Effect of Nematode on Tobacco

Effect of Nematode on Tobacco Tobacco 1.0 Introduction Tobacco (Nicotina tabacum L.) is one of the most important non- food crop and widely grown commercially (Akerhust, 1981). This plant had a high economic value and widely demanded throughout the world for the usage of the nicotine, cigarettes, cigars and other tobacco product (Akerhust, 1981). Nowadays, in Malaysia, Tobacco industry is very crucial in uplifting the socio-economic status of farmer in Kelantan, Terengganu, Kedah and Perlis. There were 20,524 farm families, 355 tobacco curers, 1300 grower and 25384 station workers. This industry generates about 150 million in income per year. 38% of the income goes to the farmers and 18% to the curers (http://www.malaysiayellowpages.net/mpi/details/TOBACCO.htm). This plant can give a stable income and therefore increased the income of farmer (Wells, 1987). Tobacco was cultivated as a rotation with the paddy for a side income (Anon, 1981). Tobacco plant can be infected by bacteria, fungus, virus, and parasitic nematode. Disease infection lower the tobacco yield and also quality. This research focused on effect of plant parasitic nematode on tobacco. Plant parasitic nematode can be found wherever tobacco is grown. The severity of the damage they caused depended on climate and soil type (Luc, Sikora et al. 2005). Nematode infection may lower the quality and yields. Annual report from North Carolina in 2008, showed Meloidogyne spp. it self cause loses around $2,505,126 in 2004, $1,596,452 in 2005, $ 1,772,819 in 2006, $1,542,864 in 2007, and $4,096,321 in 2008 while other nematodes cause $146,297 in 2004, $2281 in 2005, $529,188 in 2006 and $208,612 in 2008 (www.dowagro.com/soil/products/tobacco/economic.htm). In Malaysia, the effect of nematode on tobacco yield reduction has not been fully understand or revealed. Therefore, the objectives of this project were: To observe the effect of nematode on tobacco. To observe the relationship of soil physical properties on nematode population density and disease severity. 2.0 Literature review 2.1 Tobacco Tobacco was one of the most important non- food crop and widely grown commercially (Akerhust, 1981). This plant originated came from South America (Tso, 1972). However according to Gerstel (1961), Nicotina tabacum not occurring in wild state it was amphidiploids which come from hybridization of Nicotina sylvestris and Nicotina tomentosiformis. This plant has a high economic value and has been widely demanded throughout the world for the usage of the nicotine such as cigarettes, cigars and other tobacco product (Akerhust, 1981). This plant also important for the research purposes (Tso, 1972). Many researches have been done by using this plant mostly in Plant physiology and Genetics (Bateman Millar, 1966; Albersheim et. al., 1969; Kosuge, 1969). In Malaysia, Tobacco was first introduced in year 1959 by Malayan Tobacco Company (now known as Malaysia Tobacco Company, MTC) in Kelantan cultivation area for 8 hectares (Anon., 1976). Nowadays, in Malaysia, Tobacco industry has been very crucial in uplifting the socio-economic status of farmer in Kelantan, Terengganu, Kedah and Perlis. There were 20,524 farm families, 355 tobacco curers, 1300 grower and 25384 station workers. This industry generated about 150 million in income per year. 38% of the income goes to the farmers and 18% to the curers (Ministry of primary industry, 2010). This plant can give a stable income and therefore can increase the income of farmer (Wells, 1987). Tobacco is cultivated as a rotation with the paddy for a side income (Anon, 1981). However, product and quality of tobacco leaf are quite low due to encountering many problems, including diseases. For Tobacco cultivation, a deep and well drained soil is needed. This is where nematodes problem develop rapid ly (Luc, Sikora et al. 2005). 