Note: Descriptions are shown in the official language in which they were submitted.
1N/PA-98
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A NOVEL FORMULATION USEFUL AS A NITRIFICATION AND UREASE
INHIBITOR AND A IvIETHOD OF PRODUCING THE SAME
Field of Invention
The present invention relates to a novel formulation comprising nitrogenous
fertilizers in
combination with castor oil and oil derived from Artemisia annua useful as
nitrification
and urease inhibitor. More particularly, the invention relates to the
development of a cheap,
and potential urease/nitrification inhibitor from essential oil (Artemisia
annua oil). The
invention also provides a method for producing the novel formulation useful as
nitrification inhibitor.
Background of the invention:
Among the agricultural inputs to augment food production, use of fertilizer,
in particular
urea plays a key role. It is estimated that the present global consumption of
fertilizer N is
in the order of 77 Tg annually which is estimated to be increased to 144 Tg by
the
beginning of next century. In India, the present consumption of fertilizer N
is about 9.5 m
tons which is estimated to be increased to 14 m tons by 2001. Utilization
efficiency of
fertilizer N seldom exceeds 50%; it is yet low in paddy.
When applied to soil, urea hydrolyses by enzyme urease to from NHS and finally
to N03
by some bacteria. The NH.; is subjected to loss by volatilization whereas N03
is prone to
losses through leaching and dentrification. Apart from increasing the cost of
cultivation in
agriculture, N losses through leaching as well as in gaseous form have direct
concern in
environmental degradation. As a consequence of leaching N03 concentration in
ground
water has been increasing at an alarming rate. Any strategy that can restrict
or retard these
processes, would reduce the loss of N. The present invention deals with
finding strategies
for retarding urea transformation and nitrification for a higher fertilizer N
use efficiency.
Researchers have tried to reduce the losses of N (i) by applying N in small
doses at
different growth stages of crop, so that the plants use it before it is lost
and (ii) by deep
placement or incorporating the urea in soil. The ammonium produced can be
retained for
sometimes in the exchange complex of soil clay particles.
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Some of the modern tools suggested are (i) use of unease inhibitors such as
phenylphosphorodieamidate (PPD), hydroxamates and benzoqauinones (Gould, WD et
al
1986, Adv. Agron. 40:209-238), which reduce the hydrolysis of urea and this
can reduce
ammonia volatilization losses, and (ii) use of nitrification inhibitors such
as N serve or
nitrapyrin (2-chloro-6(tricholoromethyl)-pyridine), DCD (Dicyandiamide), AM (2-
amino-4-
chloro-6-methylpyrimidine), KN3 (potassium azide) and thiourea (Sahrawat,
1989, Adv.
Agron 42:279-302, Prasad and Power, 1995, Adv. Agron 54:233-81).
These compounds have been successful in some parts of the world. However,
because of cost
and poor availability these could not be made commercially popular to common
farmer. An
interesting discovery was the nitrification inhibitory properties of neem seed
cake (prasad et
al, 1971, Adv. Agron 23:337-381) and neem cake coated urea and has found some
favour
among Indian farmers. Neem bittern/extracts such as NiminTM, NeemexTM, etc.
for treating
urea are in Indian market. More recently a neem oil urea emulsion adduct has
also been
reported (Prasad et al, 1998, Curr. Sci. 75:15). Many of the fertilizer
mixture, coated materials
especially synthetic chemicals are put to limited use such as in turf or in
high price agriculture
such as commercial floriculture and that to in developed countries and have
not reached the
general farmers due to high costs.
Objects of the Invention
The main object of the invention is to develop a novel unease and
nitrification inhibitor from
natural source, more particuarly the evaluation of unease and nitrification
inhibiting properties
in Artemisia oil.
Another object is to develop novel unease nitrification inhibitor formulation
comprising urea
and other nitrogenous fertilizers, castor oil and oil derived from Artemisia
annua.
Yet another object is to develop cheap and eco-friendly unease and
nitrification inhibitor
which is easily decomposable and leave no adverse influence in the soil.
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3
Still another object is to provide a method for producing nitrification
inhibitor formulation.
