Biological Nitrogen Fixation ( BNF ) is an effectual alternate natural beginning of N made available to the dirt. The entire one-year terrestrial inputs of N from BNF, ranges from 139 million to 175 million metric tons of N ( Burns and Hardy, 1975 ; and Paul, 1988 ) , with the N repairing symbiotic association turning in the cultivable land accounting for 25 to 30 % ( 35 million to 44 million metric tons of N ) . Even though the truth of these figures is problematic ( Sprent and Sprent, 1990 ) , they illustrate the comparative importance of BNF in cropping systems and the magnitude of possible chances available to replace the 80 to 90 million metric tons of nitrogen-bearing fertilisers applied yearly to agricultural land ( Peoples et al 1995 ( a ) ; and Peoples et Al 1995 ( B ) . ) . Among the assorted beings involved in the arrested development of N to the dirt, the symbiotic association, particularly that of the leguminous workss with the Rhizobium has the greatest quantitative impact on the N rhythm ( Brockwell et al 1995 ; Peoples et Al 1995 ( a ) ; and Tate, 1995 ) . However, several environmental conditions are restricting the efficient growing and activity of nitrogen-fixing workss. As the most serious menace faced by agribusiness in waterless and semi-arid parts is salt ( Rao and Sharma, 1995 ) , choosing a salt tolerant rhizobial strain or a salt tolerant leguminous plant, the chance for the success of the partnership has been a failure. This is because legume-Rhizobium mutualism and nodule formation on the leguminous plants is more sensitive to salt or osmotic emphasis than the Rhizobium or the works ( Zahran and Sprent, 1986 ; El-Shinnawi et Al 1989 ; Velagaleti et Al 1990 ; and Zahran, 1991 ) .
The salt hurt on the symbiotic interaction non merely inhibits the formation of the nodules, but besides thereby leads to the decrease of the growing of the host works. Other effects of salt on the nodulation, includes formation of non-functional nodules with unnatural construction, and debasement of peribacteroid membrane ( Bolanos et al 2003 ) . Bacterial chemotaxis, colonisation, root hair curving ( Zahran, 1986 ; Zahran and Sprent, 1986 ; and Tu, 1981 ) and distortion ( Singleton et al 1982 ) , decrease in nodular respiration ( Delgado et al 1994 and Walsh, 1995 ) , leghemoglobin content ( Delgado et al 1993 ) and impaired N-fixing activity ( Bordeleau and Prevost, 1994 ) have besides been observed as inauspicious effects of salt. Hence, testing for a salt tolerant symbiotic legume-rhizobium system is more of import.
Among the dietetic leguminous plants of the universe, black-eyed pea, Vigna unguiculata L. , stands 6th ( National Academy of Sciences, 1994 ) in ingestion. Cowpea has been categorized as a salt tolerant works with ECe of 5.0 dS/m ( California Fertilizer Association, 1980 ) , ( Ayers and Westcot, 1985 ) and, so it does non get away from salt hurts wholly. A lessening in the shoot dry affair, shoot/root ratio, nitrate consumption, leaf-nitrate reductase ( NR ) activity and shoot-nitrate content of the works ( Silveria et al, 1999 ; and Maas and Poss, 1989 ) under salt emphasis, though rhizobial strains isolated from Vigna unguiculata L. nodules are known to be tolerant upto 450 millimeter NaCl ( Mpepereki et al, 1997 ) . However, really small is known on the consequence of salt on the alterations in the form and distribution of nodules in the different parts of the root system, and on their efficaciousness to set up a symbiotic association, in other words, the ability of the Rhizobium-legume to develop a partnership for organizing nodules, a depository for repairing N that can be used by the leguminous plant and energy resources for the bacteria. Consequences of the work carried out in these positions are discussed in this paper.
Materials and methods:
Study sites and species
Rhizobial strains were isolated from Indian potato ( Arachis hypogaea L. ) , a cross-inoculating leguminous plant assortment for black-eyed pea, and a widely cultivated leguminous plant in the territory, were collected from different Fieldss in and around Dindigul, Tamil Nadu, India. Cowpea seeds for pot civilization were besides collected from attested resources.
Screening of salt tolerant rhizobial strains:
Rhizobial strains were isolated from Arachis hypogaea L. nodules, a cross inoculating group of black-eyed pea. The stray strains were characterized and authenticated utilizing standard microbiological and biological methods. Pure civilization of the stray strains, GRI I, GRI II, CP I, CP II and CP III, were maintained till the terminal in YEMA angles.
Strains were classified as slow-growers and fast-growers utilizing a biochemical method, by turning them on Bromothymol Blue ( BTB ) medium, yeast extract mannitol agar supplemented with 0.5 % alcoholic solution of bromothymol blue.
