ABSTRACT provides a valuable source of protein and thereby

sustaining the nutritional balances of low-income

Rhizobia are nitrogen-fixing diazo-trophic bacteria
populations (Appunu et al., 2009). It contains about
that grow inside legume root nodules and fix
40 % protein (Bhangu and Virk., 2019). Soybean
atmospheric nitrogen. A series of studies were
products are cholesterol free, high in calcium,
performed in Soil Microbiology Laboratory in Soil
phosphorous and fiber, and have one of the lowest
Science Division, Bangladesh Agricultural Research
levels of saturated fat (BIDCO., 2005).
Institute (BARI) and a field study was performed at
Subarnachar, Noakhali in 2022 with the objectives to
Rhizobia are soil bacteria that are gram negative,
isolate of potential rhizobia species from different
non-pathogenic, and helpful. Frank developed the
areas of Noakhali, Bangladesh. Then characterize
term rhizobia in 1889 because they produce nodules
isolated stains, produce bio-fertilizer and field
on the roots and stems of leguminous plants. (Spaink
performance of those effective bacteria on soybean.
et al., 2012). Rhizobium generally enters the root
For isolating Rhizobium bacteria, root nodule
hairs, multiplies there, and produces nodules because
samples were collected from 50 different fields
it is resistant to a wide range of temperatures (Nehra
location of Noakhali, Bangladesh using a global
et al., 2007). Rhizobia convert free atmospheric
positioning system (GPS) record along with crop
nitrogen into useable forms of nitrogenous
history. The characters of 10 bacterial isolates along
compounds and feed them to the host legume
with one reference strain were studied following the
through a symbiotic connection, while the bacteria
standard microbiological and biochemical methods.
profit from the host plants' carbon sources and other
The cells of Rhizobium isolates were examined under
nutrients (Mcneill and Unkovich., 2007).
stereo-microscope and found that the cells were rod
shape, motile and gram-negative bacteria. The
A biofertilizer is a product which contains live
biochemical characters such as Congo red, Gram
microorganisms that colonize the rhizosphere, or the
staining were investigated. The isolates were
inside of plants, and boost plant development by
observed to lack the ability to absorb Congo red from
increasing the availability of nutrients to the host
YEMA medium. Seedlings were grown in glass
plant when given to seeds, plants, or soil. (Vessey.,
tubes to check the infection ability of Rhizobium and
2003). They increase soil fertility by fixing nitrogen
found nodule formation in seedling roots within 30-
from the atmosphere, solubilizing insoluble
50 days after inoculation. A field experiment was
phosphates, and generating plant growth-promoting
conducted at subarnachar, Noakhali and the
chemicals (Mazid and Khan., 2015). These
experiment was laid out (cv. BARI Soybean 6) in
biofertilizers have been pushed to take use of the
Randomized Complete Block Design (RCBD) with
naturally occurring biological system of nutrient
three replications. Rhizobium biofertilizer inoculated
mobilization, which vastly improves soil fertility
soybean plants exhibited better performance in
and, as a result, crop productivity (Pandey and
nodule numbers, nodule weights, root weight, shoot
Singh., 2012). Microorganisms play a critical role in
weight, 100 seeds weights, pod length, pod plant -1,
the agricultural eco-system by fixing, solubilizing,
plant height, stover yield and seed yield than non-
mobilizing, and recycling nutrients. These bacteria
inoculated plants, indicating that all Rhizobium sp.
are naturally found in soils, although their numbers
effectively enhanced nodulation and growth
are frequently few. Rhizobium species linked with
parameters on soybean plant than non-inoculated
leguminous plant root nodule symbiosomes
plants revealing the positive impact of Rhizobium sp.
endophytically fix molecular nitrogen (N 2) through
on yield parameters of soybean in the Noakhali area.
nitrogenase enzyme-catalyzed processes (Flores-
Tinoco et al., 2020; Jangir et al., 2020). Legumes
INTRODUCTION absorb biologically fixed nitrogen to provide
nutritional needs, especially when nitrogen is scarce
Soybeans [Glycine max (L.) Merrill] are one of the (Basu and Kumar., 2020).
most important food crops in the world. To produce
large soybean yields, a lot of nitrogen is required. Nitrogen is an important plant nutrition element since
With growing usage in food and industrial items, it is a necessary component of all amino acids and
soybean demand has expanded substantially nucleic acids. Despite the fact that 78.1 percent of the
throughout the world (Trostle., 2010). Growing per atmosphere includes N2 gas, plants cannot utilize it
capita meat consumption has increased demand in unless it is transformed into a useful form (Ferguson
addition to growing goods and soybean use as cow et al., 2010). Biological N2 fixation (BNF) is a free
feed (Trostle., 2010). The most cost-effective source source of nitrogen that resource-strapped farmers
of nitrogen for soybean is the biological fixation of may use to boost crop yields (Ladha et al., 2013). In
atmospheric N2 via a symbiotic connection between symbiotic legumes, the production of root nodules
the plant and soil bacteria, most of the genus requires intricate molecular communication between
Bradyrhizobium (Campo et al., 2009). Soybean the bean host and the rhizobial microsymbiont

1
(Oldroyd et al., 2011). Rhizobia differentiate into N2-
fixing bacteroids, which use the nitrogenase enzyme
to convert atmospheric N2 to ammonia (Terpolilli.,
2012). While many rhizobia infect roots by the
infection thread, some infect roots through "crack
entrance" or root epidermal cells (González-Sama et
al., 2004; Ardley et al., 2013; Bianco., 2014).
Chemical fertilizers can affect the microbial flora in
the soil and also add high acidity to the soil. As a
result, the pH of the soil changes, which can destroy
some important bacterial types. Due to their high
water solubility, these fertilizers can also be
dissolved into the groundwater without benefiting the
plant. Chemical fertilizers may also promote plant
diseases. The soil becomes harmed if chemical
fertilizers are applied often and continuously to the
same soil (Mishra et al., 2013). Chemical fertilizers
contain chemical substances whereas biofertilizers
contain living microbes and other organic matter,
which contain the essential nutrients required for the
growth of plants. Biofertilizers are beneficial for the
environment, are an excellent source of nutrients,
enhance soil texture, eliminate pathogenic
components that cause illness, and assist to maintain
soil fertility. Overall, biofertilizers are a good source
of the nutrients needed by plants to increase
production and produce healthier harvests.