Summary of the Invention
The present invention comprises of development of unease and nitrification
inhibitors from
essential oil and its derivatives coated with urea/ammonium producing N
fertilizers (0.5-1.0%
on w/w basis of fertilizer). Nitrogenous fertilizer is initially coated with
castor oil at 1% (w/w
basis of fertilizer). The coating resulted in retarding of nitrification by 29-
36% as against 31-
42% with dicyandiamide (DCD), which is taken as the reference. This
formulation was found
superior to DCD with respect to inhibition of unease activity (retarded by 32-
35% as
compared to DCD; 5-15%). The activity of both the nitrifying organisms i.e.
Nirosomonus
and Nitrobacter significantly decreased with this formulation. This natural
product
formulation augmented apparent N recovery to 56% against 37% with uncoated
urea and 54%
with DCD.
Detailed Description of the Invention
Accordingly, the present invention provides a novel formulation useful as a
nitrification and
unease inhibitor; said formulation comprising an effective amount of
nitrogenous fertilizer,
caster oil and oil derived from Artemisia annua, in an amount sufficient to
enhance the
nitrification inhibitory activity of the formulation.
In an embodiment of the invention, the amount of caster oil can be selected
from 0.2 to 2%
(w/w basis of fertilizer), preferably 1.0% (w/w basis of fertilizer).
In another embodiment, the amount of oil used in the composition, which is
derived from
Artemisia annua may be in the range of about 0.5-5% (w/w basis of fertilizer).
In a further embodiment of the invention, the oil derived from Artemisia annua
may be
coated, mixed or doped with the nitrogenous fertilizer.
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In another embodiment of the present invention, the nitrogen fertilizer may be
selected from
urea or ammonium sulphate and other ammonium producing fertilizer.
In yet another embodiment of the present invention, oil derived from Artemisia
annua may be
selected from Artemisia oil or its derivatives.
In another embodiment the invention provides a method for producing
nitrification inhibitor
formulation comprising the step of applying an effective amount of castor oil
(1%) and oil
obtained from Artemisia annua to nitrogenous fertilizer.
In an embodiment, the nitrogenous fertilizer may be coated, doped or even
mixed with castor
oil and oil obtained from Artemisia annua or its derivatives.
Yet another embodiment of the invention relates to a method of using Artemisia
oil as urease
and nitrification inhibitor which comprises of coating urea granules or any
other ammonium
forming fertilizer with castor oil 1.0% w/w/ basis of fertilizer as a primary
coating (as fixative
material) followed by coating with Artemisia oil in the range of 0.5%-5.0%
(w/w basis of the
fertilizer).
Nitrogen is one of the most vital plant nutrients in agriculture. It is
estimated that present
global consumption of fertilizer N is of the order of 77 Tg annually which is
likely to increase
to 145 Tg by the beginning of the next century. India consumes about 9.5
million tones, which
is estimated to be increased to about 14 million tones by 2005. Fertilizer
urea comprises of
more than 80% of the total consumption of nitrogenous fertilizers. Out of the
total quantity of
N applied to soil the recovery seldom exceeds 50%. It is further less in
submerged conditions
(rice). Fertilizer urea when applied to soil is hydrolysed by enzyme urease to
form NII4 which
is prone to losses through volatilization. The NH4 further oxidizes to NOz and
finally to N03
by nitrification which are generally lost through leaching
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and denitrification. Any process that slows down these transformation
processes can
reduce loss of N to a great extent. Urease and nitrification inhibitors play a
vital role in
retarding these processes, thereby increasing the nitrogen use efficiency.
A series of chemicals have been reported as urease and nitrification
inhibitors. However,
use of many of these has been restricted to research level because of their
high cost, low
availability, chemical nature, phytotoxicity and environmental consequences.
Natural
products like those from Karanj (Pongamia glabra), neem (Azadirachta indica)
etc. are
reported to have nitrification inhibitory properties. However, commercial
exploitation of
these materials had not been possible because of one reason or another.
The present invention relates to development of a urease and nitirification
inhibitor from
Artemisia oil. This is observed to be as effective as chemical inhibitors like
DCD, cost
effective, as required in very small quantity and ecofriendly (expected to
have minimum
residual effect).
Urea is the main source of nitrogen fertilizer used in the world due to its
high N content,
high solubility and non-polarity. However, it is suceptible to various N loss
mechanisms,
viz. ammonia volatilization, leaching and denitrification. Urease and
nitrification inhibitors
are used to resist fertilizer N losses by retarding urea hydrolysis and
nitrification. A series
of synthetic chemicals are used as urease and nitrification inhibitors to
restrict urea
hydrolysis and denitrification, respectively. These compounds are successful
in some parts
of the world. However, many of these synthetic chemicals have not been found
to be
commercially viable because of one reason or another. The present invention
deals with
the discovery of urease and nitrification inhibitory properties of Artemisia
annua oil which
being a natural product is ecofriendly and cheap as compared to synthetic
products. A
series of laboratory and greenhouse experiments were conducted to workout the
antimicrobial and N regulatory properties of the oil taking dicyandiamide
(DCD) as the
reference check. Artemisia oil has been found as effective as DCD as
nitrification inhibitor.
