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1 23
Sugar Tech
An International Journal of Sugar Crops
and Related Industries
ISSN 0972-1525
Sugar Tech
DOI 10.1007/s12355-015-0386-x
Effects of a Formulated Bio-product
Containing the Nitrogen Fixing Bacterial
Strain Enterobacter oryzae 3LSO1 on
Sugarcane Growth
Nutthawut Meesilp, Prasit Jaisil, Nipa
Milintawisamai & Suwanna Niamsanit
1 23
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RESEARCH ARTICLE
Effects of a Formulated Bio-product Containing the Nitrogen
Fixing Bacterial Strain Enterobacter oryzae 3LSO1 on Sugarcane
Growth
Nutthawut Meesilp
1
•Prasit Jaisil
2
•Nipa Milintawisamai
3
•Suwanna Niamsanit
1
Received: 10 February 2015 / Accepted: 9 June 2015
ÓSociety for Sugar Research & Promotion 2015
Abstract Nitrogen (N) is an essential element for plant
growth, widely applied as chemical N-fertilizer to improve
yield of sugarcane. The elevated doses of fertilizers may
have negative and unpredictable effects on the environ-
ment. Bio-fertilizers containing nitrogen fixing bacteria
(NFB) are alternative and potential way to reduce appli-
cation of chemical N-fertilizer. The NFB strain Enter-
obacter oryzae 3LSO1 isolated from rhizosphere soil of
sugarcane can fix atmospheric dinitrogen (N
2
), produces
indole-3-acetic acid as plant growth promoting substance.
Therefore, this effective strain was developed as a vermi-
culite-based bio-product to enhance sugarcane growth. The
bio-product was compared storing conditions at 10 and
30 °C for 30 or 60 days after formulation (DAF), and then
its performance was evaluated. Result stated that the bio-
product could be kept at 10 and 30 °C for up to 60 DAF
without a decrease in the NFB population or a loss in its
ability to fix N
2
. In a greenhouse experiment, commercial
sugarcane variety Khon Kaen 3 applied with the E. oryzae
3LSO1 bio-product and planted with or without nitrogen
fertilizer could increase the tiller number, plant height,
fresh and dry weight, NFB population in soil and sugar-
cane, compared with the control. This E. oryzae 3LSO1
bio-product not only used as a bio-nitrogen fertilizer for
sugarcane crops, but also reduced chemical N-fertilizer
application for sustainable agriculture.
Keywords Nitrogen fixing bacteria
Enterobacter oryzae Sugarcane Bio-product
Formulation
Introduction
Sugarcane (Saccharum species hybrids) is one of the major
economic plant crops. Chemical fertilizers applied are to
increase the crop yield. Nitrogen is the important
macronutrient for plant growth, but it is easily lost by
several factors such as de-nitrification and soil surface
wash in rainy season. Chemical N-fertilizers are usually
applied at high dose in fields. However, these are becoming
more scarce and costly. N
2
-fixation is one of the possible
biological alternatives to save N-fertilizers and leads to
more productive and sustainable agriculture without
harming the environment (Dobereiner and Urquiaga 1992).
Several commercial sugarcane varieties such as U-thong
(series 9–13), K84–200, Khon Kaen 3 are cultivated in
Thailand depending on weather and soil types.Khon Kaen
3 is the most important and high yielding commercial
sugarcane planted variety in the Northeast of Thailand
including Khon Kaen province. This variety exhibits many
dominant characteristics such as resistant to red rot disease,
high commercial cane sugar (CCS), high cane yield, low
flowering and growing well in non-irrigated sandy soil.
NFB are the bacteria containing nitrogenase enzyme that
enable N
2
-fixation to occur in nature. Some sugarcane
varieties have been recorded to fix up to 70 % of their
nitrogen requirement through biological nitrogen fixation
&Suwanna Niamsanit
suwan_ni@kku.ac.th
1
Department of Microbiology, Faculty of Science, Khon Kaen
University, Khon Kaen 40002, Thailand
2
Department of Plant Science and Agricultural Resources,
Faculty of Agriculture, Khon Kaen University, Khon Kaen
40002, Thailand
3
Department of Biochemistry, Faculty of Science, Khon Kaen
University, Khon Kaen 40002, Thailand
123
Sugar Tech
DOI 10.1007/s12355-015-0386-x
Author's personal copy
(Barraquio et al. 2000). NFB strain Enterobacter oryzae is
one of the plant growth promoting bacteria containing N
2
-
fixation ability (Ruppel and Merbach 1995), produce phy-
tohormones such as auxin compounds as indole-3-acetic
acid, solubilize calcium phosphate and inhibit the growth
of some phytopathogenic fungi (Hardoim et al. 2013). This
strain has been isolated from agriculturally important
grasses including sugarcane. E. oryzae was reported the
rhizosphere soil and also in the interior regions of the plant
roots and shoot without causing disease symptoms (James
and Olivares 1997). Many workers reported it could pro-
mote the growth of various plant species such as cereals,
wheat and sugarcane (Ka
¨mpfer et al. 2003; Remus et al.