2.2 Nematodes related with the Tobacco Plant parasitic nematode can be found wherever tobacco is grown. The severity of the damage they caused may depended on climate and soil type (Luc, Sikora et al. 2005). Many tobacco producing countries are near or within the inter-tropical zone. The dominant nematodes that parasitize tobacco plant were Meloidogyne spp. (a root-knot nematode). Most of important species from this genus were M.arenaria, M.incognita, M.javanica, and M.hapla. M.incognita and M.javanica were important species in Malaysia. Other Meloidogyne spp., were rarely reported. Similarly, Pratylenchus spp. were also dominant species that parasitize tobacco plant (Kimpinski and Thompson 1990). Apart from Meloidogyne spp. and Pratylenchus spp., Tylenchorhynchus spp., Globodera spp., Ditylenchus dipsaci and Aphelenchus ritzemabosi were reported to parasitized tobacco plant in certain restricted area. Other nematodes such as Helicotylenchus, Rotylenchus, Scutellonema, Rotylenchulus sp., Tetylenchus and Crinomella sp. hav e been found to infect tobacco plant but not normally associated with losses. Some nematode species such as Xiphinema, Longidorus, Trichodorus, and Paratrichodorus have been reported to transmit viruse to tobacco (Luc, Sikora et al. 2005). Nematodes also may cause disease complex. For example Meloidogyne spp. a root-knot nematodes has been proved to increase the incident of Fusarium wilt even when their population were incapable to cause direct damage to the tobacco plant (Webster, 1972). Another example was interaction between Pratylenchus brachyurus (lesion nematode) and Phyptopthora parasitica var. nicotianae (cause black shank disease). Inagaki and Powell (1969) found that P. brachyurus caused more severe and rapid diseased development of black shank symptom than when the fungus alone. 2.3 Root-knot nematodes, Meloidogyne spp. 2.3.1 Distribution Meloidogyne spp. are always important parasites in tobacco cultivation, wherever the climate favours them (Nusbaum, 1960; Daulton, 1964; Barker et al., 1981; Rich et al., 1982). There were 61 species and two subspecies in this genus at the end of 1988 (Eisenback, 1985; Eisenback Hirschmann, 1991). Nowadays until year 2000 there were 80 species have been describing (Carneiro et al., 2000). Parasitism of Meloidogyne spp. was first reported by Tisdale (1922) in Florida. This genus was also a serious pest in Southern Africa in the late 1920s (Jack, 1927; NaudÑ?, 1929). Meloidogyne incognita and M. javanica were mostly found parasitize the tobacco plant. Their infection was very relying on the climate, since M.javanica had a higher tolerance towards drought and high temperature compared with M.incognita (Daulton Nusbaum, 1969, 1962; Taylor et al., 1982). Meloidogyne arenaria and M.hapla were the next mostly found to cause infection on tobacco plant. Meloidogyne hapla was reported to be found in the cooler parts of the world. Report from fields survey in Florida showed M.javanica was found in 65% of fields survey area, M.incognita 33% and M.arenaria was rarely found (Rich Garcia, 1985). Report from North Carolina showed M.arenaria population had increased gradually although M.incognita was the predominant species there. This observation also showed the same in South Carolina (Fortnum et al., 1984; Schmitt Barker, 1988). Apart from that, M.javanica and M.hapla was reported to be found in North Carolina. Reported showed that there were 64% of M.incognita and 29% of M.javanica to be found in Philippines (Madamba, 1981). Meloidogyne incognitagraham, M.microcephala, M.mayaguensis, M.cruciani, M.enterolobii, M.ethiopica, M.platani, M. themesi were also reported to parasitize reproduce tobacco plant but their importance was very restricted (Cliff hirschmann, 1984; Jepson, 1987; Rammah 1988; Rammah and Hirshmann, 1988). 2.3.2 General morphology The morphology of this genus were almost all same the except for some characteristic which usually were very useful for species identification. They were usually sexually dimorphic. Adult female have swollen, saccate bodies (pear shape like body). The size of female ranged in median length 0.44-1.30 mm and width about 0.33-0.70 mm (Eisenback, 1985). They have protrudes neck anteriorly while vulva and anus were located terminally. The female of this genus have pearly white body with moderately thick cuticle. Stylet were short, moderately sclerotized and protrusibly hollow. The stylet size was 10-24ÂÂ µm in length which consists of cone, shaft and knobs. The morphology of the stylet was quite varying between species in this genus. The morphology of stylet should be one of the supplemental characteristic to be observed for species identification. The stylet functions like hypodermic needle which was moved by protractor muscles. The shaped of the cone, shaft, and knobs also differ a mong female species in this genus. At the posterior of stylet knobs, there was dorsal esophageal gland orifices (DEGO). DEGO was the two sub ventral gland orifices open into the esophagus lumen. DEGO had a varied distance among species which also can