Nematodes In Tomato Crops Biology Essay

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Chapter 1INTRODUCTION

Tomato (Lycopersicon lycopersicum Karst) belonging to family Solanaceae is the most popular and widely consumed vegetable crop (Khoso, 1988). Out of 15 vegetables, tomato ranks at second in terms of total world annual production. In Pakistan it is cultivated over 47.10 thousands hectares with the annual production of about 50.23 thousands tonnes (MINFAL, 2007). It is grown all over Pakistan in different seasons as the climatic and adaphic conditions of Pakistan are favorable for producing high quality tomatoes (Chaudhary, et al., 1995).

Historical evidences showed that tomato is of Tropical American origin and said to be first domesticated in Mexico. From Tropical America, it was then introduced in Europe, West Africa, Tropical Asia and throughout the tropical regions all over the world. Whatever may be its early history of cultivation, the tomato has got tremendous popularity from the middle of 19th century to the present time (Tiwari and Choudhury, 1986). In Pakistan, two crops of tomato are produced annually, first in spring and second in autumn. However, in most areas of Sindh tomatoes can be grown throughout the year. It is the major vegetable cash crop in Sindh, Punjab and Balochistan (Badshah el al., 1997).

As compared to other vegetables, it is rich in nutritional value and available comparatively at low prices. In every home tomato is used in different ways like salad, ketchup, chatni and in other delicious dishes. Proximate composition of tomato fruit shows that 100 grams of edible portion contains 93.76 g of water, 0.85 g protein, 0.33 g total lipids, 4.64 g carbohydrates, 1.1 g total dietary fiber and 0.42 g ash. A fresh raw tomato also contains an exceptional amount of vitamin A (623IU/100 g) and B1/Thiamin (0.059 mg/100 g). It is an excellent source of vitamin C/ascorbic acid (19.1-23 mg/100 g), which is commonly more deficient in our diet as compare to vitamin A or B. in addition , it contains Riboflavin (0.048 mg/100 g) and Niacin (0.628 mg/100 g).100 g of edible portion of tomato also contains Mg (11 mg),P (24 mg), Na (9 mg), Fe (0.45 mg), K(222 mg) and Ca (5 mg). Tomato is said dieter dream in its natural state being very low in caloric fruit containing 21 kcal/88 kJ.

In addition to nutritional value, tomato fruit act as nutraceutical having high medicinal value. Its pulp and juice act as digestive stimulant, mild laxative, a promoter of gastric secretion and blood purifier. It is also considered to be intestinal antiseptic and useful in cancer of mouth and sour mouth. It stimulates torpid liver and is good in chronic dyspepsia (Tiwari and Choudhury, 1986). Tomato is a major source of lycopene, which act as anti-oxidant and anti-cancer agent. Tomatoes are linked in particular to lower rates of pancreatic cancer and cervical cancer. It appears that the consumption of tomatoes (high in vitamin C) confers benefits in preventing and treating cancer.

The average yield of tomato in Pakistan is very low as compared to other tomato growing countries of the world. There can be several reasons for this low yield such as changing climates conditions, conventional methods of cultivation, uncertified low yielding varieties and biotic factors. The biotic factors that affect the productivity and quality of tomato are fungi, bacteria, viruses, nematodes, weeds and insects. Among these biotic factors root knot nematodes are known to cause severe damage to tomato in different parts of the world (Dropkin, 1980) and tomato is considered as the most favorable host for root-knot nematodes (Lamberti, 1979).

Nematodes, belonging to kingdom Animalia, are wormlike in appearance, having wide host range and induce a variety of diseases. Nematodes are the most numerous multicellular animals on earth (Kimpinski and Sturz, 2003). Several hundred species of nematodes feed on living plants, obtain their food with spears or stylets and cause a variety of plant diseases. Nematodes cause 8 to 20% losses to major crops, fruits and vegetables worldwide, for a total of over $87 billion annually (Sasser and Freckman, 1987).

Among the nematodes, causing different plant diseases, root knot nematodes (Meloidogyne spp.) are the most devastating and damaging. These nematodes reduce world crop production by about 5%. Root knot nematode belongs to order Tylenchida, sub-order Tylenchina, super family Heteroidea, family Meloidogynidae and genus Meloidogyne. Amongst all the nematode genera, Meloidogyne genus ranked first and considered very important plant pathogen (Dropkin, 1989).

