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Abstract The present work aims to isolate bacteria belonging to pink pigmented facultative methylotrophic bacteria(PPFMs) and to study the ability of nodule formation and nitrogen fixation by them with some legume plants , as well as their relation to enhancing the growth yield and yield quality of some legume crops. The results of the present work could be summarized as follows: I- Isolation, purification and Identification of methylotrophic bacteria: I. 1. Ninety four isolates of methylotrophs were obtained from green leaves and root nodules of legume plants . I.2. Five PPFMs, PPFM Lt, PPFM.Sb, PPFM.Tr, PPFM.M and PPFM.D isolates belonging to Lupine , Soybean , Egyptian clover, Alfalfa and Handaqooq legume plants had the ability to grow on YEM media with or without 0.05% methanol whereas other PPFM isolates were failed to grow at the same used medium. I.3. Three PPFM isolates , PPFM.Lt, PPFM.Sb and PPFM.D were able to induce legume root nodules for all tested plants (Lupine , Soybean , Egyptian clover and Alfalfa) , as compared to PPFM.Tr and PPFM.M, which failed to induce any legume root nodule for the same tested plants. II-Comparitive studies between nodulating methyotrophic bacteria and Rhizobia . 1.Morphological studies indicated that, PPFM isolates nearly had the same morphological and cultural characteristics, as compared with the reference rhizobial strains used. As well as , microscopic examination indicated that all isolates were short rods ,Gram negative and motile. II.2. Physiological characteristics cleared that, the three isolates (PPFM.Lt, PPFM.Sb and PPFM.D) and two reference rhizobial strains (USDA 110 and ARC 408) were catalase positive . II.3.Utilization of different carbon sources showed that , all isolates and strains can utilize sucrose and D-glucose, except PPFM.Lt, and utilized SUMMARY 95 Heba O.M.A.Orf,Ph.D.,2014 citrate and methanol, except both rhizobial strains (USDA 110 and ARC408). II.4.Antibiotic sensitivity test indicated that, the three PPFM bacteria were resistant to Ampicillin ,Gentamycin and Colistin antibiotics and were susceptible to Kanamycin and Streptomycin antibiotics, while the two rhizobial strains were resistant to all antibiotics tested . II.5.Molecular biology studies indicated the following: 5.1. Protein pattern using SDS-PAGE of PPFM isolates and rhizobial strains noted that, Cluster analysis of protein marker data placed the bacterial PPFM isolates and rhizobial strains into two main groups and similarity between the three PPFM isolates and the two rhizobial strains was 65.01 %. 5.2. Random amplification of the DNA placed the three PPFM isolates and the two rhizobial strains into two major groups, which were divided into minor clusters, giving different degrees of polymorphism according to the used primers. The similarity; a) at cause of using primer No. (2) ranged from 51.4% to 83.87%. b) At cause of using primer No. (4) Similarity between the two main groups (PPFMs isolates and rhizobial strains) ranged from 54.57% to 85.85% . c) At cause of using primer No. (6) the similarity between the two main groups ranged from 37.38% to 92.29%. III. The relationship between methylotrophic bacteria (PPFMs) and some legume plants as compared to rhizobia under greenhouse and field conditions. III.1. Greenhouse experiments: 1. PPFM.D isolate recorded the lowest nodule number per plant (28 nodules plant-1), as compared with PPFM.Lt , PPFM.Sb and the SUMMARY 96 Heba O.M.A.Orf,Ph.D.,2014 mixtures and these numbers were 50-56 , 67-36 and 53-33 for soybean and lupine, respectively. 2. PPFM.Sb and PPFM.Lt recorded high nodule numbers and nodule dry weights, compared to USDA 110 and ARC 408 and such increases were 81 ,40 % and 49 ,41 % for soybean and lupine respectively. 3. There were no significant differences for shoot dry weights (g.plant-1) between application of PPFM.Sb and PPFM.Lt, as compared with both USDA 110 and ARC 408 rhizobial strains. 4. Application of rhizobial strains (USDA 110 and ARC 408) had the higher values of plant N-content but no significant differences were found when compared to the applied of PPFMs(PPFM.Sb and PPFM.Lt). 5. The values of nitrogen derived from air (Ndfa %) and N2-fixed ranged from 84.82 to 93.75 % and 86.95 to 93.37 % for soybean and lupine plants, respectively. 