2000; Ruppel et al. 2006).
Improvement of sugarcane growth under greenhouse
conditions by using NFB as plant growth promoting agent
was reported by soaking the cane setts with bacterial
suspension before planting (Suman et al. 2005). However,
this method is impractical due to difficulty in handling,
transport, storage and large-scale application (Rabindran
and Vidhyasekaran 1996). Several researchers developed
the formulation of efficient bacterial strains with carrier
materials such as talc, lignite, coir dust peat and vermi-
culite. However, the development of a formulation of
NFB used as bio-nitrogen fertilizer has not been reported.
For example, Pseudomonas fluorescens was formulated
with carrier materials to control damping-off of sugar
beet, a disease caused by Pythium spp. (Moe
¨nne et al.
1999). Vermiculite-based formulations has been reported
for supporting the survival of bacteria during storage in
different conditions (Rabindran and Vidhyasekaran 1996;
Graham-Weiss et al. 1987).
In a previous study, the NFB isolate 3LSO1 was iso-
lated from the rhizosphere soil of field-grown sugarcane
on N-free LGIP medium and identified as E. oryzae
(based on biochemical test and 16S rDNA). In this study,
the NFB strain displayed a high efficient nitrogenase
activity to fix N
2
investigated with flame ionization
detector gas chromatography (acetylene reduction assay)
and produce indole-3-acetic acid (colorimetric method).
When it was applied to commercial sugarcane variety
Khon Kaen 3 as a bacterial suspension planted in defi-
ciency N soil with 3 levels of N-fertilizer (0, 75,
151 kg N/ha), it greatly enhanced sugarcane growth with
N at rates 0 and 75 kg N/ha. Thus, the effective strain E.
oryzae 3LSO1 was developed as bio-products used ver-
miculite-base for supporter. During storage the bio-pro-
duct, a N
2
-fixation ability and survival of NFB population
were measured. Moreover, the bio-product containing E.
oryzae 3LSO1 was also proved able to promote growth of
a commercial sugarcane variety Khon Kaen 3 with or
without N fertilizer under greenhouse condition.
Materials and Methods
Bacterial Preparation
The E. oryzae 3LSO1 inoculant was prepared by growing
in Luria–Bertani medium (LB, quantities per liter; 10 g
Tryptone, 5 g Yeast extract, and 5 g NaCl) with shaking at
30 °C for 8–10 h. Bacterial cells were harvested by cen-
trifuging at 65009gfor 20 min at 4 °C, and then re-sus-
pended in 600 mL fresh LB medium to make the inoculum.
The turbidity of the cell suspension was measured as
optical density at 620 nm (0.08 910
8
cfu/mL) with a
spectrophotometer.
Formulation
The vermiculite-base was packed in polypropylene bags
before autoclaving at 121 °C 15 lb/in
2
for 30 min (2 days
consecutive). A sub-sample of the carrier material was
analyzed to determine its initial characteristics [total N
(Kjeldahl), total P, K, Ca, and Mg (HNO
3
–HCLO
4
diges-
tion)]. To prepare each bag of bio-product, a 600-mL ali-
quot of the prepared bacterial inoculum was added to 2.5 L
sterile vermiculite, and mixed well under sterile conditions.
Storage and Analyses of Bio-product
All of the NFB bio-product bags were stored at 10 °C and
30 °C for 30 to 60 DAF. For analyses, a 5 g sample of each
NFB bio-product were collected at 0, 30, and 60 DAF and
suspended following serially diluted in 0.85 % normal
saline. Then, 0.1 mL aliquots of appropriate dilutions were
spread on N-free LGIP medium (quantities per liter; 0.2 g
K
2
HPO
4
, 0.6 g KH
2
PO
4
, 0.2 g MgSO
4
7H
2
O, 0.02 g
CaCl
2
2H
2
O, 0.002 g NaMoO
4
2H
2
O, 0.01 g FeSO
4
7H
2
O,
5 ml of 0.5 % bromothymol blue in 0.2 N KOH, 100 g
sucrose, 15 g agar, and pH 7). The number of bacterial
colonies formed after 48 h incubation at 30 °C was used to
calculate as log cfu/g.
