RESEARCHES of THE FIRST INTERNATIONAL CONFERENCE
(BABYLON AND RAZI UNIVERSITIES) ( 2011)
Effect of Landuse in the Evaluation of Activity of Nitrifying Bacteria and
Viability of some types of Fungi in the Dissolving Phosphate Compounds
during different Cropping Periods at AL- Mishkhab Region
Nihad Habib Mutlak AL- Ezerjawi1
Abstract :
This study was conducted at Al-Mishkhab region – Al-Najaf AL- Ashraf governorate, in
the rice research station, which included four land uses, , the first was a research station of rice,
and the second represents the soil of a farm cropped with Mongbean, and the third site was a soil
planted with date palm and fruit, and the fourth location was un cropped soils (Fallow), soil
samples were collected from these sites for the chemical , physical and biological analysis.
The main aim of this study was to evaluate the effect of landuse on some
chemical,physical and biological properties of the soil under land uses in addition to evaluate the
activity of nitrifying bacteria (Nitrosomonas and Nitrobacter) during the different Cropping
periods and the ability of some species of fungi in the dissolving phosphate compounds during
the different periods of cropping.
Rice soils have ahighly activity for the nitrifying bacteria Nitrosomonas and Nitrobacter at the
three periods of the study in compared to of other land uses, that reached 5.57, 3.39 colony
forming unit. gm -1 respectively in the surface horizon soils of rice , whilst the fallow soils were
poor in its nitrifying activity ,it has reached 1.13, 0.80 colony forming unit. gm -1 respectively.
Studying soils were contained much of the phosphate-dissolving fungi, as Aspergillus
niger, Trichoderma harzianum which isolated from Rice soils were the most efficient in the
dissolving the phosphates on the Martin media ,its halo diameter reached 4.2,4 mm respectively
, while the Aspergillus niger and Trichoderma harzianum which isolated from Mongbean soils
achieved
significant increase in dissolved phosphate, as the halo diameter were reached 4.5, 4.3 mm
respectively, also the results showed that the best period of incubation, which verify abest
efficiency of the fungus in the phosphate dissolving, was 120 hours.
Introduction
Land is an important natural resource with a growing need for population growth, and
therefore, the best management of the land of those that take into eye-sight in the use and
understanding of current and future uses that associate with environmental and economic
changes.
The prolonged use of actionable soils for agriculture and the continued operations of tillage
leads to a lack of content of the soil organic matter with an increase in nitrification( Newgi &
Yadav, 1994) as noted (Stenberg et al, 1998 ) to changes in the biological characteristics in the
planted soils with grains and other non- planted in the content of nitrification bacteria.
A metal nitrogen comprizes less than 2% of the total nitrogen in the soil and synthesizes by
organic compounds in the soil of ammonium form, nitrate and nitrite (NH+4, No-3 ,No-2) and
1
College of Agriculture - University of Kufa
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RESEARCHES of THE FIRST INTERNATIONAL CONFERENCE
(BABYLON AND RAZI UNIVERSITIES) ( 2011)
other forms, but at low rates( Bremner 1965 ), nitrate and ammonium were important in fertility
sides. The process of the nitrification as biological oxidation of ammonium and the formation of
nitrates with the formation of a intermediate composite known as nitrite which means that
ammonium is the main focus of this process and the nitrification process occurs in two-part in
the soils with the presence of nitrification bacteria (Nitrobacte, Nitrosomonas), which follow the
family Nitrobactericeae (Strict aerobes , Chemo-auto trophics and spherical spiral bacteria
(Alexander 1977),that involved in one physiological properties , particularly in their getting of
energy, (Rachidi 1987) also it is not composed of the spores.
This process is completed into two steps:
The first step: Oxidation of ammonium to nitrite by Nitrosomonas bacteria with free energy
necessary for the growth of these microorganisims as follows.
1- NH4 + 3/2 O2
NO2- + 2H+ + E(66Kcal)
Second step: Oxidation of nitrite to nitrate by Nitrobacter bacteria with the liberation of energy
also for the growth of these microorganisims. ( Gray , Williams 1971)
2- NO2- + 1/2 O2
NO3- + E(175 Kcal )
In submerged soils, it occurs in the surface layer of oxidation that confrontation to immersion
water in depth 5-20 cm (Cox et al 1980) because of the availability of oxygen in this layer and
the fact that the revival of the soil microorganisms responsible for those process air are strict
aerobes. (Laanbroock et al, 2002) was remarked to increasing the total density of the biomass of
nitrification bacteria in the root zone of soils planted with grain than it is in the fallow soils, The
numbers of bacteria and fungi increased with increasing decomposition of organic matter
(Hanafy etal, 1990), as observed (Boyle & Keane, 1989), that the speed of transformation of
nitrite to nitrate greater than the transformation of ammonium to nitrite in the good drainage soils
and moderate to low, so it's normal to accumulate nitrate in the soil and is widely used by plants.
