Jan. 2014. Vol. 1, No.3
ISSN 2311 -2476
International Journal of Research In Agriculture and Food Sciences
© 2013 - 2014 IJRAFS & K.A.J. All rights reserved
http://www.ijsk.org/ijrafs.html
THE EFFECTS OF HELPING BACTERIA IN NITROGEN BEANS
FIXATION AND NODULATION WITH RHIZOBIUM
LEGUMINOSARUM BV.PHASEOLI
Hassan Rezaei : Naser Aliasgharzadeh: Behnam Tahmasebpour
University of Tabriz , Tabriz , Islamic republic of Iran .
Abstract
Some batteries settle in the Rhizosphere of legume plants and enhance the performance of ribosome batteries to
nitrogen fixation and nodulation. In this paper, we used four isolated from two species of Sodonamous containing
P.putida, P.flouresence Chao, P.Flouresence Tabriz, P.flouresence B119 and Rhizobium leguminosarum
bv.phaseoli. In a factorial experiment with complete accidental blocks base and 5 levels of helping bacteria and two
Rhizobium levels were inoculated with four repetition of Jamaran418 green bean. At the end, nodulation indexes,
growth and plant’s nitrogen indexes were measured. The results showed that all above mentioned helping bacteria
enhance the growth and performance of green bean. It should be said that P.putida had 130% effect on the green
bean nodulation increase along with Rhizobium and it was 83, 63 and 17% about P.Flouresence Tabriz, P.
florescence Chao and P. florescence B119, respectively. Also, we observed 45, 33, 22 and 8% performance increase
under the effect of P.putida, P. florescence Chao, P.Flouresence Tabriz and P. florescence B119, respectively.
Keywords: helping bacteria, Rhizobium, green bean, nodulation, Sodonamous
Some PGPR bacteria with synergistic effect on the
Rhizobium can enhance the nodulation and fixation
of nitrogen in legumes. According to conducted
researches, most of the bacteria which are effective in
this field belong to Phosphate solubilizing
microorganisms (Pisney and Azcon, 1987 and Ming
and Alexander, 1988). Rasi poor and Ali Asgarzade
(1381) investigated the role of four phosphates
solubilizing and isolated bacteria, p.flourescens,
P.putida
and
Areomonas
hedrophila
with
Bradyrhizobium japonicum on the soya nodulation
and performance and concluded that these bacteria
enhance the phosphor absorption in soya, comparing
with control group. The results show that P.putida
treatment had the most effect on the soya nodulation,
nitrogen fixation and phosphor absorption,
comparing with the other treatments along with
Bradyrhizobium japonicum.
Introduction
The widespread use of chemical fertilizers and
pesticides is discussed a lot in today world because of
their adverse effects on the environment and on the
health risks of living. The natural process of
rhizobacters is a suitable way for plants protection
which enhances the growth and protection of plants
(Delip Koomar, 2001).
Private and public investment has increased to mass
production of biologic fertilizers and especially
microbial ones after the more attention in this area.
Rhizobium inoculants are one of the most important
and widespread of these fertilizers which are used for
different legume plants.
Along with the extensive researches in Iran and
world to introduce the most efficient ways of these
bacteria performance, further research is underway to
improve the quality of these fertilizers. Some
evidences show that the bacteria like Sodonamous,
Strepto misses and bacilious as a helping one
enhance the nodulation and fixation performance of
Rhizobium bacteria (Ming and Alexander, 1988).
The results are patent and the Rhizobium inoculants
producers companies access them.
Ali khani and Saleh Rastin (2006) announced that the
Phosphate solving bacteria enhance the performance
of different rhizubioms in Iran soils.
Rozras et al (2002) had done a farm experiment on
the soya and investigated the mutual effects between
soya bacteria and Sodonamous Piotida phosphate
solving bacteria. They reported a significant increase
in the stem nodulation, Nitrogen fixation and soya
1
Jan. 2014. Vol. 1, No.3
ISSN 2311 -2476
International Journal of Research In Agriculture and Food Sciences
© 2013 - 2014 IJRAFS & K.A.J. All rights reserved
http://www.ijsk.org/ijrafs.html
‘shoot weight in the inoculation. Also in a farm
experiment in India, the effect of phosphate solving
bacteria, Sodonamous Steriata on the Beradi
Rhizobioum Gaponickom and soya symbiosis and
some measures like nodules dry weight and plant dry
weight were investigated. Their joint inoculation with
Sodonamous Sterila resulted in the significant
increase of nodule dry weight and number (Vasole et
al, 2002).
We used turbidimetry and Mac Farland to identify
the number of bacteria in the suspension.
Microbial carrier preparation
First, we milled processed vermiculite and passed it
from 100 micrometer sieve. Its 50 g was transported
to each flask and was sterilized.