The root knot nematodes are sedentary endoparasites and are among the most destructive agricultural pests attacking a broad range of crops and cause high levels of economic losses (Mai and Abawi, 1987). They are found in tropical and subtropical areas of the world and infect more than 2000 plant species including almost all cultivated plants (Hussey, 1985). More than 60 species of root-knot nematodes have been reported so far but M. incognita, M. javanica, M. arenaria and M. hapla are relatively more destructive accounting for 95 percent of all root-knot nematode infestations in field crops. The proportion of Meloidogyne incognita in the agriculture soils is 52%, M. javanica 31%, M. arenaria 8%, M. hapla 7% and other species are about 27% (Hussey and Janssen, 2002).

Among the species, M. incognita is widely prevalent, common and damaging 102 plant species in Pakistan. The infection starts when second stage juveniles (J2) penetrate in roots. These nematodes establish feeding sites within the roots where they induce roots galls or knots. Deprive plants of nutrients and cause cellular, metabolic and structural changes within plant tissues. These physiological and physical changes to the plant can reduce crop yield and quality drastically. Nematodes attacking susceptible plants at seedling stage cause heavy losses and may result in complete destruction of the crop. But infections of older plants may show only minor effects on yield or may reduce yields noticeably (Agrios, 2005).

It is estimated that 24-38% tomato plants are affected by these nematodes. The degree of damage caused by nematodes increases in combination with other soil borne pathogens. Tomato plants can be killed when Fusarium oxysporum f. sp. lycopersici is present with Meloidogyne spp. (Sikora and Fernandez, 2005). The adequate control of these biotic factors is directly related to reliable productivity and good quality tomato fruit (Csizinsky et al., 2005).

Root knot nematodes are very difficult to cope with because of their high reproductive potential and polyphagous nature. In order to avoid the subsequent increase of damaging populations, 99.9 percent control should be ensured (Whitehead, 1998). The losses due to root knot nematodes can be reduced by soil fumigation, application of nematicides and by cultivating resistant or non-host plants. But the conventional use of chemical control is causing many severe effects upon the human health as well as on the environment thus the development of unconventional and environment friendly control methods are of great importance, the biological control is one of them. There are more than 200 species of biological control agents, which mostly include fungi, viruses, bacteria and predatory invertebrates (Dasgupta, 1998).

Many compounds have now been withdrawn from use, promoting the need for new, safe and effective options (Zuckerman and Esnard, 1994). Considering all the constraints in sight, the environmental effects, cost of chemicals and health hazards, present study was carried out to test the efficacy of biological control agents Trichoderma harzianum and Azadirachta indica against Meloidogyne incognita on tomato.

The main objective of current study was:

To study the individual and combined effect of T. harzianum and A. indica on M. incognita

Chapter 2 REVIEW OF LITRATURE

Root-knot nematodes are most important and damaging pests having wide host range. They cause huge economic losses to almost all vegetables (Back et al., 2002). So, root knot nematodes are needed to control. A great emphasis has been given on biological control because of its effectiveness and environmental safeness. A brief review of biological control of root knot nematodes is given below.

Biological control by Fungi

Among the biocontrol agents that parasitize or prey on nematodes and lessen nematode populations by their antagonistic actions, fungi hold very important position and some of the fungi have shown great potential as biocontrol agents. Fungi continuously demolish nematodes in almost all types of soils because of their constant association with nematodes in the rhizosphere. The most important genera which trap or prey nematodes include Paecilomyces, Verticillium, Arthrobotrys, Hirsutella, Drechmeria, Fusarium, Nematophthora and Monacrosporium. Applications of these fungi have given outstanding results against nematodes (Siddiqui and Irshad, 1996) .

When different concentrations of T. harzianum were used root knot nematode infection and other parameters were significantly decreased in a green house experiment. Nematode egg hatching level decreased significantly after the penetration T. harzianum to nematode egg mass matrix (Sahebani and Hadavi, 2008). The potential of T. harzianum to control the root-knot nematode M. javanica and its ability to colonize eggs and second-stage juveniles was evaluated. The application of T. harzianum resulted in reduction of root galling and increased top fresh weight in nematode-infected tomatoes (Sharon et al., 2001). Acremonium strictum and T. harzianum both alone and in combination showed promising performance by improving the health of the tomato plant with a remarkable reduction in M. incognita population (Goswami et al., 2008).