Nitrification process was affected by several factors, including ammonium ion, pH, humidity ,
temperature, oxygen, soil texture, soil salinity, organic matter and period of incubation.
The nondissolvant compounds of metal phosphorus is not available to plants, however, that
many organisms can convert them to their soluble states and thus take advantage of them, it was
found that (10-50%) of the fungal isolates that isolated from the soil has a propensity to dissolve
the phosphorus compounds such as calcium phosphate including fungi Aspergellus, Fusarium,
Pencillium, Trichoderma, and that these fungi can grew on a media contain Apatite Ca2 (HPo4) 2
(Alexander,1982),wheras these fungi able to synthesize phosphorus and converse large part of
Ca3 (PO4) 2 into the dissolved formula (binary or monadic phosphate) that may exceed the needs
of plant, as evidenced by the ability of these fungi in the dissolving phosphorus through the
appearance and measuring the transparent halo diameter, that surrounding the fungus colony
which was grown on the media which contains non-dissolvent metal source of phosphorus.
While in the flooded soil that planted with arice crop, the Ferric phosphate compounds were
converted into soluble forms by reducing ferric phosphate salts into adissolved ferric salts which
lead to produce asoluble phosphate ,thus this increasing in the dissolvent forms of available
phosphorus in the flooded soils can interprete the reason for the lack of needs of rice crop in this
land to fertilization with phosphate fertilizers in compared to the needs of same crop if planted
with dry methods (Naimi, 1989). On the other hand in submerged soils, the fungi including
Trichoderma , Aspergillus, Fusarium and Pencillium can analysed proteins and amino acids and
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release large quantities of ammonia which play asignificant role in the process of nitrification
(Clay & Clapp, 1990).
This diversity in land use should achieve higher productivity and improve achemical ,
physical and biological properties of soil ,so to verify that, these properties should be subjected
to examination and comparison, therefore this study should aimed the following:
1 - Estimate the numbers of nitrifying bacteria (Nitrobacter, Nitrosomonas) of soils differ in the
system of management.
2 - Comparision some of the isolated fungi from these soils in their ability to dissolve phosphate
compounds and the best incubation period to reach asignificant dissolving of the phosphate.
Materials and methods :
The studying area :The current studying soils were included four different land uses for a period exceeds five
years for each of these uses, the study has been carried out to identify the effect of these uses in
the different characteristics of these soils.
The search area is one of the richest areas in the cultivation of rice, as it stretches to the
borders of agricultural exploitation of AL-Najaf Sea, as well as cultivation of mongbean and
vegetables, a palm orchard as well as untapped agricultural areas (fallow).
soil samples were taken from rice, mongbean , orchard and fallow soils for chemical , Physical
and
analysis
for
the
three
periods
of
the
study,
as
follows:
1 - The begining of season.
2 - Mid-season.
3 - The end of the season.
24 soil samples were studied in the search region at a rate of two riplicates for each samples due
to the type of analysis.
Laboratory procedures:Soil preparation
The samples were collected in nylon bags and dried in the laboratory of departement, then
grounded, passed through asieves (diameter 2 mm), kept for Physical analysis, chemical and
learned for each site on sampling of the three periods, while the samples for biological analysis
were taken directly and saved directly into the refrigerator to ensure that no contamination and
no additional growth of microorganisms.
Physical and chemical characteristics:1 - Distribution of the soil texture - was estimated by way hydrometer (Day 1965).
2 - The degree of soil reaction (pH): - measured for the saturated paste extract in the method
described in (Richard 1954).
3 - The degree of electrical conductivity (ECe): - estimated to saturated paste extract by the
method described in (Richard, 1954).
4 -Available phosphorus: - It has been estimated in a method (Olsen & Cole, 1954) using a
spectrophotometer).
5 - Nitrate and nitrite: - according to the method described by (Bremner & Keeney, 1965).
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RESEARCHES of THE FIRST INTERNATIONAL CONFERENCE
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Biological Analysis
Soil samples were taken from the surface - as shown for each type of analysis in the tables
and appendices for each analysis.
Nitrifying Bacterial Count .