Rhizobium is a common component of biological
fertilizers (Saeed et al., 2015). So, the objectives of
this study were (i) to isolate, identify and
characterize effective rhizobial strains collected from
soybean growing areas, (ii) to produce biofertilizers
by those effective strains (iii) to evaluate their
efficacy on growth and yield of soybean at field level
of soybean growing area of Noakhali region of
Bangladesh.
MATERIALS AND METHODS
 Collection and Isolation of Rhizobium from
nodules:
Nodules from soybean root (50 samples) were
collected from different fields of Subarnachar,
Noakhali, Bangladesh using global positioning
system (GPS) record along with crop history (Fig. 1,
Table 1). The collected nodule samples cover the
areas of Char Wabda, Char Aminul Haque, Vatior
Tek, Char Kaji Mokhlec, Char Jubli, East Shoulokia,
Char Jabber, East Char Jabber, 3 No Char Wabda,
Char Aman ullah, Char kata Bunia, Char Bata,
Tajpur, Middle Char Bata and North Koccopia
villages of Subarnachar, Noakhali District (Table 1).
The nodules samples which collected from soybean
plants in different locations taking the information of
farmer’s name, father’s name, and address with
mentioning mobile number, and existing crop
information. The major cropping pattern of these
locations are Fellow-Rice-Soybean and the existing
2
major crop is soybean. The land type of maximum
areas was medium lowland (Table 1).
Storage of collected Soybean root nodules:
When nodules were collected from soybean root
mature soybean plants were selected for nodule
collection and the nodules were separated from those
plants to count their numbers. The counted nodules
are then taken in cotton silled tubes containing silica
gel and then preserved in the refrigerator at 4oC
temperature.
 YEMA media preparation
Yeast Extract Mannitol Agar (YEMA) media were
prepared for bacteria culture. Media compositions
were properly measured and taken in distilled water
containing a conical flask and then kept in a
magnetic stirrer (a magnetic stirrer is a device widely
used in laboratories containing of a rotating magnet
or a stationary electromagnet to create a rotating
magnetic field). The pH of the solution was also
detected by using a pH meter and needed to maintain
the range of pH 6.5-7. To increase the range of pH
adding NaOH and to decrease the range of pH adding
diluted H2SO4 should be needed. Then the media
suspension needed to be autoclaved at 1210C 20 min
at 15 PSI. At the final stage, media suspension was
poured into petridish by maintaining asceptic
condition and the media was prepared after 20-30
minutes.
 Isolation of Rhizobium from nodules:
Collected nodules were then crushed by maintaining
highly aseptic conditions inside the laminar air flow
at the highly restricted microbiology lab of
Bangladesh Agricultural Research Institute (BARI).
The following steps should be maintained during the
crushing of the nodules.
1) Nodules are soaked in sterilized
distilled water for 2-3 hours before
crushing.
2) Wash the nodules 2 to 3 times by
sterilizing distilled water in an
autoclaved petri dish.
3) Soak the nodules in ethanol (just 5
seconds).
4) 6 to 7 minutes soak in calcium
hypochloride.
5) Ten times wash with sterilized
distilled water to remove traces of
calcium hypochlorite.
6) Little amount of distilled water is
taken in petridish with a
micropipette.
7) Glass rod burns in the fire and then
crushes the nodules.
 8) 0.1 ml of nodules suspension was
taken in a yeast extract mannitol
agar (YEMA) plate by using a
micropipette and then spread into
the whole plate with a spreader.
9) YEM agar plates have been
incubated at 300C for 2-3 days for
specific bacterial growth.
 Culture and subculture of Rhizobium from
mother culture.
YEMA plate growing bacteria (mother culture) took
by a sterile loop to sub-culture 1 plate and
after two times sequentially sub-cultured, a single
colony of Rhizobium bacteria was obtained from 2nd
time successfully subculture plate. The isolated
strains were purified by the streak plate method.
 Slunt Preparation
Slunt was prepared with the corresponding plating
media technique without adding congo red dye. After
mixing slunt composition with sterile distilled water
it burned over bunsen burner until boiled for better
mixing. Then, the cooled suspension was poured into
slunt tubes and then autoclaved it. The autoclaved
slunt tubes were put in laminar air flow till the
suspension turned as slunt form.
 Purification and preservation of the isolated
strains:
A final selection of the bacterial strains was done by
comparing morphological (colony) characteristics
and transferring them to agar slants. In this process,
Rhizobium bacteria took from sub culture purified
plate by using a sterile loop and then put into in slunt
media by maintaining aseptic conditions. The
cultured slunt was then incubated for 3-4 days at
280C±2 for bacterial growth. The purified slants
strains were separated and preserved as stock culture
in a refrigerator at 40C. In addition, culture mixed
with 50% (v/v) glycerol was kept in -20 0C for short
time and -800C for long time preservation.
 Preparation of broth cultures
Broth was also prepared as like plating media
preparation. Congo red or ager didn’t add in broth
culture. Congo red was caused for radish media
color and ager made the media sticky as well as
solid. The Rhizobium strains were initially sub-
cultured on plates from the test tube stock to create
the broth cultures. Erlenmeyer flasks containing
yeast mannitol broth medium were sterilized for 20
minutes at 121°C and 15 PSI of steam. A little
amount of Rhizobium culture was aseptically
transferred from the plates to the liquid medium in
the flasks with the use of an inoculating needle after
the media had been cooled. The flasks were then
3
placed on an electric shaker and shaken at 150 rpm
speed to enhance rhizobial growth. Depending on the
growth rate (fast or slow growing), the broth culture
was ready after 2–7 days or when the media in the
flasks exhibited dense development. It is known as
"mother culture." After being examined for purity
and healthy development, the culture was moved
from tubes to big flasks with sterile liquid media for
2 to 7 days. The rhizobial broth was raised in large
flasks on a horizontal shaker using YEM medium.
The broth was examined considering healthy growth
and if contamination is found it will be discarded.
We utilized broths when viable cell counts were
found more than 109/ml.
Morphological characterization of Rhizobium
isolates:
Motility test method:
Bacterial morphology was detected by microscope.
The stereo microscope can be used to view motility
in a wet mount by reducing the amount of light that
passes through the specimen. The small amount (20
µl) of bacteria culture were taken in slide and put
small amount safranine to fixed by coverslip. Then
the slide was seen under stereo microscope. This is
the most common method of observing motility. It is
quick and inexpensive method.