Halotolerance of the strains were determined based on the exponential growing and comparative salt tolerance of the strains utilizing turbidometric growing technique ( Singleton et al 1982 ) . Isolated strains were inoculated in YEMB in 9:1 ratio and incubated at 22 & A ; deg ; C for 48 hours. The diluted civilization was so transferred to 250 milliliters Erlenmeyer flasks until the initial optical denseness of the YEMB read 0.08 nanometers, and this was considered 0 clip. Inoculated flasks were so incubated at 26 & A ; deg ; C with changeless shaking ( 70 revolutions per minute ) utilizing an Orbital-shaking brooder. All readings were recorded at an interval of one hr for 8 hours, at an optical denseness of 600 nanometers.
Comparative readings of the growing of the strains under different salt interventions were taken by supplementing YEMB with different salt ( NaCl: CaCl2 in 7:3 ratio ) concentrations runing from 0 millimeters, 100 millimeter, 200 millimeter, 300 millimeter, 400 millimeter and 500 millimeter. Turbidometric readings therefore obtained were used for ciphering the exponential growing rate ( & A ; micro ; ) and the doubling clip ( t-d ) of the single strains utilizing equations given by Schlegel ( 2002 ) , and a Na response curve was derived utilizing these values.
Salt tolerance index of the works:
Cowpea workss were grown in in vitro utilizing Knop ‘s solution ( Rachel and Ravindran, 2006 ) supplemented with nitrate in Erlenmayer ‘s flask, with differing salt concentrations adjusted to 0, 1, 2, 3 and 4 dS/m, harvested on the 15th twenty-four hours after seeding, and salt tolerance index of the genotype was calculated utilizing the expression adopted by Garg and Singla, 2004.
Pot civilization experiments:
The consequence of changing concentration of salt on the symbiotic association was evaluated in pot civilization experiments. Circular fictile pots were filled with a mixture of exhaustively sifted and sterilized dirt, sand and farmyard manure in proportion of 2:2:1 by volume. Seeds were surface sterilized with 0.1 % mercurous chloride for 2 proceedingss, so washed in unfertile H2O thrice and germinated in pots. The pots were treated with saline solutions ( prepared from a mixture of NaCl, CaCl2 and Na2SO4 in the ratio of 7:2:1 ( w/v ) of changing electrical conduction, 0, 1, 2, 3 and 4 dS/m, incrementally. The pots were treated with the salt solution three yearss prior to seeding until the coveted degree of the salt has been attained and these degrees were maintained throughout the turning period by fortificating the dirt with saline solutions at hebdomadal intervals. The electrical conduction of the pot dirt was measured utilizing a Systronic conduction metre ( Type 302 ) following the methodological analysis of Ryan et Al 2001, originally done by Richards, 1954. The controls were irrigated utilizing tap H2O entirely. Surface sterilized seed were sown with 1 milliliters aliquot of 24 hours old civilization solution. They were so treated with 1 milliliter of the aliquot solution 24-hour civilization solution consecutively for three yearss. Initially five workss were sown, on the fifteenth twenty-four hours, two of the five workss were removed, and three workss of unvarying size were maintained in each pot. Plants were sampled and analysed on the 15th and the thirtieth twenty-four hours after seeding, and the pots were indiscriminately shuffled on a day-to-day footing.
Triplicates of each intervention were maintained and the undermentioned parametric quantities were analysed:
- Salt tolerance index of the works ( Garg and Singla, 2004 )
- Mean root length, figure of secondary roots, nodule fresh weight, entire figure of nodules formed, figure of nodules formed on the primary and the secondary roots and form of the root nodules.
- The symbiotic efficaciousness ( efficiency to nodulate ) on the primary or the taproot and the secondary roots of the works was calculated utilizing the derived expression:
Consequences and Discussion:
Screening for aura tolerant strains:
Five rhizobial strains, viz. GRI I, GRI II, CP I, CP II and CP III were isolated from peanut works nodules. All strains were fast agriculturists expect for CP II, which is a slow agriculturist. Based on the consequences obtained from the aura tolerance experiments, CP II was found to be the aura tolerant strain and was used for farther surveies. The public presentation of the other strains when subjected to salt emphasis were in the undermentioned order – GRI I and GRI II, CP I and CP III as evident in the Na response curve for exponential growing rate during logarithmic stage ( Fig. I. ) . The exponential tendency line shows that CP II shows an upward tendency with increasing salt concentrations unlike GRI I and GRI II that has more or less similar growing rate in all concentrations of salt supplemented to the medium ; and CP I and CP III, perversely showed a downward tendency.
Even though the salt tolerance of the rhizobial strains are normally calculated based on their doubling clip, in this instance, for the convenience of calculating how successfully a symbiotic association can be established, the influence of salt emphasis on the exponential growing rate is calculated. The rhizobial strains has to be in their exponential stage of growing for a longer period of clip, so that they can set up themselves, in the leguminous plant rhizosphere ( Schlegel, 2002 ) ; while, a higher doubling clip is favorable to get the better of the effects of salt. This is because in civilization solutions the aggressive Rhizobium isolated from insignificant cultivars, has been more tolerant to salt than the slow-growing Rhizobium. Hence, a strain that can strike a balance of these two characteristics, was found to be observed in CP II, and was therefore selected for farther rating.