The method of most probability number (MPN) adopted and described by (Alexander &
Clark, 1965) in the counting of bacterial nitrification by Nitrosomonas, Nitrobacter and using the
culture media mentioned components in (Appendix 1) by incubation the tubes which containing
the culture media at a temperature of 28 C ° for a period of four weeks and detection for nitrate
using the detector Griss-losvay where is the formation of purple- red color, at adding
reagent,mean positive indicator for the presence of nitrate.
The ability of isolated fungi in dissolving the phosphorus.
Martin media used for this purpose, mentioned in (Appendix 2) with the addition of a drop of
Rose Bengal , preparationatwo sterilizing solutions consist of 10% of each of the CaCl 2,
K2HPO4. Where took 5 ml of solution I and 10 ml of solution II and added to the culture media
after mixing together mixing together and adding to petridish in order to harden ,then planted
with isolated fungi on the medium using attaping and incubated at a temperature of 28 C ° and
for (120 , 96,72,48,24) hours and argued on their ability to dissolve the phosphorus through the
transperancy haloformation which sourrounds the funji colon.
Results and Discussion
Chemical and Physical properties:- The results of table(1) were showed that:1- The distribution of soils texture volumes (soil texture of rice and mongbean)
were Silty
Clay Loam.
2- The rate of the degree of soil reaction (pH) for rice and mongbean were 7.49, 7.45,
respectively.
3- The rate of degree of Electrical Conductivity (ECe) for the soils of rice and mongbean were
2.5, 2.8, dSm-1 respectively.
4- The rate of available phosphorus for rice and mongbean were 14.9, 11.4 m g.kg-1,
respectively.
5- The rate of nitrate and nitrite for the soils of rice and mongbean were 0.04, 0.042, gm.kg-1
respectively.
Nitrifying bacterial count:The required samples were taken to account the nitrification bacteria in surface layers for
each of the studied sites (P1, P2, P3, P4) and through the cropping periods(before ,mid and after
cropping), and accounting their numbers (using MPN )in different land uses.
The most probable number of ammonium-oxidizing bacteria:- Nitrosomonas
The Figure (1) Shown a clear effect of land use in the numbers of Nitrosomonas bacteria ,
that rates ranged of three studying periods 5.57,4.55 , 2.48, 1.1 colony forming unit. gm -1 in the
surface horizon soils of rice, mongbean, orchard and fallow respectively Table (3).
When comparing the numbers of these bacteria between different land uses for all studying
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Nitrosomonas
The logarithm of the number of bacteria Netrosomonas
(the logarithm of colony formation units gm -1 - dry soil)
periods were observed that their numbers were increasing during the the planting (Figure 1), 1t
have increased significantly in the rice soils during amid- cultivating period inspite of immersion
conditions and the limited availability of oxygen, this is consistent with what referred by (Bock
et al, 1989,) in addition to availability of the substrate of these bacteria in the reduction layer
which occures from the decomposition of organic matter, also the mongbean soils were showed
significant superiority in the numbers of these bacteriadue to asame reasons . While the orchard
soils were given a notable effect on the numbers of Nitrosomonas bacteria, while the fallow
soils did not give any significant effect on those numbers between,three.periods.
When comparing the effect of different land uses on the numbers of Nitrosomonas
bacteria,noted that the rice , mongbean and orchard soils gave significant effect in increasing the
number of these bacteria at the level of significance( P <0.05 ), while the fallow soils were not
given any significant effects, when comparing their bacterial numbers among land uses. On the
other hand, the results show that the lowest density for the numbers of Nitrosomonas bacteria,
were in the fallow soils, especially in after cultivation stage, as those numbers did not exceed
above one colony formation unit. gm -1 dry soil, this is consistent with what (Palojarvi et al,
1997) referred that there are significant differences in the content of the cultivated soils of
nitrification bacteria when compared with the uncultivated soils.
7
L.S.D.0.05=0.239
6
L.S.D.0.05=0.134
5
4
3
2
1
0
1
Rice
2
Mongbean
3
Palm orcard
Fallo4
Figuar(1) The most probable number of nitrifying bacteria Nitrosomonas in
land use
studied soils during
the cultivation periods
Nitrosomonas
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The logarithm of the number of bacteria
Nitrobacter (the logarithm of colony formation
units gm -1 . dry
soil)
Nitrobacter
The most probable number of nitrite-oxidizing bacteria, Nitrobacter.
