THE EFFICIENCY OF PREPARED BIOFERTILIZER FROM LOCAL ISOLATE OF BRADYRHIZOBIUM SP ON GROWTH AND YIELD OF MUNGBEAN PLANT A

Bradyrhizbium sp. was isolated from 40 legumes roots and soil samples collected from various agricultural areas of Iraq by growing them on yeast extract mannitol agar (YEMA) and identification based on culture and microscopic characteristics, biochemical and physiological test and PCR technology. A split plot experiment was carried out to study the effect of biofertilizer prepared from this isolate on the growth and yield of mungbean plant in sterile and non-sterile soil. The results showed a significant increases in all studied growth and yield traits due to biofertilization. The increase percentage for the biofertilized treatments was 159.35% and 266.66% compare to the nonfertilized treatments in the number and dry weight of nodules, respectively. The increase in dry weight of root and vegetative part were 114.28% and 86.57% respectively , while the increase in the nitrogen concentration in the vegetative part was 16.94%. The results showed that the biofertilized treatment was significantly superior in number of plant pods, total yield and protein % in seeds compare with non-biofertilized, the percentage increase was 49.44%, 90.59% and 16.86% respectively. As for sterilization, the results showed no significant differences between sterilized and non-sterile soil in all growth and yield parameters of mungbean plant. The effect of interaction was significant, the biofertilized and sterilized treatment gave the highest value for all studied traits except the concentration of nitrogen in the vegetative part.


INTRODUCTION
Vigna radiate is one of the important seed legumes grown in tropical and subtropical regions.The edible plant seeds are rich in proteins 20-25%, fatty acids 1.0-1.2%,vitamins A, B1, B2, C, sugars, organic and amino acids and minerals for example potassium and phosphorus (19 ,15).Farmers suffer from low yields of Mungbean, especially in the summer.This may be due to severe environmental conditions such as drought, low soil fertility or low nitrogen fixation by indigenous rhizobial bacteria, which play a role in supplying plants with optimal nitrogen through the symbiotic relationship (26).So farmers always tend to use excessive chemical fertilizers, including chemical nitrogen fertilizers that might have adverse effects due to environmental pollution, as well as large amounts are could be lost by leaching and volatilization.Researchers are looking for effective and safe alternatives which contribute to the preservation of human health and the environment in which they live as well as its inexpensive price, such as biofertilizer.Biofertilizer can defined as a solid or liquid substance containing beneficial organisms that is added with seeds, or to soil or plant seedlings in order to improve plant growth and increasing its yield by supplying the plant with nutrients ,hormones and siderophores or by increase the nutrients absorption efficiency, resistance to adverse environment conditions and diseases, and ultimately for clean environment and sustainable agriculture.The symbiotic relationship between Rhizobia and legumes has a significant impact in the success of leguminous crops cultivation of and was found that these bacteria have the potential to convert nearly 20 million tons of N 2 to NH 3 , 50-70% of the world's bio-fixed nitrogen, and the high bio-fixed nitrogen can determine the success of the symbiotic relationship between Rhizobia and legumes (20).The arid and semiarid regions of the world suffer from a lack number of indigenous rhizobia in the soil with poor efficiency of nodulation and nitrogen fixation (2).Researchers in an Australian study of Mungbean found that it did not nodulate when not inoculated with Bradyrhizobium bacteria (14).Therefore the treatment of legume seeds with bio-fertilizer containing root nodules bacteria is economically necessary of whether it relates to soil fertility and productivity or yield.The objective of this study was to test the effect of bio-fertilizer prepared from Bradyrhizobium sp.Which was diagnosed according to protocol including morphological, microscopical, biochemical and molecular methods on growth and yield of mungbean.

MATERIALS AND METHODS
Forty roots of legumes and soil samples were collected from different agricultural sites in Iraq: Samara, Kirkuk and Sulaymaniyah.Pure culture of isolated bacteria were identified on the Yeast Extract Mannitol Agar(YEMA) by several cultural phenotypic tests including color, shape and colony height according to Collins et al. (7) and Microscopic tests using a Gram stain to identify its shape, arrangement and response to the dye under oil lens of the microscope as well as to determine its movement with the hanging drop method, and biochemical tests using Congo Red stain to distinguish the Rhizobia species from the genus Agrobacterium.Bromothymol blue was also used to identify the genus of the root nodule bacteria, whether it is fast or slowgrowing such as Rhizobium, Bradyrhizobium, Sinorhizobium etc., as well as physiological tests (pH and heat), then molecular identification, was done depending on extracted DNA (6) with some modifications, and multiply them using the initiators of nolXWBTUV gene, namely the (Primer 5'-TGGGAAGCGACGCTGCCGG-3 ') and the primer (R primer 5'-GGCGGAGAAATAGGCGCCGT-3') with polymerase chain reaction to distinguish between Rhizobia species as reported by Videira et al. ( 24).