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IN/PA-98
This being a natural source and having both urease and nitrification
retardation properties,
would be very effective, cheap, safe (eco-friendly) doping material to
regulate urea N
transformation in soil.
The invention is described in detail hereinbelow with reference to the
following examples
which should not be construed as limitations on the inventive concept proposed
in the
invention.
To evaluate the performance of the proposed natural product, incubation
experiments were
conducted to follow the changes in regulation of urea -N transfomation and the
influence
of the material on soil microbes and microbial activity. Subsequently,
greenhouse
experiments were conducted to evaluate the influence of the material on
apparant N
recovery by plants.
The following examples are illustration in nature and should not be construed
to limit the
scope of invention
Example
1. Incubation experiment
A series of laboratory and greenhouse experiments were conducted to evaluate
the
efficacy of the proposed material as potent urease and nitrification
inhibitor. Artemsia oil
was used as the natural product which was compared with dicyandiamide (DCD) as
the
standard reference. Urea (46% N) was coated separately with the Artemisia oil
and DCD
using castor (Ricinus communis) oil as the fixative material. For this, first
coating of urea
granules was done by physical mixing of castor oil (1%) followed by the
natural product
and DCD at 0.50% and 1.00% with urea (w/w basis). After 24 hours of the mixing
is done,
the coated urea was mixed with 500 g soil (sandy loam, pH 8.2, available N 65
mg kg's )
and was kept in plastic container. A control (with urea alone, no coating) was
used as the
reference check. The soils under all the treatments having four replications
each were
incubated at 25° C after maintaining at 60% of the maximum water
holding capacity of
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soil. In one set of experiment, soils were analysed for urease activity and
urea N after 24
hours of incubation following methods of Tabatabai and Bremner (1972, Soil.
Biol.
Biochem. 4 : 479-487).
Representative soil samples were analysed for different forms of N i.e. Urea
N, NH4 and
N03-N at regular intervals to asses the nitrification inhibitory properties of
the materials in
question following standard procedures (Douglas and Bremner, 1970, Soil Sci
Soc. Am.
Proc. 34 : 859-862). Another set of experiment was conducted to assay the
total bacterial,
actinomycetes and nitrifying bacterial (Nirosomonus and Nitrobacter)
population
(Alexander and Clark, 1965, In Methods of Soil Analysis Part II (C.A. Black
Ed.) Am.
Soc. Agron. Inc. Wisconsin, USA; KenKnight and Torariko, 1973, Agrokhimiya 7 :
3)
2. Greenhouse experiment
To confirm the results of the incubation experiment, a pot culture experiment
was
conducted subsequently, taking Japanese mint (Mentha arvensis) as the test
crop. Eight kg
soil (2 mm) were taken in plastic container. The same coating materials and
fertilizer
(urea) as were in incubation experiment, were imposed, except that instead of
two levels of
coating materials, it was applied at 1.00% (w/w basis). The experiment was
laid in a
completely randomized design with four replications. The soils were analysed
for different
forms of N at regular intervals. In addition the plant materials were analysed
for N
accumulation and apparent fertilizer N recovery.
N accumulation - N accumulation
in treated pot(mg/kg) in control pot (mg/kg)
(no fertilzer N)
Apparent N recovery - x 100
Amount of N applied (mg/kg)
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Influence of the Coating materials on N transformation (mineralization),
urease
activity, nitrification and fertilizer N recovery:
Results from the incubation experiment (Table 1 ) indicate that the natural
product delayed
the hydrolysis of urea as evidenced by ~a higher concentration of urea-N on
day 1 after
imposition of treatments. This effect persisted till day 2. Artemisia oil was
as effective as
DCD with respect to regulation of urea -N transformation to NH4 and N03. There
was a
significant retardation of N03 formation both in DCD and the natural products
at both the
level of coating. At day 1 N03 formation retarded by 19-31 % with DCD and 13-
44% with
Artemisia oil as compared to urea alone.