be supplemental character for species identification. The excretory pore of the Meloidogyne spp. female situated anterior to median bulb valve plat and usually near stylet base. They also have two convoluted genital tracts. The major part of the total body content consists of two gonads which were very long and greatly convoluted. There were ovary with germinal zone and growth zone, narrow oviduct, globular spermatotheca and long uterus in each gonad. Spermatotheca were differing among species. Therefore this character can also be use for species identification. Apart from that, the cuticle in the perineal region of female from this genus forming a finger print-like pattern (the perineal pattern) which also had been use for species iden tification. This is because, the perineal pattern hold most characteristic of female such as tail terminus, phasmids, lateral lines, anus, and vulva which surrounded by cuticular striae or folds. They also have six large unicellular rectal glands situated in the posterior body region. These rectal glands were connected to the rectum. This gland produce very large amount of gelatinous matrix material. This material was excreted through the rectum and act as protective egg sac (Nickle, 1991). Different with the female, male of Meloidogyne sp. are vermiform. The size of the body vary between species which are about 700-2,000 ÂÂ µm (Eisenback, 1985). This is because the varying environmental condition existing during their development. Body of the male usually twisted through 180ÃÅ'Ã…   upon heat relaxation. The male stylet vary in size which are about 13-30 ÂÂ µm. The stylet and head of male from this genus are robust. Apart from that, size and shape of the stylet cone, shaft, and knobs can be use for species identification (Eisenback and Hirschmann, 1981). The location of DEGO is 2-13 ÂÂ µm posterior to the stylet knob base. The isthmus is short and most of the species have ventrally two overlapping gland lobe instead of normally three esophageal nuclei. The hemizonid located at the front to excretory pore. However some species the hemizonid located at the posterior of excretory pore. In normal male there is only one gonad while in sex-reversed males have two gonads. There is long vas deferens packed with developing sperm in the gonad. Among the species, the size of the spicules range from 19 to 40 ÂÂ µm. The spicules usually robust and the bursa are absent. Tail is short (hemispherical shape). There is also variation of tail shape between species (Nickle, 1991). A second stage juvenile was the infective stage of Meloidogyne sp. It has varied body length from 290 to 912ÂÂ µm (Eisenback, 1985). The head of second stage juvenile basically just same with the male. It has a delicate stylet with 8 to 18ÂÂ µm in length. The DEGO distance are varied among species with the distance mostly 2 to 8ÂÂ µm. The esophagus of the second stage juvenile is narrow with faintly outline procorpus. The median bulb is well defined. Median bulb has a large valve plate and three long ventrally overlapping glands that are use for molting and feeding. The second stage juvenile has a varied position of excretory pore. The hemizoid located posteriorly to the pore. The tail length of second stage juvenile varied among species. Usually the length is 15 to 100ÂÂ µm. At the end of the tail there is hyaline terminus. In this genus, second stage juveniles are group base on the tail length and tail shape (Whitehead, 1968; Jepson, 1984). Jepson (1987) showed that differences in either mean tail and or mean hyaline terminus are very large. These vast differences can be very useful to distinguish species within groups (Nickle, 1991). 2.3.3 Life cycle Meloidogyne sp. shows sexually dimorphism, which is the female are pyriform or saccate, while the males vermiform (Eisenback, 1987). The differences in body shaped between female and male occurred during the postembryonic development of Meloidogyne sp.. From the embryonic development, the egg hatched once to become first-stage juvenile and then molted as a second stage juvenile. The second-stage juvenile was infective stage. It moved into the soil and entered the root of suitable host plant. This second-stage juvenile then formed host-parasites relationship with the plant when it find preferred feeding site. The morphology of second-stage juvenile changed to flask-shape as it feeds on the special nurse cell. Then, without further feeding it molted three times into the third and