy grain, farm yard manure, banana pseudo stem and banana dried leaves) and applied as dried formulation to the soil, the population of the T. harzianum (race Th 10) could increased from 10 4 to 10 13 CFUs/g of soil within 60 days which also effectively controlled the Fusarium wilt of banana.Results of present study are partly in accordance with the previous work of Thangavelu et al. (2004) as during present investigation the increasing trend of P. fluorescens population at maximum level was up to 60 days.The results of another previous group of scientists that is, Nosir et al. (2010) also fully support the findings of present study as they have reported that the population of T. harzianum and Aneurinobacillus migulanus could increase from days 30 to 60 followed by a decrease until by day 150 after inoculation, no T. harzianum CFU were recovered afterward.
There was little difference in the level of population dynamics between unsterilized soil planted with tomato and sterilized soil planted with tomato.In case of unsterilized soil planted with tomato, although neem and jatropha cakes could support the population dynamics up to 120 days but in general, level of population dynamics was comparatively lower than the population dynamics recorded due to different substrates in case of sterilized soil planted with tomato.The possible reason behind this trend may be that in unsterilized soil planted with tomato possible presence of diverse microbial population may have pose a competition for nutrition and space for P. fluorescens but there have been no such competition in sterilized soil planted with tomato, because sterilization process of soil had certainly killed all microbes, hence no competition for nutrition and space and that is why the population of P. fluorescens could have been comparatively higher in sterilized soil and comparatively less in unsterilized soil.

Conclusion
Deoiled cakes of two tree born oilseeds (TBOs) viz., neem and jatropha were tested as solid substrate for their suitability for mass multiplication of P. flourescens and T. harzianum.P. flourescens and T. harzianum grown on jatropha cake and neem cake either alone or mixture of these two were applied to sterilized and unsterilized soil and planted with tomato.From these soil, population of P. flourescens and T. harzianum were monitored at each 15 days interval up to 120 days.Root length, shoot length and fruit yield of tomato were also measured in the plants grown in these sets of treatments.Salient findings of these studies have been summarized in following paragraphs.Tomato plants grown in sterilized soil applied with P. fluorescens grown on neem cake also resulted in very high level of recovery of P. flourescens after 60 days of application.Tomato plants grown in sterilized soil either applied with a mixture of P. fluorescens grown on jatrofa cake+ T. harzianum grown on jatropha cake resulted in highest recovery of T. harzianum after 45 days of application.
Application of mixture of two bioagents resulted in comparatively lower recovery of P. fluorescens but it was better for root and shoot growth enhancement.Tomato plants grown in sterilized soil either applied with a mixture of P. fluorescens grown on Jatropha cake or + T. harzianum grown on Jatropha cake or applied with a mixture of P. fluorescens grown on neem cake + T. harzianum grown on neem cake, showed highest root length, highest shoot length and highest fruit yields also.Root length, shoot length and fruit yields of tomato grown in sterilized soil without any amendments were comparatively lesser than those plants grown in unsterilized soil without any amendments.Application of either mixture of P. fluorescens grown on Jatropha + T. harzianum grown on Jatropha cake or mixture of P. fluorescens grown on Neem cake + T. harzianum grown on neem cake to the sterilized soil resulted in thrice increase of root length 1.5 times increase in shoot length and five time increase in fruit yields of tomato.
(1) For soil application P. fluorescens grown on jatropha cake should be preffered over neem cake for longer survival of P. fluorescens with higher viable counts in soil after application.
(2) Mixed application of P. fluorescens + T. harzianum to the soil resulted in suppression of CFUs of both the organisms than their single application.
(3) Mixed application of P. fluorescens + T. harzianum to the soil was effective in enhancing root growth, shoot growth and fruit yields of tomato.(4) Deoiled cakes of Jatrofa is comparatively better than deoiled cakes of neem for supporting fast multiplication of P. fluorescens , enhancing plant growth, vigour and also fruit yield.
(5) Deoiled neem cake is better than deoiled jatrofa cakes in supporting fast multiplication of T. harzianum.

Table 1 .
Grown of the CFUS of P. fluorescens (PF), P. fluorescens and T. harzinaum (TH) on neem cake and jatropha, in the rhizosphere of tomato plants with and without application of bio agent in sterilized and unsterilized soil.

Table 2 .
Root and Shoot length of Tomato plants sterilized and unsterilized tomato plants with P.fluorescens and Trichoderma harzianum.

Root length in cm Increasing percentage Shoot in length in cm Increasing percentage
6 after 15, 30, 45, 60,  75, 90and 120 days respectively.Serilized soil planted with tomato without any amendments (T-9) did not show any recovery of either P. fluorescens or T. harzianum, whereas in unsterized soil (t-10) there was recovery of both the bioagents but comparatively quite less than

Table 3 .
Weight of tomato fruits sterilized and unsterilized soil with P. fluorescens and T. harzianum.