Greenhouse Experiment
Soil Preparation
Sandy soil (Oxic Paleustult; Yasothon series) was used in
these experiments. The soil was air dried for 7–14 days and
a representative sample (1 kg) was analyzed to determine
organic matter (Walkey and Black wet oxidation), total N
(Kjeldahl), available P (Bray II), exchangeable K, Ca, Mg
(ammonium acetate at pH 7), electrical conductivity (soil:
water, 1:10), and pH (soil: water, 1:1). The soil was ster-
ilized by fumigation with 40 % (v/v) formalin for 1 week
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in a closed system. The formalin was allowed to evaporate
before the soil was used in the experiments and each pot
contained 13 kg soil.
Cane Preparation and Treatment
The commercial sugarcane variety Khon Kaen 3 was
selected for improving the effect of E. oryzae 3LSO1 bio-
products. Cane sett with a single bud (about 5 cm) was
planted horizontally in each pot. After 7 days, the E.
oryzae 3LSO1 bio-products that had been stored at 10 or
30 °C for 30 or 60 DAF was applied at a rate 151 kg/ha
(0.151 g/kg soil). Sterile vermiculite was added the same
rate for the control treatment. The experiments were
separated into twice of plantation by conducting in a
completely randomized design with three replicates. First
crop (December 23, 2011 to April 23, 2012), sugarcane
was applied with 30 day-old NFB bio-products containing
6 treatments (bio-products stored at 10, 30 °C and con-
trol 92 N levels). Second crop (January 23 to May 23,
2012), the plants were applied with 60 day-old NFB bio-
products also containing six treatments. Chemical fertil-
izer, urea nitrogen was applied at rates of 0 (PK) and 75
(NPK) kg N/ha (0, 0.075 g/kg soil, respectively), single
super phosphate at 0.3 g/kg soil, and potash at 1.6 g/kg
soil were applied to 30 day-old sugarcane plants in pots.
Plants were grown under greenhouse conditions at Faculty
of Agriculture, Khon Kaen University under natural light
and the moisture content of soil was adjusted with chlo-
rine-free water.
Effect of E. oryzae 3LSO1 Bio-products
after Planting
Detection of NFB Population
The NFB population of plants and soil after planting was
measured on N-free LGIP medium. Shoot and root of
plants were washed with tap water and surface-sterilized
with 5 % (v/v) chloramine T followed by 70 % (v/v)
ethanol, and finally rinsed three times in sterile distilled
water. Only shoot, the cane peel was removed with a sterile
sharp knife, and then tissue pieces were rinsed with 95 %
ethanol and flamed. Sterile shoot and root were rolled on
LB and N-free LGIP medium to verify appropriate surface
sterilization and then macerated in 5 % (w/v) sucrose
solution. Soil and extracted plant solution were diluted in
0.85 % normal saline followed by spreading on N-free
LGIP medium. The NFB population was counted after 48 h
incubation at 30 °C.
Measurement of Plant Growth and Soil Properties
Tiller number and plant height was recorded until 120 day
after planting (DAP). Above-ground plant parts were har-
vested close to the soil surface, and roots were dug from
the soil. The fresh weight of each sugarcane plant was
measured immediately upon collection, and the dry weight
was measured after oven drying to constant weight at
80 °C. Moreover, shoot and root of plants were examined
the accumulation of total N (Micro–Kjeldahl method), total
P, and total K (Wet oxidation method) contents. Soil
samples were collected near the sugarcane root and ana-
lyzed to determine organic matter (Walkey and Black wet
oxidation), total N (Micro–Kjeldahl method).
Statistical Analysis
All measurements were subjected to analyses of variance
(ANOVA) to determine the least significant difference.
Results and Discussion
Initial Vermiculite and Soil Characteristic
The physical and chemical properties of vermiculite and
Yasothon soil were presented in Table 1. The initial pH of
the carrier material was approximately neutral. The soil
had low nutrient contents, especially total N.