fourth stage juvenile, and finally become an adult. The saccate adult female resumed feeding on the special nurse cell shortly after the last molt and continued to do so for the remainder of her life. The repr oductive system of both female and male of this genus developed into functional gonads during the postembryonic development (Triantaphyllou and Hirschmann, 1960). From the number of the gonad, we can differentiate the sexes. Females always have two gonads while males usually have one. During fourth-stage juvenile, the shape of saccate male juvenile changed to the vermiform adult males. The metamorphosis occurred in which the body elongates from saccate to a vermiform shape. Fully developed male emerges after the final molt of enclosed fourth-stage male which enclosed within the cuticles of second-stage and third-stage. The adult male leaved the root and move freely through the soil and it does not feed. The mode of reproduction determined the function of the male for mating. Depending on particular species reproduction whether amphimixis or parthenogenesis, the male enters the root searching for the female to mate or just remain in the soil and die. Temperature plays a vital role fo r the length of the life cyle. For example, the first adult female of M.incognita on Tomato appear 13-15 days after root penetration at temperature approximately 29 ÃÅ'Ã…  C, the female laid the first egg about 19-21 days after penetration (Triantaphyllou and Hirschmann,1960). The life span of female is much longer than the male from 2 to 3 month. 2.3.4 Effect of Meloidogyne spp. on Tobacco plant Meloidogyne sp. caused formation of galls on Tobacco root. Usually, second stages juvenile entered via behind the root cap which involves mechanical penetration by using stylet (Linford, 1942). According to Bird et.al, (1975), the penetration also involve some enzymatic action (cellulolytic or pectolytic) which secreted by esophageal gland. Then, the second-stage juvenile moved through the cortex to the region of cell differentiation. This differentiation cell was the feeding site for them which later transformed into highly specialized feeding cells called giant cells. This cell was the permanent feeding site for them (Hussey at al., 1994). According to Dropkin (1972) and Hussey (1987), the multinucleate giant cell was the result of the introduction of secretion produced by subventral esophageal gland cells of the feeding second stage juvenile. Giant cells serve as sourced of food. The nutrient from giant cells was transferred to the nematode (Jones and Northcote, 1972). According t o McClure (1977) these cells act as metabolic sink. These giant cells affected the function of the root as it caused extensive distortion and blocked of the vascular tissue which slowed water and nutrient transport. Therefore, the absorption of nutrient and water greatly reduced. Plant growth and yield may be suppressed as photosynthates were mobilized to the giant cells. Above- ground symptoms showed chlorosis of foliage and temporary wilting (premature wilting) when water stress occurred usually during drought or sunny day. Plant was stunted and the leaves were yellow and thin. The formation of gall was due to the root tissues around nematode and giant cells undergo hyperplasia and hyperthrophy. The worse was when secondary larval invasion occurred which caused the gall to coalesce and finally the root begins to decay (Nickle, 1991). Nematode also had the ability to form disease complex with other plant pathogens. The giant cell produced by root-knot nematode was highly suitable f or development of Fusarium wilt ( Porter and Powell, 1967). 2.4 Root lesion, Pratylenchus spp. 2.4.1 Distribution Pratylenchus spp. is migratory endoparasites root-lesion nematodes. This genus was just slightly less economic important compare with Meloidogyne spp. in the tropical and subtropical regions. However, some species from this genus were responsible for significant yield loss in some tobacco cultivation area. Pratylenchus pratensis, P.negletus, P.brachyurus and P.zae have been reported to parasitized tobacco in North America while in South Africa P.hexincisus, P.thornei, P.vulnus, P.brachyurus, P.minyus, and P.zae have recorded on tobacco (Milne, 1961; Honey, 1967). In Hungary, P.pratensis had been reported to parasitize tobacco cultivation. Pratylenchus penetrans was responsible to cause yield loss in Iraq. In some region in Canada, P.penetrans, P.crenatus, and P.neglectus were mostly found in tobacco fields (Mountain, 1954; Kimpinski et. al., 1976). Canter-Vissher (1969) had found Pratylenchus penetrans in New Zealand while Singh (1974) has found Pratylenchus zae in Trinidad. In gener al Pratylenchus brachyurus and P.zae are mostly found in tropical areas while P.penetrans, P.thornei, and P.minyus are common species in temperate regions (Webster, 1972). In Malaysia, this Pratylenchus sp. was locally important. However their distribution were not clearly report (Luc, Sikora et al. 2005). 