الفهرس 
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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
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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.