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Green revolution technologies arrived in Pakistan in the 1960's through the introduction of high yielding varieties (HYV's). After about 4-5 years this technology proved ineffective because those HYV's need irrigated conditions, costly chemical fertilizers, pesticides and insecticides. Frequent irrigation caused severe damage to soil conditions in the form of salinity over a wide area and cause significant genetic erosion (Yasin, 2007). Field concentrations of fungicides have induced negative affects on inoculated microorganisms and their activities (Ayansina and Oso, 2006; Mubeen et al., 2006). The consistent use of pesticides to control pests cause resistance among pests and vectors and led to adverse effects on non-target organisms.
About 40-60 percent of applied nitrogen in form of urea is lost by volatilization, run-off, and denitrification and leaching. The leached nitrate causes insect pests and diseases susceptibility, crop lodging due to extensive growth and reduces seed quality. The continuous application of phosphorus-based fertilizers may result in trace metal contaminants and potassium fertilizers decreases vitamin C and carotene contents in fruits and vegetables while chemical fertilizers lead to malnutrition due to the degradation of carbohydrates and proteins (Rehman and Chaudhry, 2006). All these resulted in increased cost of production, poverty and food insecurity (Yasin, 2007) and threat to the lives and livelihood of millions of people (Mubeen et al., 2006). Hazardous affects of chemical fertilizers have forced the scientific fraternity to look for alternatives. A remedy to this problem is the environmental friendly biofertilizer, now used in most countries.
Biofertilizers are microbial preparations containing primary sufficient number of active strains of microorganisms having an explicit role to provide better rhizosphere for plant growth (Pal, 1986). Biofertilizer can be divided into nitrogen fixing bacteria, phosphate solubilizing and mobilizing micro-organisms and organic matter decomposers etc. some of these micro-organism are also being reported to produce plant growth promoting substance. Benefits attributed to the use of biofertilizer includes germination increase up to 20 percent, improves seedling emergence and growth, increases yield from 10 to 40 percent, improves fruit quality, saving of 25 to 35 percent inorganic fertilizers, increase availability and up take of N and P in plants, improves soil fertility and crop productivity. Higher population of beneficial micro-organism in soil increase nutrient retention and availability leading to improve yield, improves nitrogen and phosphorus fertilizer efficiency and resolves increased salinity of the soil and chemical run-offs from the agricultural fields (www.geepeebiofert.com). Thus, bio-fertilizers are important if we are to ensure a healthy future for the generations to come (Mubeen et al., 2006). Bio-fertilizers have been proved to be effective and economical alternate of chemical fertilizers with lesser in put of capital and energy (Hafeez et al., 2002).
Food legumes belong to family Fabaceae. Legumes are important crop of Asia. They account for approximately 20% of global food production (Broughton et al., 2003). It constitute about two- third of the total dietary intake in Asia. Food legumes contain 20-30% protein therefore, regarded as natural substitute for meat. World production of pulses is given in table 1.1. About 15% of the nearly 20,000 species in the Fabaceae family have been examined for N2-fixation. In legumes, bacterial species of genus Rhizobium are responsible for nitrogen fixation.
Chickpea (Cicer arietinum L.) and garden pea (Pisum sativum L.), the two most utilized food legumes in Pakistan were chosen to characterize their symbiotic partners.
1.2.1. Chickpea (Cicer arietinum L.)
Chickpea also known as Bengal gram and Garbenzo beans ranks top most on the basis of utilizable protein content. Chickpeas are a good fiber source that helps to lower cholesterol and restore blood sugar levels, making them a great food for diabetics and insulin-resistant individuals. Chickpea beans are an extremely low-fat, complete protein food and offer a good supply of zinc, magnesium, folic acid and molybdenum, a trace mineral needed for the body's mechanism to detoxify sulfites and pangamic acid and a free nucleotide. It is also diuretic, anti-stress, anti-hyperlipidemic and stamina building.. It is a stimulant, tonic, aphrodiasic, anthelmintic, and useful in bronchitis and biliousness. It is also useful in leprosy and other skin diseases. The powdered seed is used for dandruff and also used as a face pack (www. DrEddyClinic.com).