2.4.2 General morphology In general the morphology of species in this genus was very similar. There was no marked sexually dimorphism in form of anterior region. Adults have body length range from 0.3 to 0.9 mm. Their body was rather stout. Because increasing of uterus volume and the presence of eggs, the gravid females were stouter than nongravid ones. The cuticle of this genus generally thin and shows fine transverse striation. There were four longitudal lines marking the lateral field. However, additional longitudal line may be present in the central zone. Because of cuticle of gravid female were quite stretch, the lateral field was indistinct. The head of this genus was low and flattened with lip region divided into two,three, or four annules. This annules was continuous with the body countour. Cephalic framework of Pratylenchus sp. was heavily sclerotized. The apical anule among most species were round except for P.brachyurus which was angular. There were three types of head structure that can be found under SEM (Corbett and Clark, 1983). The stylet of Pratylenchus sp. were quite short around 11-25 ÂÂ µm. The stylet was stout with well-developed basal knobs. There was tapering procorpus in the pharynx which was usually roundish median bulb. The isthmus was short which overlapped with the anterior end of the mid-intestine on the ventral side. There were three unicellular glands in the lobe. The length of the ventrosublateral was unequal (Seinhorst, 1971). At 2-4 ÂÂ µm behind the stylet base, there was orifice of the dorsal pharyngeal gland duct. There was no deirids in this genus. The oesophagus of both male and female was equally developed. The tail of male was short and dorsally convex-conoid. Female of Pratylenchus spp. are monoprodelph. The genital branch of most species in this genus occurred as a short sac which usually undifferentiated. The uterus of female often tricolumellar (Nickle, 1991). Different with male, female tail usually two to three anal body diameter long. The bisexual species in this genus, have oval or round spermatheca which was filled with sperm (Luc, Sikora et al. 2005). 2.4.3 Life cycle Some species in this genus reproduced sexually while most of them parthenogenetic. This migratory endoparasitic root lesion nematode fed and laid eggs in the root cortex. Most of them can be found in roots, rhizomes, or tubers and somehow can also be found in stem or fruits. Usually after penetrate the root; this endoparasitic nematode will multiply to very large numbers (10,000-35,000 specimens per 10 g of root). All the stage starting from second stage juvenile entered the root. However with unknown reason, they moved in the soil for some time and goes for a new host root. The female laid the eggs in the root and starting from there their whole life cycle is in that root. Usually, the life cycle was completed in 50-60 days (Nickle, 1991). 2.4.4 Effect of Pratylenchus sp. on Tobacco plant Pratylenchus sp. usually moved and fed on the root cortex. This activity caused disintegration of root cortex and leading to browning of the root tissue. This was known as brown root rot (Mountain, 1954). Symptoms of this disease were pruning-root, water soaked, and lesion on the root. If the infection occurred under aseptic conditions the symptoms showed less severe in the certain experimental condition (Mountain, 1954). The above ground symptoms showed that the stunted plant wilt prematurely and in worse condition died. Inagaki and Powell (1969) reported that this genus caused disease complex with the other plant pathogens. Pratylenchus.brachyurus showed to increase infection of Blackshank by wounding the root which served as entry site. 3.0 Material and method: 3.1 Soil sample: 24 soil samples were collected from Terengganu, Perlis and Kelantan state. Collected soil sample were naturally infested with nematodes and Fusarium spp. Soil samples were store in polyethylene bags. Soil sample were kept in moist condition and out of direct sunlight. 