When the two maize hybrids were grown with and without M. arenaria in T. harzianum and T. koningii treated soil the shoot and root growth of both hybrids was significally enhanced and the reproduction of M. arenaria on the susceptible maize hybrid was suppressed, whereas the root knot resistant hybrid was not affected (Windham et al., 1989) Paecilomyces lilacinus strain 251 was evaluated for its potential to control the root-knot nematode (M. incognita) on tomato. Pre-planting soil treatment reduced root galling by 66%, number of egg masses by 74% and the final nematode population in the roots by 71% compared to the inoculated control in growth chamber (Kiewnick and Sikora).

Trichoderma harzianum and Trichoderma viride reduced the incidence and pathogenicity of Meloidogyne incognita on tomato in In vitro experiments. All the isolates were found effective and caused mortality in second-stage juveniles (J2) as compared with the control (Al-Fattah et al., 2007). Amendment of the culture filtrate of a T. harzianum to P. fluorescens growth medium improved root-knot nematode biocontrol in in vitro and glasshouse conditions. When the T. harzianum and Pseudomonas fluorescens medium was applied to unsterilized soil great reduction in nematode population densities was observed in tomato roots (Siddiqui and Shaukat 2004).

Trichoderma harzianum isolated from soil and root samples of tomato collected from root knot nematode-infested fields of different localities of Malakand and Swat (NWFP) was tested against Meloidogyne javanica in the in vitro. Isolates collected from Jabban and Shamozai (Th1 and Th9), were more aggressive against M. javanica. Different concentrations of culture filtrates (CF) of T. harzianum significantly inhibited egg hatching of the nematodes (Khattak et al.,2008).

The application of Trichoderma harzianum, T. hamatum and T. koningii culture filtrates significantly reduced the egg-masses of reniform and root-knot nematodes. Trichoderma species inhibited the nematode activity and movements in vitro. The effect of culture filtrate was more significant on Meloidogyne javanica egg than on larvae. The toxic metabolites of Trichoderma species directly inhibited nematode penetration and developments (Bokhari 2006).

Biological control by Bacteria

When freeze-dried cyanobacterial powder was incorporated into potted Weld soil at the rate of 0.2%, 0.4%, 0.6%, 0.8% and 1.0% (w/w) 5 days before tomato planting, reduced root galling and increased vegetative growth of tomato plants and root-mass production. At high doses 0.8% and 1.0% (w/w) root galling and nematode population decreased by 68.9% and 97.6% respectively compared with control (Khan et al., 2007). When rice roots were treated with Bacillus megaterium 40% reduction was observed in root knot nematode penetration and gall formation as compared with control. B. megaterium colonization of rice roots decreased migration of M. graminicola to the root zone by nearly 60% compared control. Hatching was reduced over 60% when M. graminicola eggs were exposed to secondary metabolites of B. megaterium compared with eggs not exposed (Padgham and Sikora, 2007).

Juveniles of M. javanica (500, 5,000 or 10,000) were added to the pots, amended with the formulations of Pasteuria penetrans, and dried neem cake and leaves, before planting 6-week old tomato seedlings. After 64 days tomatoes were sampled, afterward second and third crops were sown for 59 and 67 days respectively without further applications of P. penetrans and neem. The root-galling was reduced significantly at the end of third crop in combined treatment of P. penetrans and neem cake and this treatment also had the fantastic effect on the growth of the tomato plants (Javed et al., 2008b).

Plant growth-promoting rhizobacteria colonize the rhizosphere and promote plant growth or protect plants against certain plant pathogens. Different commercially available rhizobacterial inoculants (Equity, BioYield, and AgBlend) and RhizoVital were selected to test the suppressiveness of root knot nematode. Treatment with each of the inoculants increased plant growth, root weight and decreased root galling significantly (Burkett-Cadena et al., 2008).

Biological control by Algae and Nematopathogenic Nematodes

Effect of nematopathogenic nematodes was measured root-knot nematodes infecting peanut and tomato. Three hundred Meloidogyne hapla eggs and 25 IJs/cm2 of Heterorhabditis bacteriophora were inoculated to peanut seedlings in growth chamber. Treatment of H. bacteriophora to peanut seedling significantly reduced invasion and egg recovery of M. hapla as compared with control. In greenhouse Peanut seedlings were infested with 5000 M. hapla eggs and treated with 25 IJs/cm2 of Steinernema riobrave S. feltiae,or H. bacteriophora 2 weeks before, 1 week before, at the same time, 1 week after, or 2 weeks after. Pre- and post-infestation applications of S. feltiae suppressed M. hapla penetration in pre- and post-application but not egg production. Tomato plants were also inoculated with 5000 M. incognita eggs and 25 IJs/cm2 of S. glaseri or H. megidis applied at the same times in the greenhouse. High rate of S. glaseri reduced egg production and low rate of S. glaseri suppressed M. incognita penetration into tomato roots (Perez and Edwin, 2004).