Biochemical characterization of Rhizobium
isolates:
Gram staining test procedure:
1. Take a clean, grease free slide.
2. Prepare the smear of suspension on the
clean slide with a loopful of sample.
3. Air dry and heat fix
4. Crystal violet was poured and kept for about
30 seconds to 1 minutes and rinse with
water.
5. Flood the gram’s iodine for 1 minute and
wash with water.
6. Then, wash with 95% alcohol or acetone for
about 10-20 seconds and rinse with water.
7. Add safranin for about 1 minute and wash
with water.
8. Air dry, blot dry and observe under stereo
microscope.
Congo red test method:
Congo red was used in Yeast Extract Mannitol Agar
(YEMA) media preparation for bacterial test. Media
compositions with 10 ml congo red for 1 liter were
properly measured and taken in distilled water
containing conical flask. The conical flask then kept
in a magnetic stirrer for proper mixing of the
compositions with congo red. The pH of the solution
was also detected by using a pH meter and needed to
maintain the range of pH 6.5-7. To increase the range
of pH adding NaOH and to decrease the range of pH
adding diluted H2SO4 should be used. Then the
media suspension needed to be autoclaved. At the
final stage, media suspension was poured into
petridish by maintaining ascetic condition and the
media were prepared after 20-30 minutes. Then the
bacteria grow in these media by maintaining plate
growing methods.
 Seedling infectivity test:
In order to assess Rhizobium's capacity to infect
soybean seedling roots, it was cultivated in yeast
extract mannitol (YEM) broth culture at 30°C for 48
hours. After that, one to two seedlings were placed in
a YEM agar slant tube (60 ml glass test tube,
diameter 3 cm) with 0.1 ml of this broth culture. The
tube was then kept at room temperature for three to
five weeks while aseptic conditions were kept in
place, and the nitrogen-free nutrient solution was
given to YEM agar slant tubes as a source of
nutrients twice in a week.
 Inoculum preparation and biofertilizer
production
A final selection of the bacterial strains was used for
inoculum preparation. Peat soil was used as carrier
materials. The bacterial strains were re-cultured in
YEM broth for 48-72 hours for sufficient growth and
20 ml broth of bacterial culture was injected in peat
soil (100 g sterilized peat bag) for preparation of
biofertilizer production. Bacterial biofertilizer
packets were incubated at ambient temperature
(28±2°C) for 3-5 days for optimum growth. Finally,
these biofertilizer packets were used for soybean
cultivation.
 Processing of inoculant materials
Peat was used as carrier material. In Bangladesh,
there was a great reserve of peat soil in the greater
Faridpur, Khulna and Sylhet districts. It was proved
that there was a great possibility to using them for
the commercial production of peat based inoculum
(Khanam et al., 1996). After collection, the peat and
the soil materials were first air-dried thoroughly. The
air-dried materials were ground to a fine powder
using a laboratory grinding mill so as to pass through
80 mesh sieve. Powdered peat was neutralized with
5% CaCO3 to raise the pH to 6.8. A weighed amount
of inoculant materials was then taken in cotton
plugged erlenmeyer flasks and sterilized in an
autoclave at 121oC and 15 psi of steam pressure for
40 minutes. Small 0.038 mm dia polyethylene bags
were sterilized with alcohol. The sterilized materials
were then transferred into the polyethylene bags and
sealed carefully to avoid any contamination.
 Inoculation, incubation, and storage
High-count broth cultures were injected into the
inoculant so as to attain at 40% moisture-holding
4
capacity materials were contained in the
polyethylene bags aseptically with the help of a
sterilized syringe in the inoculation chamber and the
puncture was sealed by adhesive tape. The contents
were mixed by rolling in hand, incubated at 28 oC for
two weeks, and then stored at 4oC. Finally, these
biofertilizer packets were used for soybean
production.
 Quality checking
After an incubation period, the inoculum quality was
checked by counting the viable rhizobia following
the drop plate method of Miles and Misra (1938)
which provides several dilutions and purely surface
colonies on one plate. The resulting product had at
least 300 million (3 x 108) rhizobia per g of peat
(Burton and Roberts., 1967). The following were the
relevant clauses for rhizobial cultures.
1. The inoculants were carrier-based.
2. The inoculants were containing a minimum
of 109 viable cells of Rhizobium/g of the
carrier on a dry-mass basis within 15 days
of manufacture and 107 within 15 days
before the expiry date marked on the packet
when the inoculants were stored at 25 o to
30oC.
3. The inoculants had a maximum period of 6
months’ expiry from the date of their
manufacture.
4.  There was no contamination of the inoculants
with any other bacteria.
5. The pH of the inoculants was between 6.0 -
7.5.
6. The inoculants were show effective
nodulation on all those species/cultivars
listed on the packet before the expiry date.
7. The carrier material was neutralized with
calcium carbonate and sterilized.
8. The manufacturer shall control the quality
of broth and maintained records of the test.
9. The inoculants were packed in 50 to 75-
micron low-density polyethylene packet or
any other suitable container.
10. Each packet was marked legibly to give the
following information:
(a) Name of the product, specifically as
Rhizobium inoculant (b) Leguminous crop
 for which intended, (c) Name and address of
the manufacturer, (d) Type of the carrier, (e)
Batch or code number, (f) Date of
manufacture, (g) Date of expiry, (h) Storage
instructions.
 Methods of using biofertilizers
Biofertilizers can be applied to the seed or directly
into the seedbed before sowing. Different methods of
inoculant application with minor variations have
been reported in the literature (Brockwell et al.,
1995). Many of the methods are quoted by Burton
(1979) and some are the U.S. patented. Generally,
peat-based inoculants were mixed with minimum
amount of water to form a slurry (sugar or gum
arabic) and seeds were added to the slurry so as to
uniformly coat the seeds with the inoculants. The
seeds were dried in shade and sown immediately.
Liquid inoculants were also used in some countries.
This method was very effective when applied by a
gravity flow applicator or a sprayer. The method
worked well with small and large experiments, and
also for commercial sowing.
Field experiment:
The experiment was conducted at Subornochor,
Noakhali from 10th January to 24th April, 2022. The
experiment was carried out in Robi season because
winter is the best time period for soybean growth.
The experimental site is located about North latitude
22.74oN and East longitude 90.61oE. The land
selected for the experiment was medium high land.