Preparation of biofertilizer
An appropriate amount of peat moss was sieved, this peat moss was produced by the FAO as a good carrier in accordance with the specifications (22).Sieved peat moss was then distributed by 250g in high temperature resistant plastic bags, and moisturized with water by 20% of its weight, the bags were sterilized at 121°C and 1500 kPa for 30 minutes, sterilization was repeated three times for three days.Randomized samples of the carrier were tested to ensure that they were free of microorganisms by taking 1 ml of the first dilution and cultured on the YEMA medium, carrier was examined for its safety according to (12).After the injection area was sterilized in the wall of each bag filled with peat moss.The liquid inoculum was then injected into the logarithmic growth phase (1.5 × 10 9 ) as demonstrated by Vincent (25), using a 20 ml medical injection in the middle of the mass of the carrier under sterile conditions, the size of the added inoculum was 200 ml to make the moisture up of sterile and inoculated carrier of 50-60% of its weight, After the injection was completed, the injection hole was closed directly using a sticky paper tape with the recorded biomass data.The inoculated carrier was incubated at 30 ° C for 7 days with daily flipping in several directions (17), The number of living bacterial cells in carrier by dilution and counting in dishes method, The total density of living bacterial cell must be (4.6 × 10 9 ) CFU per gm carrier , which proved the results of the diagnosis that it belongs to the type Bradyrhizobium sp.

Implementation of experiment
Field experiment was conducted in 15-5-2017 in Salahhuddin Governorate samara district, Table 1 show some physical, chemical and biological characteristics of soil, experiment carried out by use split plot, the land was divided into two parts representing the sterilization factor (without sterilization, with sterilization of soil), each part of the land was divided in to three replicates the distance between each replicate was 1m, and each replicate was divided in two plots representing the biofertilization factor (without biofertilizer, with biofertilizer) the area of each plot was 1m x 1 m and the distance between plots was 1m.

Soil sterilization
The soil was taken at a depth of 30 cm from surface of soil and was sieved with 2 mm metal sieve.Then it was transferred to the laboratory and air dried in laboratory.Soil samples were sterilized by autoclave at a temperature 121 ° C and 1500 kPa for one hour, the sterilization was repeated 3 times to ensure the elimination of the microorganisms, and then the soil was returned with bags to the site of the experiment to be distributed to the experimental units.Urea fertilizer (46% N) at 40 kg N ha -1 was added in two batches before planting and at flowering (17), triple super phosphate( 21% P) at 160 kg P ha -1 ,and potassium sulphate (41.5% K) at 160 kg k ha -1 , which were added once before planting (1).

Seed surface Sterilization
The seeds were washed several times and placed in sterile glass conical flasks and submerged in a sufficient amount of Sodium hypochlorite at 2.5% concentration for 3 minutes then washed with sterile water for at least 6 consecutive times to get rid of the effects of the sterilization material Seeds treatment with bio-fertilizer and planting: The seeds were treated with biofertilizer according to the method described by Somasegaran and Hoben (22).The arabic gum (40%) solution was prepared and used as an adhesive materials, then the sucrose solution was prepared to increase the vitality and efficiency of the bacteria in the formation of the root nodules by dissolving 15 g sucrose in 100 ml sterile distilled water, and added to inoculum on peat moss in ratio of 1:3 respectively before use .mungbean seeds were processed for cultivation by mixing 100 g of seeds with 4 g of bio-fertilizer (10), then the inoculated seeds were spread on blotter paper in order to dry them in the shade away from the sun light.The mungbean seeds were planted on 15/5/2017 in lines at a distance of 15 cm between lines and depth of 2-3 cm , and after planting the experiment unit was irrigated directly.After 10 days of germination, the seedlings were thinned in densely areas.The distance between one plant and another was 15 cm and by 5 plants in the line .All management of the crop, such as hoeing, weeding, irrigating and others were conducted as required.