The effect of inoculum density and application time of Mononchoides fortidens against Meloidogyne arenaria on tomato plants, grown in pots containing 500g Weld soil inoculated with 10 second stage juveniles/g soil, was measured. Treatment of pots with different concentrations (0, 50, 100, 150, 200, and 250 per pot) of M. fortidens 7 days before tomato planting significantly reduced root galling and the final population of M. arenaria and improved vegetative growth of tomato plants and root-mass production, compared with control (Khan and Kim, 2005).

Before transplanting tomato seedling, roots were dipped in different concentrations of culture filtrate of Microcoleus vaginatus for 30 minutes. Root-dip treatment reduced the root galling, population of M. incognita and improved vegetative growth of plants as compared to control. as the concentration of culture filtrate was increased the beneficial effect of root-dip treatment also increased. Root galling and final nematode populations were reduced by 65.9% and 97.5%, respectively when treated at the highest concentration (Khan et al., 2005).

Biological control by Botanical Toxicants

Soil amendments with various formulations of Neem (A. indica) such as sawdust, leaf powder and oilseed cake decreased plant-parasitic nematodes significantly relative to control plots. All treatments increased fresh and dry weights and the height and number of pods on chickpea plants (Akhtar, 1998). Second stage juveniles of M. incognita were exposed to 500 ppm of each plant extract for 24, 48 and 72 h. leaf extracts of, Aristolochia bracteolate, Solenostemma argel and Ziziphus spina-christi and the seed extracts of Datura stramonium, Aregimone mexicana and A. indica caused high mortality rates upto 94-80% after 72 h of exposure. For most of the extracts, the mortality rate increased with increasing exposure time (Elbadri et al., 2008).

M. incognita eggs were exposed to root extracts of Dharek, Neem, Castor and Datura. One hundred percent inhibition of egg hatching and larval mortality was observed standard root extracts of Neem and Dharek. Decrease of egg inhibition and larval mortality was observed with an increase in the dilution of the extracts. Similarly juvenile mortality was also increased with an increase in exposure time (Kayani et al., 2001).

When AM-fungi was combined with different oil cakes (R. communis, Brassica campestris and A. indica) resulted in reducing the galling and nematode multiplication thus improving the plant growth and yield. The best results regarding, reduced root infection, nematode reproduction and plant growth and yield were obtained with the combination of AMF and R. communis oil cake (Bharadwaj and Sharma, 2006).

Raising mycorrhizal seedlings of tomato in soil amended with neem (A. indica) cake enhanced plant vegetative growth and reduced root galling and final population of M. incognita signifacantly. (Rao et al., 1995). Exposure of Second stage juveniles of M. javanica to aqueous extracts of Neem (leaves and cake) at 2.5%, 5%, and 10%w/v and a refined product, Aza at 0.1% w/v. The 10% extracts of Neem leaf and cake caused 35% and 28% mortality and 83% and 85% immobility, respectivelyas compared to control. (Javed et al., 2008a).

Soil application with crude and refined formulations of neem in protective and curative manner had significant effect on number of egg masses. Protective application of neem leaves and cake did not reduce the invasion of juveniles whereas aza at 0.1% w/w did. On the other hand curative application significantly reduced the number of egg masses and eggs per egg mass as compared with the control (Javed et al., 2007a). Five concentrations of water soluble extracts of Ocimum sanctum, Ricinus communis, Carica papaya, Tagetes patula and A. indica were filtered, added to petri dishes and infested with the eggs of M. incognita. O. sanctum showed best results with no hatching within 48 h vs. 34.8% in the control (Bharadwaj and Sharma, 2007).

The aqueous extracts of neem, castor and pagomia cakes were evaluated as substrate for mass production of T. harzianum and in the management of M. incognita in egg plant. Castor cake extracts at 10%gave maximum growth of mycelial mat and spore production of T. harzianum compared with moderate growth in 10% pagomia cake and 5% castor cake extracts. Application of plant based formulations of T. harzianum was effective in producing vigorous seedlings with least root galling (Rao et al., 1998).

Exposure of M. incognita eggs to concentrations of root extracts of Siam weed, Neem, Castor bean and Lemon grass significantly reduced egg hatching and increased larval mortality. 100% concentration of root extracts of Siam weed and Neem exhibited 100% egg hatch inhibition and larval mortality. While with same conc. of root extracts of Castor bean and Lemon grass resulted in 93 and 95% inhibition of egg hatch and 62.1 and 75% larval mortality respectively. An increase in the dilution of all the extracts, egg inhibition and larval mortality was decreased. Similarly juvenile mortality was also increased with an increase in exposure time (Adegbite and Adesiyan, 2005).