Pot Culture Experiments:
Salt tolerance index of the works as evaluated on the 15th and 30th twenty-four hours after seeding, showed that the genotype used in this experiment had a tolerance to salt up to 3 dS/m ( Table -1 ) . However, workss were still subjected to a higher concentration of salt of 4 dS/m, in order to prove the public presentation of the symbiotic system.
Consequences of the pot civilization experiments showed that the symbiotic system tolerated salt up to 2 dS/m on the 15th twenty-four hours after seeding, but the degree of tolerance started to diminish from 3 dS/m. In contrast to the controls, the symbiotic system succumbed to salt emphasis by the thirtieth twenty-four hours after seeding at 1 dS/m.
The incursion of the pat root system into the dirt besides seemed to cut down under saline conditions, with a considerable lessening in the primary root length when the salt concentration was increased to 4 dS/m on the 15th twenty-four hours after seeding, and a farther diminution on the thirtieth twenty-four hours after seeding likely owing to decease of the tissue. On the reverse, an addition in salt concentrations resulted in an addition in the figure of secondary roots, though does non seemed to be pronounced as the figure of the secondary roots on the 15th and the thirtieth twenty-four hours after seeding did non increase with increasing salt concentrations ( Table -2 ) .
In malice of the consequence of salt hurt on the works seemed to be marked on the thirtieth twenty-four hours after seeding, the consequence of salt on the nodules formed does non look to correlate with the salt tolerance of the works on the 15th or 30th twenty-four hours after seeding, as there was no fluctuation in the size, form and fresh weight of the nodules. However, there was marked salt imposed displacement in the spacial distribution of nodules that thereby act uponing the efficiency of nodules formed, that is, the symbiotic efficaciousness ( Table-3 ) . The efficiency for nodule formation on the taproot of the works is on a worsening tendency with increasing salt concentration ( 2 dS/m ) on 30th twenty-four hours after seeding ; and besides, there was a difference in the coloring material of the nodules formed in the primary and secondary roots ( Table-2 ) . However, there was a reversal tendency in the symbiotic efficaciousness on secondary roots of the workss on the thirtieth twenty-four hours after sowing, which besides seemed to fall in line with the salt tolerance degree of the symbiotic system, a diminution in the efficaciousness get downing from an addition in salt concentration from 2 dS/m to 3 dS/m. Furthermore, the distance of discrepancies in the symbiotic efficaciousness on the 15th DAS and 30th DAS has increased about twice, from 0.4 to 0.9, as observed from the F-test computations, thereby demoing a time-dependant addition in the displacement of tendency towards nodule formation on the secondary roots.
The salt tolerance of the works and the halo tolerance of the symbiotic system seemed to be differing from 3 dS/m to 2 dS/m that is a fluctuation of 1 dS/m. However, for measuring the efficiency of the symbiotic system the initial observation is on the ability of the symbiotic systems that can organize efficient nodules under saline conditions. Yet another interesting characteristic that has been observed is the addition in the figure of nodules formed in the secondary roots than in the primary roots under increasing salt concentrations. But this tendency does non prevail when the salt tolerance of the symbiotic system decreases, as it is obvious from the consequences that the symbiotic efficaciousness on secondary roots decreases at 3 dS/m while the salt tolerance index of the works is up to 3 dS/m.
Nodule formation and its distributional form on the different parts of the roots have been observed and assorted decisions and generalisations have been made under non-saline conditions. The formation of nodules, per Se, has important consequence on the decrease in the growing of the primary root, and induces the formation of laterals or nodules. In fact, the formation of nodules on the taproot is considered as a desirable characteristic than on the secondary roots, as the nodules formed on the primary roots are efficient. At this occasion, it is besides of import to take into consideration that the underlying mechanisms in sidelong root induction and nodule formation are more or less similar ( Lim, 1963 ; Dart and Pate, 1959 ; and Nutman, 1959, 1948 ) .
However, the formation of nodules on the sidelong roots has besides been observed in soybean workss during cod filling phases ; and, Lim ( 1963 ) observed a similar consequence when the figure of bacteriums in the leguminous plant rhizosphere decreased. Hence, the plausible accounts for this displacement of nodule formation from primary roots to sidelong roots could be due to a decrease in the figure of bacteriums over clip owing its failure to set up in the leguminous plant rhizosphere due to reduced sludge organizing capableness ( Nilson, 1957 ) , or decreased surface country for induction of infection. This is because of the addition in the figure of secondary roots, a “ cross tolerance ” mechanism ( Malash and Khatab, 2008 ) to the osmotic emphasis or drouth emphasis, thereby cut downing the surface country exposed for the induction of infection and colonization by the Rhizobium sp. However, the existent mechanism involved can merely be better explained merely when more penetrations and cognition is gained on the physiological and the biological mechanisms on the constitution of the Rhizobium in the leguminous plant rhizosphere, induction, formation, and constitution, and efficiency of the nodules therefore formed are understood.
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