Table 1 Characteristics of vermiculite and soil
Sample pH EC
a
Percentage of element accumulation
OM
b
NP K MgCa
Vermiculite 7.7 – ND ND* 0.44* 5.02* 1.31* 0.93*
Soil 4.8 0.038 0.66 0.028* 15.31** 244.97*** 5.55*** 70.22***
ND not detected
a
Electrical conductivity (mS/cm)
b
OM organic matter
* Total, ** available; *** exchangeable
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E. oryzae 3LSO1 Population in Bio-products During
Storage
The result of counting NFB population in bio-products is
presented in Table 2. Before storing at 10 or 30 °C(0
DAF), each bio-product contained the E. oryzae 3LSO1
population average in log 7 cfu/g. The population rate of
this NFB strain in bio-products extremely increased (log
8–9 cfu/g) in the first 30 DAF, since the bacterial cells
were centrifuged and re-suspended in fresh LB medium
that might be the culture substrate to prolong shelf life of
NFB during stored. The populations at 60 DAF showed a
little bit increased from 30 DAF. Due to the properties and
structure of the vermiculite-base were inorganic and multi-
lamellate, it provided superior aeration and space for
microbial proliferation. Although the vermiculite base did
not support microbial growth because of its mineral nature
(Graham-Weiss et al. 1987), but NFB bio-products sup-
plemented with fresh culture substrate (LB medium) could
promote the growth of E. oryzae 3LSO1.
Effect of E. oryzae 3LSO1 Bio-products on NFB
Population
The populations of NFB after planting (120 DAP) counted
on N-free LGIP medium is shown in Fig. 1. Soil, shoot and
Table 2 E. oryzae 3LSO1 population in the bio-product at 0, 30, and
60 DAF
Treatment E. oryzae 3LSO1 population (log cfu/g)
0 DAF 30 DAF 60 DAF
Bio-product 10 °C 7.42 9.35 9.44
Bio-product 30 °C 7.83 8.22 8.50
DAF days after formulation
0
2
4
6
8
10
Vermiculite
+ PK
Vermiculite
+ NPK
Bio-product
10 °C + PK
Bio-product
10 °C + NPK
Bio-product
30 °C + PK
Bio-product
30 °C + NPK
log cfu/g
(A)
Soil Shoot Root
0
2
4
6
8
10
Vermiculite
+ PK
Vermiculite
+ NPK
Bio-product
10 °C + PK
Bio-product
10 °C + NPK
Bio-product
30 °C + PK
Bio-product
30 °C + NPK
log cfu/g
(B)
Soil Shoot Root
Fig. 1 Effects of 30 (a) and 60
(b) DAF E.oryzae 3LSO1 bio-
products on NFB population at
120 DAP
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root of the treatments without inoculation the bio-products
(both 30 and 60 DAF) revealed a low NFB population not
more than 100 cfu/g. Generally, some species of NFB have
been widely discovered in un-inoculated sugarcane, but with
limited N
2
-fixation efficiency (Barraquio et al. 2000).
Planting sugarcane with bio-products, NFB population
detected in soil was higher (log 7–8 cfu/g) than in shoot (log
4–5 cfu/g) and root (log 3–5 cfu/g) of plant. Most bacterial
colony morphology found in soil and plant samples inocu-
lated with bio-products were found similar to E. oryzae.The
result revealed that E. oryzae could infect and colonize into
shoot and root playing role as endophytic bacteria. Cracks at
the root branching points and wounds on the root system
were probably the entry points for the bacterium to enter and
establish inside the plant (Sevilla et al. 1998,2001;James
2000). Studies have shown that soil microbial biomass
nitrogen increased during crop growth, with the highest
values at maturity (Shukla et al. 2013).