Studied characteristics
Number of root nodules (nodule plant -1 ), dry weight of the nodules (gm), dry root weight (gm plant -1 ), Dry vegetative weight (gm plant - 1 ) , Number of pods in the plant, Seed yield (tons ha -1 ), Nitrogen concentration in the vegetative and seed parts (8).The experimental results were statistically analyzed using ANOVA, and the averages was compared with the least significant difference of LSD at the level of 0.05 based on the program (18).Sterilization did not effect on the number of root nodules.The effect of interaction between inoculation and sterilization was significant in the number of root nodules of Mungbean, the biofertilized and sterilized treatment was significantly higher in root nodules number.The biofertilized and sterilized treatment was superior on other three interaction treatments, and this treatments gave the highest average of nodules number with 24.42 nodules plant -1 , and this can be due to the effectiveness of introduced bacteria strains in infection and formation of nodules, this result agree with Gupta et al. (9) in their study on Mungbean.Table 3 shows that the inoculation has a significant effect on the dry weight of root nodules, as the biofertilized treatments exceeded the non-biofertilized and the fertilized treatments giving average dry weight of 0.11 g nodule -1 , while the non-biofertilized which gave 0.03 g nodule -1 , with an increase of 266.66%.This increases may be attributed to the ability of the introduced isolate to compete with the microorganisms, which is reflected in its ability to cause infection and the formation of the largest number of root nodules, as indicated by Rahima et al. (16).As for the sterilization treatments, no significant differences were found in the dry roots nodules.The interaction effect was significant, the biofertilized and sterilized treatment being superior to the other interaction treatments.

RESULTS AND DISCUSSION
The biofertilizered and sterilized treatment gave an average for dry root nodules weight reached of 0.09 g nodule -1 compared with the non-biofertilized and sterile treatment with 0.00 g nodule -1 .Table 4 shows that the biofertilized treatment significantly exceeded the non-biofertilized treatment, and the biofertilized treatments gave the average dry root weight of 1.20 g plant 1 , while the nonbiofertilization gave the average 0.56 g plant -1 , with an increase of 114.28%, these results came in agreement with Kaur and Khanna (11) in their studies on mungbean.As for the sterilization factor, there was no significant effect on the weight of the root weight of the mungbean plant, the results are agreement with the Alkurtany and Ali (3) in their studies on soybean, there was no significant difference between the sterilization treatments and non-sterilization treatment.The interaction between the bio fertilization of Bradyrhizobium sp. and soil sterilization was significantly higher for the dry root weight, the fertilized and sterilized treatment was significantly higher than the other of the interaction treatments, the biofertilized and sterile treatment gave an average dry root weight of 1.50g plant -1 , compared with nonfertilized and sterile which gave average of 0.43 g plant -1 , with a percentage increase of 248.83%.. Table 5 show the effect of inoculation with Bradyrhizobium sp., sterilization and interaction in the dry weight of vegetative part during flowering stage.Results indicate that the inoculation has a significant effect on the dry weight of vegetative part, the biofertilizered treatments gave dry weight reached 12.65 g plant -1 ,while the nonbiofertilized treatment gave 6.78 g plant -1 , with an increment of 86.57%.This increase may be attributed to the fact that the use of nitrogen-fixing bacteria leads to an increase in the accumulation of nitrogen and converting it into amino acids and thus protein compounds that benefit the plant in the formation of different tissues (17).These results agreed with Al-jourany and abass (13) and Bhushan et al. (5) in their studies on Mungbean.Sterilization did not show a significant effect on the dry weight of the plant, these results agreed with Alkurtany and Ali (3), who confirmed there was no significant difference between sterile and non-sterile treatments in the dry weight of the soybean plant.The interaction between biofertilization and sterilization was significant, the biofertilizerdsterilized treatment gave the highest vegetative dry weight of 15.71 g plant -1 , while the nonbiofertilized and sterile treatment gave the lowest mean of 6.11 g Plant -1 .The results in table 6 show that the bacterial inoculation has a significant effect on the nitrogen concentrations(%) with biofertilized treatments gave the average of 4.90%, while the non-fertilized treatments gave 4.19% with un increment of 16.94%.This may be due to the increase in the number of Bradyrhizobium that came from inoculation and its efficiency in the incidence of infection on the root and the formation of active root nodules, which increases the amount of fixed nitrogen and thus increases the concentration of nitrogen in the plant.These results in agreement with Badar and Qureshi (4).Table 7 shows the result of the number of pods at maturity stage, the bacterial inoculation significantly affect the number of pods giving 9.46 pod plant -1 , compared to nonbiofertilizered treatment with 6.33 pod plant -1 with an increase of 49.44%.The effect of interaction between the inoculation and sterilization was significant on pods number and the biofertilizered-non sterile treatment gave the highest value of number of