Table1.1. World production of pulses (million tones)
Latin America & Caribbean
Source: Food and agriculture organization (FAO) of the United Nations (2007)
1.2.2. Green pea (Pisum sativum L.)
Green pea (Pisum sativum L.) is also known as Garden pea. It is an annual self pollinated, herbaceous plant. Green pea generally grows best between 10Â°C and 20Â°C. It can be included anywhere in a rotation (Whytock and Frame, 1985a). Rhizobial inoculation of seed was beneficial to nodulation, plant growth and nitrogen fixation on acid soils where peas had not previously been grown (Sparrow et al., 1993).
Its nitrogen-fixing ability estimates vary but usually amounts up to 70 kg N/ha (La Rue and Patterson, 1981). However, Kucey (1989) recorded 117 kg N/ha in western Canada. Garden pea is a valuable source of protein. A typical analysis of the cut material prior to ensiling is: crude protein (16-20%); DOMD (proportion of digestible organic matter in the dry matter) 60-65% and metabolizable energy (ME, 10.0-10.5 MJ/kg of dry matter). It can be grazed by a range of livestock. Vitamin K, vitamin C, thiamin (vitamin B1) manganese, dietary fiber and folate are present abundantly in green peas. Green peas also contain ample amount of vitamin B6, riboflavin (vitamin B12), protein, niacin, vitamin A, copper, phosphorous, zinc, iron, potassium and magnesium.
Rhizobia are strictly aerobic, rod shaped cells, 0.5 - 0.9 Âµm Ã- 1.2 - 3.0 Âµm in size, non- spore forming mobile by a single polar flagellum or two to six peritrichous flagella. They formed white pigmented, circular, convex, semi-translucent raised mucilaginous colonies. They are chemo-organotraphic in nature. Due to nitrogen (N2) fixation ability Rhizobium sp. is widely used as nitrogen bio-fertilizer for leguminous plants. The species can fix up to 220 pounds of N2 per agricultural acre per year. On nutrient basis, one tonne of Rhizobium inoculants is equivalent to 100 tonnes of inorganic fertilizer (www.geepeebiofert.com). The root nodule bacteria are a diverse group of ubiquitous soil inhibiting Gram-negative bacteria (O' Hara, 2001).
In Brazil, soybeans inoculated with Rhizobium are responsible for $1.3 billion per year savings in production costs (Coutinho et al., 2000). One species is generally effective with only one species, but few exceptions are also present when more than one strain is associated with the same host.
Earlier rhizobia were divided in to two groups. Slow growing rhizobia i.e., Rhizobium japonicum and fast growing bacteria i.e., R. leguminosarum, R. phaseoli, R. ciceri (Stanfield et al., 1989). Recently on the basis of sequences of the small subunit of 16s ribosomal RNA taxonomy and systematic have been revised and this genus is divided in to four genera namely I) Rhizobium (R. leguminosarum, R. tropici, R. etli); II) Sinorhizobium (S. fredi, S. moliloti, S. teranga, S. saheli); III) Mesorhizobium (M. huakuii, M. ciceri, M. tianshanense, M. medeterraneum) and IV) R. galegae (Young, 1996).
1.4. Rhizobial diversity
To achieve maximum biological nitrogen fixation (BNF) out of any legume-rhizobium association it is necessary to properly characterize and identify rhizobia before they are made commercially available for field application (Sahgal and Johri, 2003). Assessment of diversity within rhizobial natural populations in various regions of the world has received increased attention (Ando and Yokoyama, 1999; Satyaprakash et al., 2006; Kucuk and Kivanc, 2008). At that time common method to differentiate strains within Rhizobium species include; generation time (Maatallah et al., 2002a; 2002b), ability to fix nitrogen in plantsÂ (Noel and Brill 1980; Duhri and Bottomly 1983, Jenkins and Bottomely 1985; Kamicker and Brill, 1986), pH tolerance, temperature tolerance, salt tolerance (Kucuk and Kivanc, 2008), resistance to antibiotics and heavy metal (Kandeler et al., 2000; Khan and Scullion, 1999; Ellis et al., 2003; Kucuk and Kivanc, 2008). Carbon source utilization patterns have also been used to distinguish isolates and strains among the Rhizobiaceae family (Kucuk and Kivanc, 2008). Shoot total protein analysis helps to establish the optimum fertilizer rate used for pulse legumes (Satterly et al., 2004); whole cell soluble protein pattern (SDS-PAGE) has been used not only to identify rhizobial strains (Fabiano and Arias, 1990; Irisarii et al., 1996) but also to differentiate among isolates within the same serogroup (Broughton et al., 1987); nodule protein profiles are used to screen salt tolerant symbiotic strains (Mhadhbi et al., 2004); nodule antioxidant enzyme studies are very critical for effective symbiotic associations as legume nodule have high potential for producing reactive oxygen species (ROS) due to their strong reducing condition (Gorgocena et al., 1995; Tejera et al., 2004; Jebara et al., 2005; Loscos et al., 2008).