The experimental details for this trial are given
below:
Field used
A farmer’s field was selected for the research trial
at Tereja pul, Subarnachar, Noakhali. The name
of the field owner was Abu Bakar. Clods were
broken, weeds and unwanted materials were
removed. Initial soil samples from the experimental
fields, taken at a depth of 0–15 cm, were collected
and subjected to normal procedures for analysis
(Table 05).
Fertilization
Chemical fertilizers were applied according to the
treatments. In the form of TSP, MoP, gypsum, zinc
sulphate, and boric acid, respectively, phosphorus,
potassium, sulphur, zinc, and boron (100-22-42-40-
5-1 kg N-P-K-S-Zn-B kg ha-1) were utilized. When
preparing the land, P, K, S, Zn, and B were all used.
Crop
The test crop was soybean and the variety was BARI
soybean-6. Bangladesh Agricultural Research
Institute (BARI) has developed and released this
variety for cultivation in the famer’s fields. This is
considered as a very popular high yielding variety. It
usually takes 90-100 days to complete its life cycle
5
and is successfully grown in most soils of soybean
growing areas of Bangladesh.
Experimental design
The experiment was laid out in a Randomized
Complete Block Design (RCBD) with 12 treatments
and 3 replications. Ten Rhizobium isolates, with a
reference strains (Rhizobium sp BARI RGm901)
obtained from Soil Science Division, Bangladesh
Agricultural Research Institute (BARI) and a control
treatment were placed in each replication. The
recommended plot size was 3m x 2m and plant
spacing was 30 cm (row to row) x 10 cm (line to
line).
Treatments combination are as follows:
T1: Rhizobium sp 01
T2: Rhizobium sp 02
T3: Rhizobium sp 03
T4: Rhizobium sp 04
T5: Rhizobium sp 05
T6: Rhizobium sp 06
T7: Rhizobium sp 07
T8: Rhizobium sp 08
T9: Rhizobium sp 09
T10: Rhizobium sp 10
T11: Rhizobium sp BARI
RGm901
T12: Control
Seed sterilization
Seeds were mixed with 5% methyl alcohol and
shaked properly. Then the seeds were drained and
washed out with sterile water. The seeds were settle
down for 5 minutes with 0.2% mercuric chloride and
drained off. Finally the seeds were washed with
sterile water by 6 times and dried in the sun.
Inoculation of seeds with Rhizobium
In field tests, the carrier-based (peat) Rhizobium
inoculum was employed. All the ten Rhizobium
strains were used in inoculum. At a rate of 1.5 kg ha -
1
, peat-based rhizobial inoculum containing 10 9 cells
g-1 inoculum was employed. Before planting, seeds
and inoculum were well mixed (20:1 ratio). 75 kg of
seeds per hectare were utilized.
Sowing of seeds
Soybean seeds were sown in line. Field was irrigated
upto satuaration to allow the soil and inoculum to
settle down in the field. After germination of seeds,
unhealthy plants were uprooted carefully.
Intercultural operation
Field was watered whenever necessary to
maintain field moisture condition. Intercultural
operations (weeding, bug management and
mulching) were done when necessary to ensure
the normal growth of the crop. The field was
observed regularly to record any change of plant
growth. No insecticide was used during the
growth period. The plants were free from insects
and diseases. Nodules were collected at the 50%
blooming stage by carefully removing five sample
plants chosen at random from each unit plot. After
being extracted from the roots, nodules were
counted, oven-dried, and
Harvesting
At maturity, information on yield and yield
components was recorded. The soybean was
harvested on 25th April, 2022. The plants were
carefully uprooted with minimum disturbance of
roots. The roots were washed with tap water.
Roots and shoots were separated with the help of
a sharp scissors and were preserved after
necessary processing for determining root mass
and percent root colonization. Shoots were dried
in an oven for 72 hours at 700c.
Statistical analysis
The data was entered and collated in a MS Excel
spreadsheet and analyzed using Statistix 10 TM
software. A RCBD design was used to statistically
examine all the data. Analysis of variance (ANOVA)
was used to examine the effects of the treatments on
the variables that were assessed, and the least
significant difference (LSD) multiple range test,
which was computed at the 5% level of probability
(𝑃 ≤ 0.05), was used to compare the treatment means
(Gomez and Gomez, 1984).
RESULT AND DISCUSSION
This chapter deals with the presentation of
experimental results along with their interpretation
and discussion in respect of the response of
Rhizobium inoculant to soybean.
Morphology and biochemical traits of Rhizobium
isolates
A total of 50 bacteria (isolates from soybean root
nodules) were collected from Subarnachar, Noakhali
area of Bangladesh (Fig. 01 and Table: 01). The
bacteria were characterized following the standard
microbiological and biochemical methods. As
standard microbiological methods, the morphology
and gram staining were investigated. The
biochemical characteristics such as congo red, gram
staining was investigated. The cells of Rhizobium
isolates were examined under stereo microscope and
found that the cells were rod shape and motile. The
isolates absorbed counter stain as they were gram
negative bacteria. Aung., (2020) stated that
Rhizobium was gram negative, rod shaped and motile
which was in line with the present study. The isolates
were observed to lack the ability to absorb congo red
from YEMA medium containing this dye where
colonies were colorless white or very faintly pink
6
colonies which is in agreement with the findings of
Mondal et al., (2016). Congo red was thought to
form colored colloidal complex with ions on the cell
surface, the colonies absorbed little dye and
remained colorless or became slightly pink after 2
days of incubation which proved all colonies
belonged to Rhizobium. By determining bacterial
plate growth activity and study of overall
morphological characteristics ten well-characterized
bacteria were selected for further studies.
Findings of motility test:
The motility test declared that all of the bacteria
were gram-negative because they contain flagella
which must be needed for their movement.
Gram staining test findings:
In case of gram negative bacteria, the cell wall also
takes up the crystal violet-iodine complex but due to
the thin layer of peptidoglycan and a thick outer
layer which is formed of lipids, crystal violet-iodine
complex gets washed off. When they are exposed to
alcohol, the decolorizer dissolves the lipids in the
cell walls, which allows the crystal violet-iodine
complex to leach out of the cells. Then when again
stained with safranin, they take the stain and appear
pink color. The bacteria in the sample were pink
when the stain reacted with them. Isolated ten
bacterial strains were showed gram negative under
stereo-microscope.