Microbial suspension inoculation on the carrier
Rhizosphere bacteria always do not enhance the
nitrogen fixation and nodulation by legumes and
sometimes can have negative or neutral effects on
these indexes. Pan et al (2002) investigated the
effects of two Rhizosphere bacteria (Serratia
Proteamawlans and Serratia liguefaciens along with
Bradyrhyzobium japonicum Strain 5326 on the two
soya spices and showed a significant difference of the
plant performance treated with Rhizosphere bacteria,
but these bacteria had no significant effect on the
nitrogen fixation and nodulation. Anjela Bergern et al
(2001) had conducted an experiment in Sweden and
investigated the effects of P.putida strain A313
symbiosis with Pea symbiotic Rhizobioum bacteria
and the results showed the reduction of nitrogen with
P.putida strain A313.
12 ml from each microbial suspension with clear
population was inserted in flasks containing material
and were stirred. The carrier material got wet but
kept the powder tissue and another flask containing
12ml sterile culture media was considered as control
treatment.
Select and prepare the soil for the greenhouse
experiment
We used the soil from agriculture faculty earth in
Karkaj. They were placed in plastic pots after passing
from 4mm sieve and were sterilized in autoclave for
2 hours.
Preparation treatments and planting pot
We should consider these items more due to
increased production of Rhizobium inoculants in the
country as a good replacement for nitrogen fertilizers
and lack of their efficiency in the farm and do
extensive researches to enhance their quality. This
study introduces the most effective bacteria between
four helping items.
After determining the required amount of soil
phosphorus
and
potassium
fertilizer
recommendations based on soil test and the beans,
and calculate the required amount of fertilizer was
applied.
To determine the amount of nitrogen-fixing
symbiosis with Rhizobium method, no nitrogen was
added to the soil in the pot. Green bean seeds were
soaked in distilled water for 2 hours and then were
placed in 95% ethanol for 30 seconds and
immediately after that were put in 0/1% acidity
Mercuric chloride for 15 minutes. Then it was put in
a solution of 1% sodium hypochlorite for 30 minutes,
until the seeds surface to be disinfected. Then it was
washed 5 times with sterile distilled water and was
put in Petries containing water-agar for 3 days. To
create the same condition for all of the pots, they
were moved every 2 days. Plants until the pods and
seeds were grown for 75 days.
Materials and Methods
Proliferation of used bacteria
In this experiment, we used P.flourescens B119 (H2),
P.flouresence Chao (H1) (Tehran Plant Pests and
Diseases Research Institute), P.flouresence Tabriz
(H3) (Tabriz university soil biology laboratory) and
one isolated from P.putida (H4) (Tabriz isolated) as
the helping bacteria and control ones (H0) along with
leguminosarum bv.phaseoli Rhizobium. King B
culture media was used to Sodonamous proliferation
and YEMB was for Rhizobioum proliferation.
Experimental design and statistical analysis
Those culture media were liquid. Sodonamous flasks
were kept for 2 days in shaker incubator in 28 °C and
120 rpm until to bacteria growth and Rhizobioum
flasks were in this condition for 3 days.
In a factorial experiment with complete accidental
blocks base and 5 levels of helping bacteria and two
Rhizobium levels were inoculated with four
repetition of Jamaran418 green bean. At the end,
2
Jan. 2014. Vol. 1, No.3
ISSN 2311 -2476
International Journal of Research In Agriculture and Food Sciences
© 2013 - 2014 IJRAFS & K.A.J. All rights reserved
http://www.ijsk.org/ijrafs.html
nodulation indexes, growth and plant’s nitrogen
indexes were measured. We used variance analysis
and comparing their means via MSTATC.
Comparing the means was done with Don Ken test in
5% probability. The graphs were drawn by Excel.
0/8% water-agar was transferred in the petri dishes
after sterile. The culture media was sterilized in 1/5
pressure atmosphere for 20 minutes and 121 °C in
autoclave. The medium was distributed in sterile petri
dishes. The bacteria were grown on solid medium
using a sterile wooden toothpick into each petri was
spot on.
Vitro biochemical tests
Sperber culture media was prepared to measure the
quantity ability of bacteria for the dissolution of
phosphate. The culture media was sterilized in 1/5
pressure atmosphere for 20 minutes and 121 °C in
autoclave. The medium was distributed in sterile petri
dishes. The bacteria were grown on solid medium
using a sterile wooden toothpick into each petri was
spot on. After putting in 48 hours of incubation,
colony diameter and the diameter of the clear zone
around it indicates that the dissolved phosphate was
measured.
The time required for maximum germination/ %
Maximum number of germinated seeds= Germination
rate (% per day)
Results and Discussion
Physical and chemical analysis of soil used in pot
culture experiment is described in Table 1.
Number, fresh weight and dry weight of nodules
Mean comparison shows that all helping bacteria
except P.flourescens B119 increased the bean
nodulation. P.putida had the more number of nodules,
fresh weight and dry weight in the bean.
Measurement potential of siderophore production
by bacteria
We used Chrome azurol-s culture media for this
purpose (Bernhard et al, 1987).
P.putida had the nitrogen percent and the least
percent belonged to the control treatment without
bacteria.
10 ml of abovementioned media and 90 ml of King’s
B were sterilized in autoclave and then Chrome
azurol-s was added to the culture media and the final
material was aired in the Petries. After 48 hours,
colony diameter and the diameter was measured and
the ratio of the diameter to the colony diameter
(Bernhard et al, 1987).