A mixture of Haplophyllum and Plectranthus oils (1:1) showed high toxicity to M. javanica by killing all nematode juveniles and inhibiting egg hatching at 12.5 mg/ml concentration after 24 h exposure time in vitro. Tomatoes grown in soil amended with a combination (1:1) of the two oils developed fewer root galls than those grown in soil treated with higher doses of either oil In the green-house (Onifade et al., 2008).

Neem cake (A. indica) and a biocontrol fungus, T. harzianum either singly or in combination were evaluated for the management of M. incognita on tomato. They significantly increased tomato plant growth and reduced the root galling. The final populations of M. incognita were observed in tomato seedlings transplanted in neem cake-amended soil incorporated with T. harzianum. Increase in colonization of T. harzianum on roots of tomato was also observed in the above treatments which indicated favorable effects of neem cake amendment on the growth of T. harzianum (Rao et al., 1997).

It is obvious from the above review that biological control agents like T. harzianum and A. indica have a great potential to reduce root knot nematodes and increase plant growth significantly.

MATERIAL AND METHODS

Chapter 3Collection of root samples

Root samples showing characteristic symptoms of root- knot nematode were collected from M. incognita infected tomato field. Roots were carefully lifted with small spade up to 20-25 cm depth from the root zone along with approximately 1 Kg of adhering soil. These samples were put in polythene bags , moistened well to ensure the adequate moisture for survival of nematodes. Samples were stored in refrigerator at 4° C until processed.

Isolation of Root-Knot Nematode from roots and soil

Root-knot nematodes were isolated from infested roots and soil by White-head and Hemming tray method (White-head and Hemming, 1965). In this method, the infected roots with egg masses were sliced and chopped after washing thoroughly under tap water. The soil and roots were kept in a tray lined with tissue paper having sufficient amount of water. After 24 hours, water was decanted off in a beaker and allowed to settle for one hour. When the juveniles were settled, the excess of water was drained out until about 100 ml remained.

Isolation of root knot nematodes from soil samples was done by Cobb's decanting and sieving method. In this method, 2-3 litters of water was added to a plastic tub having 250-500 g of soil sample, thoroughly mixed until all clods were broken up. Heavy soil particles, roots and rocks sunk to bottom and were drained out from water by hand. The supernatant water was poured through the coarse sieve (36- mesh) into the second bucket. A liter of water was added and above step was repeated to get optimum number of nematodes. The water suspension was stirred, allowed to settle and dispense the supernatant water from the second bucket gently through the fine sieve (100-mesh) and allowed it to run down the drain. The sieve was washed with a gentle stream of water to remove fine particles. The same process was repeated by using fine mesh sieve (275 and 325 fine mesh sieve). Finally the material left on the 325-mesh sieve was transferred into a 500 ml beaker. The suspension was allowed to settle down to the bottom of beaker for about one hour. Excessive water was drained off with the help of siphon tube and counting of nematodes in 1 ml water was made in three replicates.

Identification of Meloidogyne spp.

Root knot nematode species was identified as Meloidogyne incognita on the basis of perineal pattern. Root gall with mature female was selected and placed in watch glass with water. The root tissue was teased apart with forceps to remove adult females. the cuticle of the female was ruptured near the neck and gently pushed the body contents out. The cuticle was placed in a drop of 45% lactic acid on a petri dish. The posterior half of the body was cut off, further the posterior piece of the cuticle having perineal patterns to square and completely remove inner tissue by flexible bristle. The perineal pattern-bearing portion was transferred to drop of glycerin on a microscope slide. The glass cover slip was gently placed and sealed with paraffin and labeled. The pattern was examined under a research microscope (Eisenback et al., 1981). Two species were dominant M. incognita and M. javanica. M. incognita was purified for the further experiments.

Extraction of Meloidogyne incognita eggs

Root knot infected tomato roots were collected and cut into 1-2 cm segments. Roots were shaken vigorously (manually) in 200 ml of a 0.5 to 1.o% NaOCl solution for 1-4 minutes. NaOCl solution was passed quickly through a 200-mesh sieve, nested over 500-mesh sieve to collect freed eggs. A stream of cold water was passed through 500-mesh sieve to remove residual NaOCl and rinse for several minutes. The remaining roots were rinsed for several times to remove more eggs and then collected in beaker by sieving (Hussey and Barker, 1973).