3.2 Tobacco seedling preparation: Sterilized seeds were sown to sterile sandy soil. (River sand). After sown, seedlings were kept out of direct sunlight. Fertilizer applied for twice a week via foliar application. After 30 days of nursery tobacco seedlings were transferred to each soil. 3.3 Inoculation of tobacco seedlings: 6 kg of soil samples (naturally infested) were transferred into plastic container (33x22x10 cm) with drains. Then, 30 days of healthy Tobacco seedlings were transferred to each soil container. Each soil samples were planted with 10 Tobacco seedlings. Fertilizer was applied twice a week via foliar application. Ground symptoms were observed everyday. Tobacco plants were all harvested after 6 weeks. 3.4 Plant observation: Harvested Tobacco plants were observed for the disease symptoms, size of the plant, number of leaf, leaf area, plant weight and disease severity index. Wet weight of Tobacco was measured by using a weigher. Plant size was determined by using ruler. Size of the plant was measured from crown up until shoots. Number of leaf was counted including the number of undeveloped leaf. Root gall disease severity index was determined by using following scale: 0= no root galls 1= 1-25% root galls 2= 26-50% root galls 3= 51-75% root galls 4= 75-100% root galls Disease severity index for root lesion was determined by using following index: 0= no root lesion 1= 1-25% root lesions 2= 26-50% root lesions 3= 51-75% root lesions 4= 75-100% root lesions Root then was stored in the FAA (Formaldehyde 100ml, Glacial acetic acid 50ml, Distilled water 850ml) suspension. 3.5 Isolation of nematode from soil samples: Isolation of nematode and soil inhabiting forms were extracted from soil samples by using Modified Baerman Funnel Technique (Hooper,1968; Viglierchio and Schmitt,1983).This was the simplest technique to isolate nematode and soil inhabiting forms. By using this technique we can avoid lack of oxygen and possibility of nematode lodging on the sloping funnel sides due to instead of using funnel we used a shallow dish. For this experiment instead of funnel a round shallow plastic container was used. A supporting gauze was put onto the plastic container with 0.5cm space between them. A milk filter paper with 50cc soil was put on the supporting gauze. Distilled water was added until the material was almost awash. After 5 days, the content of the dish was transfer into test tube. FAA was added to prevent population changes during storage. 3.6 Nematode counting: Nematode suspension collected via Modified Baerman Funnel was shaked. Then, 1ml was taken and transfer onto disposable plastic Petri dish. The number of all nematodes and parasitic nematodes were counted under a dissecting microscope by 5x to 10x magnification. Counting was repeated for three times. Percentage of parasitic nematodes was calculated. 3.7 Isolation of nematode from root: Nematode from root part was isolated by direct isolation. For root-knot nematodes especially female, the root tissue was carefully tease away with forceps and a fine needle to release the head and neck. Infected plant part was put onto slide and squash to check for the existence of nematode. The nematode then was stain with Phyloxine 1%. 3.8 Isolation of Fusarium spp. The root part was washed with running tap water to eliminate remaining soil particle. Then, the root was cut including healthy part (0.5cm). After that, the pieces of root were dipped in 70% ethanol for 1 minute. Then, the pieces were transferred into 5% sodium Hypochlorite solution to sterilize its surface for 3 to 5 minutes. The pieces then were transferred to sterilized distill water to rinse the pieces for 3 times each for 1 minute. After that, the plant pieces was put on sterile filter paper to eliminate excess water and then, were put on the acidified water agar medium. Finally, the dishes were sealed with parafilm and were incubated for a few days. Growing colonies were observed. 3.9 Soil pH: The soil pH was determined using a soil suspension (Rowell, 1994). 10 ÂÂ ± 0.1 gram of air dry soil sample was used in this experiment. 25 ml of water was added to the soil sample. Then, soil suspension was shacked occasionally by hand over 15 minutes period. The pH meter was calibrated at pH 4 and then pH 7 consistent reading. The soil suspensions were stirred and insert the electrodes. The pH was recorded after 30 second. 