6. Application of rhizobial strains led to scored the higher amounts of nitrogen content by fixed atmospheric N2 ,which attained 151.80 and 81.6 mg plant-1 for soybean and lupine plants and scored higher percentage (12.4 and 7.0 %) , compared to apply PPFM.Sb and PPFM.Lt, respectively. from the above mentioned results it could be stated that, isolate PPFM.Lt could be related to Methylobacterium nodulans. IV. Field experiment: A field experiment was carried out, in two different locations, as follows: IV.1.Sandy soil: 1. Growth stage: Heba O.M.A.Orf,Ph.D.,2014 1.1. Inoculation with specific Rhizobium scored higher significant increases as compared to fertilized treatments with 15 kg N Fed-1, in both nodules number (14-5) 3-folds and nodules dry weight (327-116)3-folds. 1.2. Inoculation with PPFM.Lt scored higher significant increases as compared to fertilized treatments with 15 kg N Fed-1, in both nodules number (15 - 5) 3-folds and nodules dry weight (355 - 116) 3-fold. 1.3. Inoculation with PPFM .Lt , specific Rhizobium , foliar application of PPFM. Lt and fertilized with 15 kg N Fed-1 scored the highest values in both parameters (19and460) for nodule number and nodule dry weight respectively as compared inoculated plants with specific Rhizobium and fertilized with 15 kg N Fed-1 (15-350). 1.4. Inoculation with PPFM.Lt and foliar application of PPFM. Lt generally caused significant difference at nodules number and nodules dry weight as compared to inoculated plants with specific Rhizobium and fertilized with 15 kg N Fed-1. 1.5. Inoculation with PPFM.Lt and foliar application of PPFM.Lt caused significant increases as compared to applied PPFM.Lt +15Kg N Fed-1 and such increases were 12.5% for plant dry matter and 17% for plant Ncontent. 2. Harvest Stage 2.1.Inoculation with PPFM.Lt in combination with rhizobial inoculation in presence of 15 Kg N Fed-1scored the highest value in total biological yield (1.105 Kg Fed-1) as compared with any other treatment as well as, the highest seed yield (0.401 Kg plot-1 and 337 Kg Fed-1) was obtained. 2.2.The highest values (82.3 g 100 seed-1) of seed index was obtained when inoculation with PPFM.Lt and rhizobium in fertilized plants with 15 Kg N Fed-1,as well as the highest value of seed crude protein percentage(27.88%) was observed . IV.2.Clayloam soil 1. Growth stage Heba O.M.A.Orf,Ph.D.,2014 1.1.Significant increases were found in case of inoculation with PPFM.Lt in nodules number and also significant increases were observed in nodules dry weight as compared to inoculated plants with specific Rhizobium and such increases were 61 and 170 % in nodules number and dry weight of nodules ,respectively. 1.2. Inoculation with specific rhizobium and fertilized with15 Kg N Fed- 1 scored the highest values in both nodules number (33) and nodules dry weight (407) as compared to any other treatments. . 1.3. Bi-inoculation with PPFM.Lt scored no significant increases in both nodules number (30-33) and nodule dry weight (395-407) as compared to inoculated plants with rhizobium and fertilized with 15 Kg N Fed-1 . 1.4.Inoculation with PPFM.Lt and foliar application of PPFM.Lt and fertilized with 15 Kg N Fed-1 recorded the highest values (8.9 g.plant-1) for plant dry matter and (231.4 mg.plant-1) for plant N-content. 1.5. Application of PPFM.Lt caused significant increases as compared inoculation with only PPFM.Lt and such increases were (11%) for plant dry matter and (33%) for plant N-content. B. Harvest Stage 1. The highest seed yield (2.874 Kg. plot-1) was obtained by fertilized lupine plants with(50 Kg N Fed-1). 2. No significant increases were observed incase of inoculation PPFM.Lt +15 Kg N Fed-1 (2.501 Kg plot-1 ) as compared to fertilized plants with50 Kg N Fed-1. 3. the highest values of seed index (107.6 g 100 seed-1 ) was observed in fertilized plants with 50 Kg N Fed-1 as compared to any other treatment but there is no significant increases between this treatment and inoculation with PPFM.Lt+ 15 Kg N Fed-1 (99.3 g100 seed-1 ). 4.The highest values of seed crude protein %was recorded when using inoculation with PPFM.Lt and specific Rhizobium in presence of 15 Kg N Fed-1 (30.94%) as compared to any other treatments. |