Effect of E. oryzae 3LSO1 Bio-products on Plant
Growth
N-fertilizer was found a key factor in control treatments
which confirmed the necessity of nitrogen element for plant
growth, since the treatment with no N-fertilizer (vermi-
culite ?PK) showed less tiller number, less plant height,
shoot and root fresh weight, shoot and root dry weight as
compared to the treatment with N-fertilizer (vermi-
culite ?NPK). On the other hand, application of E. oryzae
3LSO1 bio-products (both 30 DAF and 60 DAF) without
N-fertilizer (PK) exhibited more tiller numbers (over 75 %
increased), plant height (over 25 % increased), shoot and
root fresh weight (over 30 and 17 % increased, respec-
tively), shoot and root dry weight (over 32 and 60 %
increased, respectively) than the control treatment with
N-fertilizer (vermiculite ?NPK), respectively (Tables 3
and 4). These results suggested that E. oryzae 3LSO1 bio-
Table 3 Effect of 30 DAF E.oryzae 3LSO1 bio-product on plant growth at 120 DAP
Treatment Tiller no. Height (cm) Shoot (g) Root (g)
Fresh weight Dry weight Fresh weight Dry weight
Vermiculite ?PK 0.0
b
156.7
b
209.53
b
74.87
b
23.86
b
12.33
b
Vermiculite ?NPK 1.0
b
176.3
b
226.32
b
87.49
b
37.94
b
12.75
b
Bio-product 10 °C?PK 7.6
a
(86.8) 256.7
a
(31.3) 585.50
a
(61.3) 142.97
a
(38.8) 88.70
a
(57.2) 39.33
a
(67.6)
Bio-product 10 °C?NPK 4.0
a
(75.0) 265.0
a
(33.5) 558.70
a
(59.5) 154.56
a
(43.4) 96.76
a
(60.8) 52.23
a
(75.6)
Bio-product 30 °C?PK 6.6
a
(84.8) 235.0
a
(25.0) 558.93
a
(59.5) 136.22
a
(35.8) 136.26
a
(72.2) 57.00
a
(77.6)
Bio-product 30 °C?NPK 6.6
a
(84.8) 246.7
a
(28.5) 581.08
a
(61.1) 139.09
a
(37.1) 92.54
a
(59.0) 44.25
a
(71.2)
DAF days after formulation, DAP days after planting
Values in the same column with different letters are significantly different (p\0.05, Duncan’s test). Figure in bracket indicate percent
improvement over control
Table 4 Effect of 60 DAF E.oryzae 3LSO1 bio-product on plant growth at 120 DAP
Treatment Tiller no. Height (cm) Shoot (g) Root (g)
Fresh weight Dry weight Fresh weight Dry weight
Vermiculite ?PK 0.0
b
178.7
b
206.00
b
74.33
b
76.57
b
12.82
b
Vermiculite ?NPK 1.3
b
195.0
b
279.12
b
88.16
b
95.51
b
13.29
b
Bio-product 10 °C?PK 9.0
a
(85.6) 290.0
a
(32.8) 563.29
a
(50.4) 156.37
a
(43.6) 119.13
a
(19.8) 37.21
a
(64.3)
Bio-product 10 °C?NPK 10.3
a
(87.4) 288.3
a
(32.4) 614.82
a
(54.6) 165.33
a
(46.7) 130.29
a
(26.7) 45.75
a
(71.0)
Bio-product 30 °C?PK 9.0
a
(85.6) 293.3
a
(33.5) 663.56
a
(57.9) 209.64
a
(57.9) 115.90
a
(17.6) 33.07
a
(59.8)
Bio-product 30 °C?NPK 7.6
a
(82.9) 286.7
a
(32.0) 439.56
ab
(36.5) 129.55
a
(31.9) 125.90
a
(24.1) 40.18
a
(66.9)
DAF days after formulation, DAP days after planting
Values in the same column with different letters are significantly different (p\0.05, Duncan’s test). Figure in bracket indicate percent
improvement over control
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products could not only promote growth, but also reduce
chemical N-fertilizer application for sugarcane plantation.
These findings indicated that bio-products were stable, and
the bacteria were capable of fixing atmospheric N into
significant amount that could be used for plant growth,
such as NH
3
?
. The phytohormones (indole-3-acetic acid)
produced by E. oryzae 3LSO1 was another factor in the
increased stem and root elongation of sugarcane plants as
reported by other workers (Koomnok et al. 2007; Arshad
and Frankenberger 1998).
Effect of E. oryzae 3LSO1 Bio-products on Nutrient
Accumulation
The results of effect of E. oryzae 3LSO1 on sugarcane
growth at 30 DAF and 60 DAF were found comparable.
Therefore, we used the sugarcane plants and soil of 60 DAF
bio-products to investigate the nutrient accumulation. All
bio-products significantly enhanced the accumulation of N,
P and K in shoot (Table 5). Every formulated NFB treat-
ment without N-fertilizer (PK) exhibited the N
2
-fixation
ability to increase N-uptake in shoot by 46–62 % over the
control (Vermiculite ?NPK). In addition, only N and P of
root inoculated with E. oryzae 3LSO1 were increased over
50 and 23 %, respectively. Soil organic matter was com-
posed of microorganisms and plant residues. A high quantity
of NFB population detected in soil after planting (log 7 cfu/
g) increased the organic matter (8.2–12.4 %) and N
(33–38 %) as compared to the control treatments (Table 6).