Figure (2)showed the numbers of Nitrobacter bacteria in the second stage of the nitrification
process were less than Nitrosomonas bacteria, where their rates in the surface horizons of rice,
mongbean , orchard and fallow soils for cultivation stages 3.3, 2.4, 1.9, 0.81 colony forming
unit. gr -1and the respectively, table (3), it was noted that the numbers of Nitrobacter were
increased in mongbean soils significantly, this is due to availability of substrate to these bacteria
which provides nitrite NO2 resulting from the oxidation of ammonium (Tappe et al, 1999), also
the rice soils were showed asignificant increasing in those numbers, although it have alower
rates of substrate, while fallow soils did not show any increase in theose numbers.
When comparing the effect of different land uses in the numbers of Nitrobacter bacteria ,noted
that the rice , mongbean and orchard soils gave significant effect in increasing the number of
these bacteria at the level of significance ( P <0.05 ), while during stage cultivation for the rice
and mongbean soils were shown significant effect in the numbers of Nitrobacter, while the
orchard and fallow soils did not show any significant effect of its numbers when compared
between the three periods of cultivation, this is consistent with what indicated by (Briones et al,
2002).
4
L.S.D.0.05=0.180
3.5
L.S.D.0.05=0.111
3
2.5
2
1.5
1
0.5
0
1
Rice
2
Mongbean
3
Palm grove
Fallow4
land use
Nitrobacter
Figuar(2) The most probable number of nitrifying bacteria Nitrobacter in studied
soils during the cultivation periods
The ability of isolated fungi in dissolving the phosphorus:
on Martin medium:The figures (3,4) were showed that the dissolving diameters that surrounding the developing
funji (A. niger, T. harzianum., A. Terrus, F. oxysporum, P.globosum) on Martin medium which
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isolated from rice soils after 120 hours were 4.2, 4, 3 , 1.7, 1.5 mm, respectively, and showed
that the A. niger which isolated from rice soils were showed a significant superiority in its
ability to dissolving phosphates in compare with other fungi table (3), while the fungi A. niger.,
T. harzianum that Isolated from mongbean soils showed a significant superiority in its ability to
dissolving phosphates in compare with other fungi table(3) , were showed that the dissolving
diameters that surrounding the developing fungi A. niger, T. harzianum., A. Terrus, F.
oxysporum, P.globosum after 120 hours were 4.5, 4.3,3.1, 1.5 , 1.3 mm, respectively, as it was
found that there are a certainly, statistical differences at the level of significance (P <0.05) in the
dissolving diameters that surrounding the colonies of these fungi on different incubation periods
up to 120 hours. The fungus P.globosum has shown the low ability to dissolve phosphorus for
both soils. It also showed that the duration of 120 hours of incubation gave a significant effect
on the ability of isolated fungi from rice and mongbean soils on dissolving phosphorus Table (4).
4.5
Incubation period (hours)
4
Between land uses
L.S.D.0.05 = 1.169
Dissolving diameter (mm)
3.5
Between plant period
L.S.D 0.05 =1.845
24
48
72
96
120
3
2.5
2
1.5
1
0.5
0
1
Aspergillus niger
2
Aspergillus tirice
3
Trichoderma
4
Pencillium
5
Fusarium
Martin
Figure (3)
the ffect of different species of isolated fungi from rice soils in dissolving
phosphorus on Martin media
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5
4.5
Between land uses
L.S.D.0.05 =1.149
Between plant periods
L.S.D 0.05 =2.168
Dissolving diameter (mm)
4
24
48
72
96
120
3.5
3
2.5
2
1.5
1
0.5
0
Aspergillus
niger
1
Aspergillus
tirice
2
3
Trichoderma
4Pencillium
5
Fusarium
Martin
Figure (4) the ffect of different species of isolated fungi from mongbean soils in
dissolving phosphorus on Martin media
Appendix (1) the components of the medium Nitrate-calicium carbonate for the
development of bacteria Nitrobacter, and the medium Ammonium-bicarbonate For the
development of bacteria Nitrosomonas
Nitrosomonas Nitrobacter
components
(NH4)2SO4
K2HPO4
NaCl
MgSO4.7H2O
FeSO4.7H2O
CaCO3
Weight
gm.00
gm00.
gm.00
gm
.0.0
gm.00
gm500
D.W
ml00...
components
KNO2
K2HPO4
NaCl
MgSO4.7H2O
FeSO4.7H2O
CaCO3
CaCl2
D.W
Weight
gm .0.0
gm00.
gm.00
gm.00
gm.0.0
gm00.
gm.00
ml00...
Appendix (2) the components of Martin medium-tested the ability of fungi to dissolve
phosphorus
Weight
Glucose
Pepton
K2HPO4
MgSO4.7H2O
Streptomycin
Agar
Rose bengal
D.W
components
gm
0.
gm0
gm0
gm.00
gm0.
gm00
gm
.0.00
ml
00...
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