In order to promote crop productivity in metal-polluted soils; recently, (PGRPs)
are being applied to restore contaminated soils. (Wani et al., 2008a; Wani et al.,2008b; Wani et al., 2009).
All these techniques with addition of genomic DNA fingerprinting using random amplification of polymorphic DNA (RAPD) (Tarco and Bezdicek 1987) has been not only used to discrimination bacterial strains (Oliveira et al., 2000), but also for analysis of genetic diversity (Young, 1996; Corich et al., 2001; Mâatallah et al., 2002 a; Kumar et al., 2006; Brigido et al., 2007).
1.5. Soil salinity
The environment has long been known to influence symbiotic nitrogen fixation. Salinity is considered a significant factor affecting crop production and agricultural sustainability in arid and semi-arid region of the world, reducing the value and productivity of the affected land ((Tejera et al., 2004; Gama et al., 2007).
Involvement of salt stress in the accumulation of free amino acids nitrate, ammonium and reduction in protein synthesis in plants are reported by many workers (Pessarakli et al., 1989). Salt stress increase non-protein-N fraction and irregularly change the protein-N fraction (Udovenko et al., 1970).
Salinity in the soil and irrigation water is an environmental problem and a major constraint for crop production. Currently, 20% of the world's cultivated land is affected by salinity, which results in the loss of 50% of agricultural yield (Zhu, 2001; Bartels and Sunkar, 2005). At present, there are nearly 954 million hectares of saline soils on the earth's surface. All these salt affected soils are distributed throughout the world. A large bulk of about 320 million hectares and land in South and South East Asia is under the grip of salinity.
Soil salinity is also a serious problem of agriculture in Pakistan. In Pakistan, about 6.30 million hectares of land are salt-affected and of which 1.89 hectare is saline, 1.85 million hectare is permeable saline-sodic, 1.02 million hectare is impermeable saline-sodic and 0.028 million hectare is sodic in nature. It is estimated that out of 1.89 million hectares saline patches, 0.45 million hectares present in Punjab, 0.94 million hectares in Sindh and 0.5 million hectares in NWFP. The substantial rise in the water table has caused salinity and water logging in large areas of Sindh, Punjab, NWFP and Balochistan. Several salt-tolerant grain, fruit and fodder species have been identified for practicing saline agriculture in the country (Alam et al., 2000).
More than 70% of the tube-wells in saline areas are pumping out brackish water. The problem is more severe in Sindh and Southern Punjab than other parts of the country (Malik et al., 1979). In-fact, these problems are threatening the whole production system of arid and semi-arid areas of Pakistan. These areas are now subjected to severe degradation and desertification. Excessive irrigation system losses coupled with inadequate drainage provisions have greatly contributed to the rise of water table causing serious problems of water logging and salinity in many areas of Pakistan. The salinity control and reclamation projects constructed to combat water logging and salinity and thereby, increase agricultural productivity have resulted in partial success (Piracha et al., 1995). In order to control water logging and soil salinity in large areas of Pakistan, the introduced SCARP (Salinity Control And Reclamation Projects) failed badly as it result in disturbed balance between the fresh-saline ground water and produced a unending problem of saline water movement towards fresh ground water zones (Qazi et al., 1996). Now a day efforts have been made to learn to live with salinity and make profitable use of saline land and water resources. Recently a safe alternative approach has been employed to combat salinity known as "Bio-saline agriculture technology". which involves the cultivation of salt tolerant species/cultivars with genetic traits to utilize salt affected soils. This technology gives economic return and provides vegetative covers to soil which reduces evaporation and hence the rate of salinization. This biological approach involves screening and selection of highly salt-tolerant plant species/varieties from the naturally existing germplasm or from these developed through breeding, hybridization and other techniques, and then introducing the selected plants for increased plant establishment and productivity in saline areas (Aslam, 2006). Many halophytes are reported to grow efficiently in saline soils (Aslam, 2006). Although chickpea (Cicer arietinum L.) and green pea (Pisum sativum L.) have not been reported to grow in saline soils but as the problem of salinity is growing there is need to test both of the crops for salt tolerance along with their symbiotic partner. Salt stress are more pronounced in arid and semiarid regions, particularly because plants grown in the areas take most of their nitrogen demands from symbiotic N2 fixation (Zahran, 1991).