Congo red test findings:
The purity of isolated Rhizobium sp. was tested
using the congo red method since various strains of
the bacteria varied in their capacity to hold onto the
dye. Ten bacterial isolates were grown on yeast
extract mannitol agar with congo red in the dark on
YEMA medium did not hold onto the dye. All of
these bacteria formed whitish color and didn’t
absorb congo red which indicates that they were
gram negative. For Rhizobia, yeast extract was a
good supply of easily available amino acids, vitamin
B complex, and auxiliary growth agent (Aneja.,
2007).
Seedling infectivity test
Infection ability was checked in a test tube culture of
soybean. Ten isolates of effective Rhizobium (strain
sp 01 to strain sp 10 of soybean) were used for the
infection ability test and all bacteria successfully
produced nodules in soybean roots in both test tubes
(Table 7, Fig. 24).
Olivares et al., (1980) found that the infectiveness of
Rhizobium meliloti and Medicago sativa formed
many nodules during the infectvity test using a
method that involves mixing tetracycline or without
tetracycline.
Rhizobium enhances nodulation of soybean:
At 50% flowering stage of soybean, the number of
nodule was recorded higher in inoculated Rhizobium
bacteria compared to the non-inoculated control. The
highest number of nodules (31.3 plant-1), was
recorded with treatment T7 which was similar to
treatment T2 but differ from all other treatments. The
second highest number of nodules (29.7 plant -1),
were recorded with treatment T 2 but differ with all
other treatments. The lowest number of nodules (13.3
plant-1), were recorded with treatment T 12.
Bradyrhizobium japonicum (strains B157) inoculated
soybean plant recorded height nodulation 45 pieces
per root compare to non-inoculated soybean
(Vorobey et al., 2022). It might be due to the
capacity of Rhizobium strains with the surrounding
favourable environment.
Alam et al., (2015) found that numbers of nodules
were higher in soybean plants inoculated with
Rhizobium sp. BARIRGm901 than in non-inoculated
plants. Rhizobium inoculated plants of all genotypes
yielded significantly (p ≤ 0.05) higher nodule
numbers and weights than non-inoculated plants in
both years (2010 and 2011) of the experiment. Alam
et al., (2015) also mention that BARI soybean6
inoculated with Rhizobium sp. BARIRGm901
produced significantly (p ≤ 0.05) higher nodule
numbers than other inoculated and non-inoculated
genotypes across all the treatments in 2010 and 2011,
with the exception of MTD10 + R (“+R” indicates a
genotype inoculated with Rhizobium sp.
BARIRGm901) and Shohag + R. In each year, the
nodule numbers on all soybean genotypes correlated
positively with stover yield (2010: r = 0.691; 2011: r
= 0.796) and seed yield (2010: r = 0.743; 2011: r =
0.810) (Alam et al., 2015).
Sultana et al., (2014) found that different rhizobial
strains significantly influenced the number of
nodules plant-1 in soybean. The highest number of
nodules plant-1 (27.58) was observed in plants
inoculated with Rhizobium strain-J43 which was
significantly higher than those of other treatments.
Strain-102 and mixed strains (J43 and 102)
inoculated plants that produced identical number of
nodules plant-1. On the other hand, the lowest number
of nodules plant-1 (13.83) was counted from control.
Nodulation might be due to the effective symbiosis
between legume and rhizobial strains. Thus, the
findings of this experiment are in agreement with
those of Slattery et al., (2004) who reported that
Rhizobium leguminosarum bv viciae is responsible
for the effective nodulation of faba bean, lentils and
field pea. Egamberdiyeva et al., (2004) investigated
the effect of inoculation with Bradyrhizobium
japonicum S-2492 on soybean nodulation. They
observed positive effects on nodule numbers.
Kavathiya and Pandey., (2000) found 69 nodules
plant-1 by inoculating mungbean seed with
Rhizobium. Different levels of N also showed
significant variation with respect to the numbers of
nodules plant-1. The highest and lowest number of
nodules plant-1 was 25.91 and 17.83 observed with
the treatment N15 and control which was identical
7
with N20. The interaction effect of different rhizobial
strains and N fertilizer on the number of nodules
plant-1 revealed that the highest and lowest number of
nodules plant-1 was 30.33 and 5.00 found in the plant
inoculated with rhizobial strain-J43×15 kg N ha-1 and
control treatment respectively. An increase in
nodules per plant due to the application of
inoculation in combination with nitrogen fertilizer
was reported by Rashid et al., (1999).
Hossain., (2018) found in Lentil (Lens culinaris
Medik.) that the highest number of nodules (10.29)
was produced in the plants treated with Rhizobium
strain 1, which was significantly different from the
other treatments. The second highest number of
nodules (7.96) was found in Rhizobium strain 2.
Mixed culture of Rhizobium strains 1 and 2 produced
the lower nodules (7.58) than single strain of
Rhizobium. Urea 50 kg N/ha produced a slightly
higher number of nodules (3.82) than control (3.14)
i.e. uninoculated treatment. The lowest nodule
number (3.14) was found in uninoculated treatment.
The maximum dry weight of nodule in gram was
obtained from Rhizobium strain 1 followed by mixed
culture of Rhizobium strains 1 and 2. The lowest
nodule dry weight (3.80 g) was observed in the
uninoculated treatment.
Correlations were examined the relationship between
nodule number with pod plant-1, nodule number with
stover yield and nodule number with seed yield of
soybean. The study revealed a significant and
positive correlation between nodule number with pod
plant-1 (Fig. 26), nodule number with stover yield
(Fig. 27) and nodule number with seed yield (Fig.
28) of soybean and followed the regression equation
y = -0.3246x + 64.564 (R² = 0.3702***), y = 0.0188x
+ 3.247 (R² = 0.0813**) & y = -0.0087x + 2 (R² =
0.396**) respectively. It indicates that the nodule
number helped to increase pod, stover and seed yield
of soybean.
Nodule weight increased by soybean inoculation:
The nodule weight was recorded higher in inoculated
Rhizobium bacteria compare to non-inoculate
control. The nodule weight was marked the highest
(0.66 g plant-1) with T11 treatment which was identical
with treatment T7 and differed with other treatment.
The second highest nodule weight (0.61g plant -1),
were recorded with treatment T 7 but differ with all
other treatments. The lowest nodule weight (0.19 g
plant-1), were recorded with both treatment T3 & T12.