3.10 Soil moisture percentage: The water content of soils was determined by drying soil samples at 105 ÃÅ'Ã…  c (Rowell, 1994). For this experiment, soil samples were air dry for two days. Then, weight air dry soil samples for 10ÂÂ ±0.1 gram (W1). Instead of using a moisture can, aluminums foil was used. The aluminum foil was weighed (Wo). Then, weighed soil samples were put on the aluminum foil and placed them in an oven at 105 ÃÅ'Ã…  C for 24 hour. The weigh of soil sample with aluminum foil was weighed (W2). To calculate the weight of soil samples after oven dry the following formulae was applied: Weight oven dry soil (W3) = (W1+Wo)-W2 To calculate moisture percentage of soil samples, the following formulae was use: Moisture percentage (%) = W3/ (W2-Wo) x 100 3.11 Soil particle density: Determination of soil particle density involves the measurement of the volume of a known mass of particles. The soil is dispersed in water and all the air is expelled from the suspension. In a known volume of suspension the volume occupied by the particles is then found (Rowell, 1994). A clean and dry 50ml volumetric flask including stopper was weigh (Wo). Ten grams of oven dry soil samples were added into the volumetric flask. The volumetric flask was filled with distilled water until one-half full. The volumetric flask (without stopper) then was put in boiling water heating with a water bath for 30 minutes and gently agitated the content to prevent loss of soil by foaming. The volumetric flask and its content then cooled to room temperature. Distilled water was added up to the 50 ml mark. Water drop on the outer-side of the volumetric flask was wiped, insert the stopper and weighed (W2). The soil particle density was determined by using the following formulae: Soil particle density (Dp) = Soil mass/Particle volume Particle volume = Conical flask volume volume of water in flask Volume of water in flask = mass of suspension -mass of soil Mass of suspension = W2-W0 3.12 Soil texture analysis: Texture of soil samples were determined by using Hydrometer method (Bouyoucos, 1962; Page, 1982). Then, texture of soil samples determined by referring to USDA Textural triangle after calculation of the percentage of each particle (Brady, 1984). For this experiment, 50g of soil samples were placed into 600 ml beaker. Then, 100 ml of 6% hydrogen peroxide was added to decompose the organic matter. The mixture was kept remaining at room temperature overnight. After that, the beaker was placed on a hot plate at 90 ÃÅ'Ã…   C for 10 minutes. Then, 50ml of 1N Sodium hydroxide (NaOH) (dispersing agent) was added to the suspension and increase the volume to 400 ml with distilled water. The suspension was left for 20 minutes. Then, beaker was placed on a stirrer and stirred thoroughly for 10 minutes. The suspension was transferred to 1000 ml measuring cylinder. Then, distilled water was added to 1000 ml mark. Suspension was allowed to equilibrate thermally and the temperature was recorded. Mo uth of the measuring cylinder was covered with a parafilm and inverted for several times until the contents are thoroughly mixed. Mixture was left in a cool, shaded place. Then, the hydrometer was immediately into the suspension and reading was taken after 40 seconds until consistent reading. Hydrometer was removed and cleaned. The temperature of the suspension was recorded with thermometer. The thermometer was removed and remixes the suspension. Then, let the cylinder sit for 2 hours. At exactly 2 hours later, the hydrometer was again placed into the suspension and data was read. The temperature of the suspension was also seconded with thermometer. The actual reading must be corrected in order to get revised value depending upon the actual temperature. a. Add 0.36 g/L to hydrometer reading for each degree >20 ÃÅ'Ã…  C b. Subtract 0.36 g/L from hydrometer reading for each c. Density reading should also be corrected from the density of the dispensing solution (NaOH+ distilled water) without soil. These reading are must be subtract with the soil solution density reading. Finally, after calculating the percentage of each particle, use the USDA Textural triangle to determine the textural class of soil samples. Readings from specific gravity hydrometer was converted to soil g/l by using converting table (http://classic.globe.gov/fsl/html/templ.cgi?conversionlang=ar). Table 3.1 Hydrometer converting table Specific Gravity Grams Soil/L Specific Gravity Grams Soil/L Specific Gravity Grams Soil/L 1.0024 0.0 1.0136 18.0 1.0247