1.5.1. Symbiotic nitrogen fixation under saline conditions
Variation among strains of Rhizobium spp. in the symbiotic performance under saline conditions has been reported by many researchers (Subbarao et al., 1990). Salt has been reported to badly affect different stages of successful symbiosis such as, survival, growth, and distribution of rhizobium in soil, limit root colonization, hang-up process of infection and nodule development or demolition of active nodule functioning (Craige et al., 1991; Tate, 1995; Jenkins et al., 1998). Salt stress has also been involved in nodule greening and responsible for diminution of the Leg-hemoglobin content inside the nodules (Delgado et al., 1993).
Biosynthesis and accumulation of several substances of plant and microbial origin for prevention against salt induced stress has been reported.To protect cell from oxidative damages, notable range of antioxidant enzymes and metabolites are usually present in plant cell that avert the formation of reactive oxygen species (ROS). Rhizonium- legume symbiosis is one of the diverse physiological processes to which antioxidants are concerned (Loscos et al., 2008).
Involvement of nodule antioxidant enzyme in effective symbiotic associations under saline conditions have also been studied because nodule always contain high level of (ROS) (Gorgocena et al., 1995) due to strong reducing condition (Tejera et al., 2004; Jebara et al., 2005; Loscos et al., 2008).
In order to improve salt ability of rhizobia to grow in saline soils, screening of salt-tolerant rhizobia from environmental rhizobial populations in saline or non-saline soils (Zahran, 1991) is cheaper and more successful than improvement to through genetic engineering (Munns et al., 1981).
Salinity tolerance among rhizobia varies species to species. R. meliloti strains tolerate 100 mM NaCl (Mashhady et al., 1998), R. leguminosarum have been reported to be tolerant to NaCl concentrations up to 350 mM NaCl in broth culture (Wahab et al., 1979; Breedveld et al., 1991). It is evident from several investigations that Symbiotic effectiveness is positively correlated with high- tolerance under saline condition (Chen et al., 1992). Therefore, there may be scope for selecting a Rhizobium-legume symbiosis that is better adapted to saline conditions
1.6. Relevance of work
Keeping in mind the host-rhizobia symbiotic specificity and bacterial adaptations to prevailing environments, it is important to characterize these microbial assets and to optimize legume use.
It is possible that Pakistani arid soils may contain resistant strains to the existing extreme environmental stresses such as salinity, high soil temperature, pH, antibiotic and heavy metal tolerance. There is great need to isolate and evaluate such strains for large arid areas of Pakistan Improvement in efficiency of legume-commercial rhizobia is likely to result increased production of protein in legumes. The symbiotic effectiveness of rhizobia that nodulate wild legumes and Rhizobium japonicum are studied extensively, but very little is known about native Rhizobia nodulating chickpea (Cicer arietinum L.) and garden pea (Pisum sativum L.) of Pakistan. This work will guide to select strains that efficiently nodulate and fix nitrogen and may be used as inoculants for chickpea and garden pea.
Therefore, present study is aimed to characterize 19 Rhizobium strains isolated from root nodules of chickpea and 11 from green pea, using soils collected from different regions of Pakistan for phenotypic, molecular and symbiotic characterization. The best symbionts for relative effectiveness, total shoot protein and in-vitro temperature, pH and salt tolerant were further evaluated for salt stress. Symbiotic characters, nitrogenase activity, whole cell protein profiles (SDS-PAGE), nodular antioxidant enzyme activities were studied under salt stress.
Phenotypic characterization of rhizobial isolates.
To study genetic diversity for plasmid profile analysis.
To determine the extent of polymorphism at the DNA level and the genetic relatedness among rhizobia using RAPD primers.
To study relative infectivity and effectiveness to identify and characterize symbiotically effective and ecologically persistent strains that could be used as inoculums for Cicer arietinum L. (Chickpea) and Pisum sativum L. (Green pea).
Screening for efficient symbionts under salt stress, effect of salt on host and its rhizobial partner in saline conditions and proteomic screening (SDS-PAGE) for salt stress protein expression of rhizobial isolates