Alam et al., (2015) reported that the weights of
nodules were higher in soybean plants inoculated
with Rhizobium sp. BARIRGm901 than in non-
inoculated plants. Rhizobium inoculated plants of all
genotypes yielded significantly (p ≤ 0.05) higher
nodule numbers and weights than non-inoculated
plants in both years (2010 and 2011) of the
experiment. Across all treatments, BARI soybean6 +
R (“+R” indicates a genotype inoculated with
Rhizobium sp. BARIRGm901) produced
significantly (p ≤ 0.05) higher nodule weight than
other inoculated and non-inoculated genotypes in
both years, with the exception of MTD10 + R and
Shohag + R. The dry weight of nodules of all
soybean genotypes were positively correlated with
stover yield (r = 0.727) and seed yield (r = 0.728)
only in 2011.
Cassol., (2017) found that Nodule dry weight was
significantly (P ≤ 0.05) affected by the interaction of
soybean variety and rhizobial strain. Soybean variety
Jalele produced significantly higher nodule dry
weight (0.20 g plant-1) when inoculated with B.
japonicum strain TAL 379 followed by variety Cheri
(0.19 g plant-1) with the same rhizobial strain. This is
in agreement with the report of Liu et al., (2011) who
observed that nodule dry weight was significantly
dependent on B. japonicum strain and soybean
variety interaction.
Sultana et al., (2014) found that the maximum and
minimum nodule dry weight was 0.19 g and 0.06 g
recorded from the plant inoculated with strain-J43
and non-inoculated plant. It is clear that there was no
statistical difference in nodule dry weight of plants
inoculated with strain102 and mixed strains (102 and
J43). Different rhizobial strains increase the
nodulation of the plant compared to control. Icgen.,
(2002) conducted a field experiment with five local
and seven standard strains of Rhizobium and the
maximum increase in nodule dry weight was found
in rhizobial infection compared to control in
soybean. Bhuiyan et al., (1998) stated that
inoculation with Rhizobium increased nodule dry
weight. When treated with different N levels 15 kg N
ha-1 showed the highest (0.154g) and control showed
the lowest (0.09g) nodule dry weight plant-1. The
results from the interaction effect of rhizobial strain
and N fertilizer showed the highest and lowest
nodule dry weight was 0.23g and 0.01g obtained
from a treatment combination of strain-J43 × 15 kg N
ha-1 and control. The rhizobial strain treatment in
conjunction with N level influenced the nodule dry
weight of the plant.
A correlation study was done to examine the
relationship of nodule weight with pod plant-1, nodule
weight with stover yield and nodule weight with seed
yield of soybean over unicoculated control. The
study revealed a significant and positive correlation
between nodule weight with pod plant -1 (Fig. 30),
nodule weight with stover yield (Fig. 31) and nodule
weight with seed yield (Fig. 32) of soybean and
followed the regression equation y = -0.4487x +
57.705
(R² = 0.0005***), y = 1.3773x + 3.1443 (R² =
0.3085**) & y = 0.1236x + 1.7365 (R² = 0.0154**)
respectively. It indicates that the nodule weight
helped to increase the seed yield of soybean.
8
Rhizobium contribution to shoot biomass of
soybean:
The shoot weight was recorded higher in inoculated
Rhizobium bacteria compareed to non-inoculate
control. The highest shoot weight (11.00 g plant -1)
was recorded with treatment T3 which was identical
with the treatment T5, T6, T7 & T9 but differed with
all other treatments. The second highest shoot weight
(10.80 g plant-1), were recorded with treatment T2
which was identical with treatment T9 but differ with
all other treatments. The lowest shoot weight (6.46
plant-1), were recorded with treatment T 12 which was
similar with treatment T11 but differ with all other
treatments.
Alam et al., (2015) found that soybean varieties and
advanced lines inoculated with Rhizobium sp.
BARIRGm901 produced greater shoot weights,
compared with non-inoculated controls in both years
of the experiment. BARI soybean6 + R (“+R”
indicates a genotype inoculated with Rhizobium sp.
BARIRGm901), BGM02026, and BGM02026 + R
produced significantly (p ≤ 0.05) greater shoot
biomass in 2010, but BARI soybean6 + R, BARI
soybean6, BGM02026, and BGM02026 + R showed
significantly (p ≤ 0.05) greater shoot biomass in 2011
compared with other treatments genotypes. All
soybean genotypes were positively correlated with
stover yield (2010: r = 0.970; 2011: r = 0.719) and
seed yield (2010: r = 0.668; 2011: r = 0.709).
Rhizobium influence to root biomass of soybean:
The root weight was recorded higher in inoculated
Rhizobium bacteria compare to non-inoculate
control. The highest root weight (1.82 g plant -1) was
recorded with treatments T6 that was identical with
treatments T4, T5 & T11 but differ with all other
treatments. The second highest root weight (1.77 g
plant-1) was recorded with treatments T4 that differ
with all other treatments. The lowest root weight
(1.05 g plant-1) was recorded with treatment T 2 that
was identical with treatment T10 but differ from all
other treatments.
Akter et al., (2019) found that root dry weight of
Mungbean was influenced significantly by the
application of phosphorus with Rhizobium. The
results of root dry weight at 30, 45, 60 and 75 DAS
have been found. The highest dry weight of root
(0.316, 0.451, 0.570 and 0.717 g) plant-1 was found
in 40 kg P ha-1 with Rhizobium inoculant and the
lowest dry weight of root (0.148, 0.208, 0.368 and
0.422 g) plant-1 was found in T1 (control) treatment.
Similar results were reported by Khan et al., (2017)
and Erman et al., (2009).
Alam et al., (2015) found that soybean varieties and
advanced lines inoculated with Rhizobium sp.
BARIRGm901 produced greater root weights
compared with non-inoculated controls in both years
of the experiment. Root biomasses were significantly
(p ≤ 0.05) higher in BARI soybean 6 + R (“+R”
indicates a genotype inoculated with Rhizobium sp.
BARIRGm901) and BGM02026 + R plants in 2010,
and in BARI soybean6 + R, BGM02026 + R,
MTD10 + R, and MTD10 plants in 2011, compared
with other treatments genotypes. Root biomass of all
soybean genotypes were positively correlated with
stover yield (2010: r = 0.983; 2011: r = 0.991) and
seed yield (2010: r = 0.771; 2011: r = 0.998).
Rhizobium contribution to plant height of
soybean:
The plant height was recorded higher in inoculated
Rhizobium bacteria compare to non-inoculate
control. The highest plant height (64.5cm) was
recorded with treatments T 10 which were identical
with treatment T3 & T7 but differ with all other
treatments. The second highest plant height (64.4cm)
were recorded with treatments T3 which were
identical with treatment T7 but differ with all other
treatments. The lowest plant height 47.7cm was
recorded with treatments T12.