Shoot weight
The treatments with Rhizobioum and helping bacteria
had the driest weight. P.putida with R.l. bv.phaseoli
created the driest weight and wet one of shoot.
Figures 2 and 3, the difference in the level of 5%,
were significantly different from other treatments and
control.
Germination and seedling growth of helpful bacteria
in the presence of green beans and Rhizobium
First, the bacteria suspensions were prepared.
Organic
pH
7/56
ECe(s/cm)
948
Neutral
carbon
matter
%
%
0/6
9/1
Moisture
of Field
capacity
Available
Nitrogen%
Phosphor
Available
Potassium
Clay
Silt
Sand
%
%
%
24
21
55
tissue
%
15
0/12
5/4
Phosphor:method of Olsen (Na2 CO3,0/5 Molar and PH=8/5) [10]
Potassium: Ammonium acetate(one normal and PH=7) [10]
Organic carbon: method of Valkely black [10]
3
232
Loamy
sand
Jan. 2014. Vol. 1, No.3
ISSN 2311 -2476
International Journal of Research In Agriculture and Food Sciences
© 2013 - 2014 IJRAFS & K.A.J. All rights reserved
http://www.ijsk.org/ijrafs.html
Table2. Mean comparing the effect of helping bacteria on the nodulation of green bean root
Dry weight
Nodule per
plant(gram)
wet weight
Nodule per
plant(gram)
Number of
Nodule per
plant
Helping bacteria
b0/024
c0/325
d*19/75
control
ab0/079
c0/405
cd23
P.flourescensB119
ab0/086
ab0/942
ab36/5
P.flourescensTABRIZ
a0/122
a1/273
a45/5
P.putida
ab0/084
bc0/782
bc31/5
P.flourescensCHAO
Different letters in each column indicate a significant level of 5% is likely.
Table3. Mean comparing of biochemical tests in helping bacteria
Sydrophore production
Phosphate solving
Diameter of halo
/the colony
Diameter of halo /the
colony
b2/015
b2/110
b1/990
a2/378
bc2/713
b3/585
a4/475
c1/974
Bacteria
P.flourescensB119
P.flourescensTABRIZ
P.putida
P.flourescensCHAO
Different letters in each column indicate a significant level of 5% is likely.
Figure1. Rhizobium bacteria and bacterial adjuvant treatment combination effect of nitrogen on shoot
4
Jan. 2014. Vol. 1, No.3
ISSN 2311 -2476
International Journal of Research In Agriculture and Food Sciences
© 2013 - 2014 IJRAFS & K.A.J. All rights reserved
http://www.ijsk.org/ijrafs.html
Figure.2 Rhizobium bacteria and bacterial adjuvant treatment combination effect of nitrogen on shoot weight
Figure3. Rhizobium bacteria and bacterial adjuvant treatment combination effect of nitrogen on dry weight
5
Jan. 2014. Vol. 1, No.3
ISSN 2311 -2476
International Journal of Research In Agriculture and Food Sciences
© 2013 - 2014 IJRAFS & K.A.J. All rights reserved
http://www.ijsk.org/ijrafs.html
Figure4. Rhizobium bacteria and bacterial adjuvant treatment combination effect of nitrogen on seed weight
R. l. bv.phaseoli had the most effect on the
nodulation, nitrogen percent, phosphor percent and
plant performance.
Weight of 100 seeds per plant
The treatments with Rhizobioum and helping bacteria
had the driest weight.
Also, we can say that the solving power of the
phosphate by P.putida and P.flourescens TABRIZ
enhance the nodulation, nitrogen fixation and green
bean performance with R. l. bv.phaseoli. But as the
results show P.flourescens B119 has the most power
of phosphate solving. Anjela Bergern et al (2001)
had conducted an experiment in Sweden and
investigated the effects of P.putida strain A313
symbiosis with Pea symbiotic Rhizobioum bacteria
and the results showed the reduction of nitrogen with
P.putida strain A313. It should be mentioned that the
positive effects of the helping bacteria on growth and
legumes nodulation is not limited to the
abovementioned mechanisms. Because, Sodonamous
has a lot of positive effects such as: HCN production,
ACC-D aminase activity, oxen production, anti biotic
production.
P.putida with R. l. bv.phaseoli had the most
performance in this field, but had a significant
difference with the other bacteria.
The results of biochemical tests on helping
bacteria
Siderophore production
Colony diameter and the diameter was measured and
the ratio of the diameter to the colony diameter.
There was a significant difference between
P.flourescens CHAO and the other ones.
Mean comparison shows that all helping bacteria
except P.flourescens B119 increased the bean
nodulation. P.putida had the more number of nodules,
fresh weight and dry weight in the bean.
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Discussion
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The results show that the helping bacteria with R. l.
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6
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Jan. 2014. Vol. 1, No.3
ISSN 2311 -2476
International Journal of Research In Agriculture and Food Sciences
© 2013 - 2014 IJRAFS & K.A.J. All rights reserved
http://www.ijsk.org/ijrafs.html
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