Conclusion
The bioproduct efficacy of E. oryzae 3LSO1 strain devel-
oped in the study was stable and maintained NFB popu-
lation during storage with potential N
2
-fixation trait. The
efficacy of E. oryzae 3LSO1 bio-product was established
with sugarcane variety Khon Kaen 3 in low N content soil.
Overall, this E. oryzae 3LSO1 bio-product not only effi-
cient as a biologically nitrogen fertilizer for sugarcane
crops, but can also reduced chemical N-fertilizer applica-
tion for sustainable agriculture.
Acknowledgments This work was supported by Khon Kaen
University and Human Resource Development in Science Project
(Science Achievement Scholarship of Thailand, SAST).
References
Arshad, M., and J.W.T. Frankenberger. 1998. Plant growth–regulating
substances in the rhizosphere microbial production and func-
tions. Advances in Agronomy 62: 45–51.
Barraquio, W.L., E.M. Segubre, M.S. Gonzalez, S.C. Verma, E.K.
James, J.K. Ladha, and A.K. Tripathi. 2000. Diazotrophic
enterobacteria: What is their role in rhizosphere of rice? In
The quest for nitrogen fixation in rice, ed. J.K. Ladha, and P.M.
Reddy, 93–118. Los Ban
˜os: International Rice Research Institute
Press.
Dobereiner, J., and S. Urquiaga. 1992. Soil biology and sustainable
agriculture. Annals of the Brazilian Academy of Sciences 84:
127–133.
Graham-Weiss, L.G., M.L. Bennett, and A.S. Pauu. 1987. Production
of bacterial inoculants by direct fermentation on nutrient-
Table 5 Effect of 60 DAF E.oryzae 3LSO1 bio-product on plant nutrient accumulation at 120 DAP
Treatment Shoot (%) Root (%)
Total N Total P Total K Total N Total P Total K
Vermiculite ?PK 0.145
b
0.107
b
1.005
b
0.249
b
0.112
b
0.354
b
Vermiculite ?NPK 0.240
ab
0.167
b
1.371
b
0.288
b
0.129
b
0.542
a
Bio-product 10 °C?PK 0.450
a
(46.7) 0.377
ab
(55.7) 2.810
a
(51.2) 0.730
a
(60.5) 0.198
ab
(34.8) 0.407
ab
(-33.2)
Bio-product 10 °C?NPK 0.378
ab
(36.5) 0.402
a
(58.5) 2.960
a
(53.7) 0.641
a
(55.1) 0.193
ab
(33.2) 0.546
a
(0.7)
Bio-product 30 °C?PK 0.645
a
(62.8) 0.455
a
(63.3) 3.027
a
(54.7) 0.736
a
(60.9) 0.172
ab
(25.0) 0.568
a
(4.6)
Bio-product 30 °C?NPK 0.676
a
(64.5) 0.438
a
(61.9) 2.369
ab
(42.1) 0.578
a
(50.2) 0.168
ab
(23.2) 0.574
a
(5.6)
DAF days after formulation, DAP days after planting
Values in the same column with different letters are significantly different (p\0.05, Duncan’s test). Figure in bracket indicate percent
improvement over control
Table 6 Effect of 60 DAF E.oryzae 3LSO1 bio-product on soil
nutrient accumulation at 120 DAP
Treatment Organic matter (%) Total N (%)
Vermiculite ?PK 0.657
b
0.0138
b
Vermiculite ?NPK 0.758
ab
0.0238
ab
Bio-product 10 °C?PK 0.846
a
(10.4) 0.0382
a
(37.7)
Bio-product 10 °C?NPK 0.826
a
(8.2) 0.0371
a
(35.8)
Bio-product 30 °C?PK 0.855
a
(11.3) 0.0369
a
(35.5)
Bio-product 30 °C?NPK 0.865
a
(12.4) 0.0356
a
(33.1)
DAF days after formulation, DAP days after planting
Values in the same column with different letters are significantly
different (p\0.05, Duncan’s test). Figure in bracket indicate percent
improvement over control
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