Alam et al., (2015) found that soybean varieties and
advanced lines inoculated with Rhizobium sp.
BARIRGm901 increased plant height, compared
with non-inoculated controls in both years of the
experiment. Plant height was significantly (p ≤ 0.05)
increased in BARI soybean6 + R (“+R” indicates a
genotype inoculated with Rhizobium sp.
BARIRGm901) and BGM02026 + R plants in 2010,
and in BARI soybean6 + R, BARI soybean6, and
BGM02026 + R plants in 2011, compared with other
treatments genotypes. Plant heights, of all soybean
genotypes were positively correlated with stover
yield (2010: r = 0.987; 2011: r = 0.910) and seed
yield (2010: r = 0.902; 2011: r = 0.911).
Sultana et al., (2014) found that different rhizobial
strains and N interaction on plant height was
significant result. The highest and lowest plant height
was 86.58 cm and 73.08 cm observed in strain-J43
and control. The variation in plant height might be
due to the rhizobial inoculation. The above results
are in agreement with the findings of Solaiman.,
(1999). When treated with different N levels the
highest (83.41 cm) and lowest (76.16 cm) plant
height were recorded from N15 and N0.
According to Tena et al., (2016), the Rhizobium
inoculants produced the tallest plants and more
branches per plant than the control. In the case of
chickpea, Maurya and Sanoria., (1986) found same
result.
9
The correlation study was done to examine the
relationship of plant height with stover yield and
plant height with seed yield of soybean. The study
revealed a significant and positive correlation
between plant height with stover yield (Fig. 36) and
plant height with seed yield (Fig. 37) of soybean and
followed the regression equation y = 0.04x + 1.3501
(R² = 0.3097**) & y = 0.0165x + 0.8339 (R² =
0.3254**) respectively. Non-significant and effects
of rhizobial strains on soybean also performed
positive correlation. It indicates that the plant height
helped to increase seed yield of soybean.
Rhizobium contribution to pod formation of
soybean:
The pod length was recorded non-significant with all
of the treatments. The lowest pod heights ranged
from 10.8 to 20.4 cm (4.3 to 8.0 inches), on average,
across all varieties at St. Adolphe. At Morris, the
lowest pods ranged from 9.7 to 17.2 cm (3.8 to 6.8
inches) from the ground. Shome et al., (2022) found
that Rhizobium and PSB significantly affected pod
length and 100-seed weight (p < 0.01). Pod length
ranged from 3.66 cm to 5.11 cm due to different
Rhizobium treatments and 4.03 cm to 4.72 cm due
to Pseudomonas treatments. The longest pod was
given by 50% of recommended N with Rhizobium
japonicum inoculant, while 75% P and Pseudomonas
striata produced that which was the longest.
Rhizobium contribution to pod yield of soybean:
The highest Pod plant -1 (62.87 plant-1) was recorded
with treatment T5 which was identical with the
treatment T8 & T12 but differ with all other
treatments. The second highest Pod plant -1 (61.40
plant-1) was recorded with treatment T8 which was
identical with the treatment T12 but differ with all
other treatments. The lowest Pod plant-1 (52.17 plant-
1
) was recorded with treatment T2.
Gedamu et al., 2021 established that the number of
pods plant-1 was affected by the inoculation of the all
EAL17, EAL1018 and EAL1035 rhizobium strains.
Accordingly, inoculation of those three faba beans
brought statistically significant (P ≤ 0.05) difference
compared to the non-inoculated treatment. This
increment in could be attributed to the increment of
faba bean plant height. The research conducted by
Yohannes Desta et al., (2015) reported that rhizobial
strain alone could significantly increase the number
of pods/plants.
Alam et al., (2015) developed the inoculation of
soybean roots with Rhizobium sp. BARIRGm901
resulted in higher pod plnat -1, compared with yields
of non-inoculated plants, in both 2010 and 2011.
BARI Soybean6 + R (“+R” indicates a genotype
inoculated with Rhizobium sp. BARIRGm901) plants
produced significantly (p ≤ 0.05) higher pod yields
(2010: 40.94 pods plant−1; 2011: 48.0 pods plant −1)
than plants from other treatments and genotypes.
Sajid et al., (2011) established that the treatments had
a significant effect on the number of pods plant -1.
However, the maximum number of pods plant -1
(79.80) was produced by inoculated plants, minimum
of pods (56.40) was produced by uninoculated plants.
Maximum pods plant-1 in the inoculated plants may
be due to the increased number of bacteria due to
synthetic inoculation. It might also be due to more
leaves and shoots plant-1 which enable the plant to
produce and sink more photosynthates/carbohydrates
to the lower parts and thus more pods plant -1 was
produced. Among the cultivars, maximum pods
plant-1 (85.30) were produced by cv. Chakori, while
less pods plant-1 (51.90) were produced by cv. KS-1.
This may be due to the genetic potential of these
cultivars.
Rhizobium contribution to stover yield of soybean:
The stover yield was recorded higher in inoculated
Rhizobium bacteria compared to non-inoculate
control. The highest stover yield (4.23 t ha-1) was
recorded with treatment T7 which was identical to
treatment T10 but differed with treatments T9 & T12.
The second highest stover yield (4.06 t ha-1) was
recorded with treatment T10 which was identical with
treatment T7 but differ with all other treatments . The
lowest stover yield (2.69 t ha-1), was recorded with
treatment T12.
Alam et al., (2015) reported that inoculation of
soybean roots with Rhizobium sp. BARIRGm901
resulted in higher stover yields, compared with yields
of non-inoculated plants in both 2010 and 2011.
BARI Soybean6 + R (“+R” indicates a genotype
inoculated with Rhizobium sp. BARIRGm901) plants
produced a significantly higher (p ≤ 0.05) stover
yield (4.83 t ha−1) than plants from other treatments
genotypes in 2010. Likewise, in 2011, BARI
Soybean6 + R and BGM02026 + R plants showed
significantly higher (p ≤ 0.05) stover yield (3.85 t ha
and 3.60 t ha−1, respectively) compared with those of
other treatments genotypes (Alam et al., 2015).
Rhizobium contribution to seed yield of soybean:
No seed pod-1:
The treatment of Rhizobium in no seed pod-1 was
recorded non-significant with all of the treatments.