Mass culturing of Meloidogyne incognita

Three weeks old tomato seedlings were transplanted into earthen pots filled with formalin sterilized sandy loam soil. After one week of transplanting, 4-5 small holes (3-cm deep) were made around each plant and 1000-1500 freshly hatched juveniles of root knot nematodes were inoculated. These holes were covered with soil to prevent drying. Plants were kept in green house at temperature range 22-35 C and watered regularly. Plants were harvested after two months to collect juveniles.

Preparation and sterilization of soil

Sandy loam soil was used for the experiment. Soil was properly mixed, air dried and sieved to clean stones and debris. After sieving, diluted formalin (1:320) was poured on a small heap of soil and was covered for seven days with polythene sheet in such a way that the fumes were confined beneath the plastic sheet.

Growing tomato seedlings

Tomato cultivar "Money Maker" susceptible to M. incognita, was raised in earthen pots containing sterilized sandy loam soil and allowed to grow for 30 days.

Transplanting tomato seedlings

Tomato seedlings grown in earthen pots were shifted to separate pots having soil amended with biocontrol agents. Seedlings were shifted from earthen to separate pots carefully and then adhering soil was removed gently by hand and then planted in pots. Nursery seedlings were handled carefully to avoid any injury.

Mass culturing of Trichoderma harzianum

T. harzianum was cultured on Potato Dextose Agar (PDA) at (27˚C±). After obtaining the pure culture, biological control agent was grown on wheat seeds. Wheat seeds (250 g) were soaked in water for 12 hours. The soaked seeds were surface dried with paper towel, placed in a 500 ml. flask and were sterilized in an autoclave for 50 minutes at 15 psi. For the preparation of spore suspension of the antagonistic fungi, 5 ml. distilled water was added to the petridish containing 10 to 14 day-old fungal colony. With the help of a sterilized loop, the surface of the fungal colony was rubbed gently to make a spore suspension. Then the sterilized wheat grains was inoculated with the spore suspension and placed in the incubator for 15 days at the room temperature. The flask was shaken every day to promote the uniform growth of the fungi. The fungi started growth after 4-5 days. After 14 days of inoculation, there was sufficient fungal growth for field application. The biocontrol agents will then be mixed in sterilized soil at the rate of 0, 4, 6, 8 and 10 gram / Kg of soil.

Preparation of Neem formulations

Neem leaves were washed carefully to remove dust and then kept in sunlight to dry. Aqueous extracts were prepared by soaking powder of dried Neem leaves for 24 h in sterilized distilled water and filtered through muslin cloth then to get clear extract, filtered through filter paper. This served as standard extract. 2.5%, 5%, 10% and 15% formulations were made by adding water in standard extract.

Evaluation of biological control agents for the control of M. incognita

3.11.1. Effect of biocontrol agents on mobility and mortality of Meloidogyne incognita

For assessment of mobility and mortality, I ml of nematode suspension containing 500 freshly hatched juveniles were exposed to different concentrations of T. harzianum ( 1:10 and 1:100), A. indica (5% and 10%) and combination T. harzianum and A. indica, in 5cm plastic plates. Plates were placed in incubator and after 24 and 48 h data was recorded regarding mobility and mortality.

3.11.2. Effect of biocontrol agents on egg hatching of M. incognita

For egg inhibition studies egg masses of uniform color and size were picked from M. incognita infected roots of tomato and carefully placed in dilutions of fungal filtrate, Neem extract both alone and in combination. Total juveniles were counted after 2, 4, 6, 8 and 10 days.

3.11.3. Effect of biocontrol agents both alone and in combination on plant growth parameters

The effect of biocontrol agents (T. harzianum and A. indica) on M. incognita was evaluated individually and in combination. One month old seedlings of tomato cv. Money Maker were transplanted to soil amended with T. harzianum (2, 4, 6 and 8 g/1kg soil) and A. indica (2.5%, 5%, 10% and 15%) individually and in combination. One week after transplantation, the plants were inoculated with freshly hatched 2nd stage juveniles of M. incognita. Each treatment was replicated five times. Inoculation of 2nd stage juveniles to water amended soils was used as control. The pots were placed in green house at 25-32 °C. After eight weeks of inoculation, data was recorded regarding plant height, fresh shoot weight, dry shoot weight, fresh root weight and number of galls per plant. The data was analyzed by using statistical packages (Steel and Torrie, 1997).

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