Gedamu et al., (2021) found that inoculation of
rhizobial strains statistically affected the number of
seeds pod-1 as compared to the un-inoculated
treatment. This finding disagreed with the finding of
Zerihun and Abera., (2014) who indicated that
inoculation of rhizobial strain did not impose a
significant difference over the un-inoculated one.
100 seeds weight:
10
The 100 seeds weight was recorded as non-
significant with all of the treatments. Shome et al.,
2022 found that Rhizobium and PSB significantly
affected 100-seed weight (p < 0.01). An 18%
increase in seed weight was observed due to the
application of 50% N with R. japonicum, while the
increase was 12% for 75% P and P. striata
inoculation. Shome et al., (2022) also mention that
treatment combinations significantly impacted the
number of seeds pod−1, in R. japonicum and P.
striata (R2P2).
Seed Yield:
The seed yield was recorded higher in inoculated
Rhizobium bacteria compared to non-inoculate
control. The seed yield (2.06 t ha-1) was recorded in
the highest with treatment T7 which was identical
with treatment T1, T2, T3, T4, T5, T6, T8 & T9 but
differ with other treatments. The second highest seed
yield (1.90 t ha-1) was recorded with treatment T4
which was identical with treatment T1, T2 & T3 but
differ with all other treatments. The lowest seed yield
(1.45 t ha-1), was recorded with treatment T12.
Alam et al., (2015) settle that inoculation of soybean
roots with Rhizobium sp. BARIRGm901 resulted in
higher seed yields, compared with yields of non-
inoculated plants, in both 2010 and 2011. Alam et al.,
(2015) also found that when inoculated with
Rhizobium sp. BARIRGm901, three of the four
soybean genotypes tested BARI soybean 6 + R
(“+R” indicates a genotype inoculated with
Rhizobium sp. BARIRGm901), MTD10 + R, and
BGM02026 + R showed significant increases (p ≤
0.05) in seed yields (3.60, 3.08, and 3.22 t ha −1,
respectively) compared with plants of other
treatments genotypes in 2010. In 2011, plants of
BARI soybean 6 + R and BGM02026 + R each
produced significantly higher (p ≤ 0.05) seed yields
(3.84 and 3.56 t ha−1, respectively) compared with
other inoculated and non-inoculated soybean
varieties and advanced lines (Alam et al., 2015).
According to Bremer et al., (1990) Rhizobium
improved lentil seed output by 135%. Seed yields
after bradyrhizobia inoculation of soybean cultivar
TGX 1485–1D at Igbariam ranged between 1.20 and
2.18 t ha–1 which had seed yields of 1.05 t ha–1 in
uninoculated plants, the poorest yield response after
inoculation with Bradyrhizobia strains was observed
in soybean cultivar M-351, with a seed yield ranging
from 0.60 to 0.98 t ha–1 (Okereke et al., 2001).
Inoculation of soybean seed with Rhizobium
significantly improved the grain yield of soybean.
Inoculated plants produced grain yield that is 35%
higher than non-inoculated ones (Adeyeye et al.,
2017).
Sultana et al., (2014) commence that when treat with
different rhizobial strains the highest grain N content
was 7.05% and maximum grain N uptake was 103.48
kg ha-1 obtained from strain-J43 and the lowest grain
N content (6.38%) and the minimum grain N uptake
was 79.38 kg ha-1 obtained from the control treated
plant. Seneviratne et al., (2000) and Zhang et al.,
(2002) reported a significant increase in seed and
shoot N of Soybean inoculated with B. japonicum.
The findings of this experiment is in agreement with
those of Kantar et al., (2003) who reported that
bacterial inoculation increased total N content (%)
significantly compared to control in chickpea. When
treated with different N levels the highest N content,
grain N uptake was 6.98%, 99.54 kg ha-1 and lowest
N content, grain N uptake was 6.24%, 80.78 kg ha-1
observed from treatment N15 and control group
respectively.
A correlation study was done to examine the
relationship between nodule number with root
weight, shoot weight and plant height. The study
revealed a significant and positive correlation
between nodule number with root weight, nodule
number with shoot weight and nodule number with
plant height of soybean and followed the regression
equation y = 0.4447x + 47.993 (R² = 0.2343***), y =
0.0358x + 8.3305 (R² = 0.0221***) & y = -0.0171x
+ 1.7987 (R² = 0.1547***) respectively in (Fig. 44).
It indicates that the nodule number helped to increase
root weight, shoot weight and plant height of
soybean which also increase soybean seed yield.
In conclusion, all the Rhizobium isolates obtained
from different soybean plants were not absorbed in
congo red and showed negative reaction with the
gram staining test. These bacteria were also rod
shaped, motile and produced nodules in soybean
roots. The conducted field experiment with soybean
(variety BARI Soybean-6), at the farmer’s field of
Tereja pul, Subarnachar, Noakhali showed results
that Rhizobium inoculation to soybean legume
increase the significant effect on plant height,
nodulation, shoot biomass, pod number, pod yield,
grain yield, 100-seed weight. The treatment T7
produces the highest number of nodules plant -1 over
all other treatment combinations. Treatment T 3 gave
11
the highest shoot weight and treatment T6 gave the
highest root weight than other treatment
combinations. The highest number of pods plant -
1 
found in treatment T5. The treatments of pod length,
number of seed pod-1 & 100 seeds weight showed
non significant results. Among the treatment
combinations, treatment T7 produced both highest
stover yield and seed yield. The application of
Rhizobium inoculum significantly increased most of
the parameters of soybean measured this study. It is
therefore concluded that Rhizobium sp 07 containing
biofertilizer application increases the growth and
yield of the soybean and Rhizobium sp 07 is the most
efficient bacterial strain. The correlation study
revealed a significant and positive correlation with
all the parameters. It indicates that the nodulation by
Rhizobium bacteria helped to increase the seed yield
of soybean compared to the uninoculated control.
The result of Rhizobium inoculation was very
encoring in respect of nodulation, growth and yield
of soybean.
SUPPLEMENTARY DATA:
Overall data finding in Statistic 10TM softwere of
soybean inoculated by Rhizobium strains.
Means followed by common letter are not
significantly different at 5% level by DMRT.
LIST OF ABREVIATIONS
CV Coefficient of Variation
SE Standard Error
LEVEL SIG. Level of Significance
EC Electrical Conductivity
OM Organic Matter
MEQ Miliequivalent
R Regression
pH Potential of Hydrogen
t ha-1 Ton Per Hectare
p p -value