Comparing on the basis of mean N03 produced, the % retardation of
nitrification at 0.5%
level of application were 31 and 29% with DCD and Artemisia oil, respectively
. The
corresponding retardation at 1.0% level of application were 42 and 36%,
respectively.
DCD maintained a higher level of NHS as compared to Artemisia oil.
Accumulation of
NH4 is undesirable as an excess of NH3 helps in increasing soil pH making the
conditions
in soil microsites congenial for NH3 volatilization.
Almost similar trend was observed with respect to transformation of urea N
(forms of N) in
the greenhouse experiment when mineral N was assessed at weekly intervals
(Table 2). As
in incubation experiment, accumulation of NH4 was higher in DCD treated soil
as
compared to Artemisia oil treated soil. Total N03 production was estimated to
be retarded
by 60% and 50% over urea alone, with coating of urea with DCD and Artemisia
oil,
respectively.
The incubation experiment further indicates that like DCD, Artemisia oil
influenced the
counts of Nitrosomous and Nitro6acter reponsible for nitrification, as well as
actinomycetes and total bacteria (Table 4). Nitrosomus population decreased by
9-14%
with DCD and 36-45% with Artemisia. Likewise, Nitrobacter population decreased
to an
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extent of 69-87% with DCD and 65-77% with Artemisia. The retardation increased
with
increasing the level of coating material.
Total actinomycetes population decreased with DCD at both the levels of
application
whereas Artemisia did not have any influence on actinomycetes population.
Finally, an
account of the total bacterial population indicate a significant antibacterial
properties of
both the synthetic and natural products.
Influence of these materials on urease and nitrification inhibitory properties
and regulation
of urea-N transformation was reflected on the apparent N recovery, total N
accumulation
by the plant and subsequently the herb yield of the test crop (Table 5). Herb
yield of
Japanese mint increased by 37%, 82% and 81% over control with urea alone
applied at
100 mg kg'1 soil, and that applied with coating DCD and Artemisia,
respectively. When
compared with urea alone, the herb yield with DCD and Artemisia increased by
29 and
31%, respectively. Both the materials augmented the apparent N recovery. The N
recoveries were 54 and 55% with DCD and Artemisia respectively, as against 37%
with
urea alone.
The present invention comprises of evaluation of urease and nitrification
inhibitory
properties in Artemisia oil. At 0.5% level of application Artemisia decreased
N03
formation to an extent of 29%. The corresponding value at 1.0% level of
application was
36%. Artemisia oil was found superior to DCD with respect to retardation of
urease
activity. Urease activity decreased by 32-35% with Artemisia oil as compared
to 5-15%
with DCD. This product inhibits the population of both Nitrosomus and
Nitrobacter;
actinomycetes population was not affected by Artemisia oil. Coating of urea
with
Artemisia oil increased apparent N recovery to 56% as against 37% with urea
alone and
54% with DCD.
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Table 1. Influence of differrnt coating materials on N transformation at
diflerrnt stages of incubation
Trcatments~ Forms of N
Day l Day 2 Day 3 Day 7 Day 14 Mean
Urea NH, NO~ Urea NH, NO~ Urea NH, NOs Urea, NH, NO~ Urea NH, NO~ Urea NH, NO~
Urea alone 4.5 42.5 99 - 6.2 218 - 16.5 16.9 - 10.5 136 - 6.9 155 - 16.5 156
(no coating
materials)
Urea +DCD
(0.50) 16.0 59.5 80 - 8.5 125 - 25.2 9.5 - 6.2 100 - 8.5 135 - 21.6 107
(1.00) 16.8 69.5 68 - 20.0 60 - 35.6 99 - 9.2 102 - 8.9 120 - 28.6 90
Urea+ Artemi.ria
(0.50) 27.5 35.0 86 7.8 5.0 120 . 22.1 112 - 3.5 109 - 9.5 125 ~ 15.0 I10
(1.00) 32.5 56.5 55 9.2 8.9 92 - 24.2 118 - 3.9 101 - 10.5 128 - 20.8 99
LSD (P=0.05) 3.45 8.90 7.50 - 1.25 10.3 - 3.90 0.50 . - 6.50 - - 7.50 - 4.50
8.50
'Coating materials added at 0.50 and 1.0°,'o w/w basis; urea applied at
100 mg kg ~ soil.
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Table 2 : Influence of different coating materials on N mineralization in soil
cropped with
menthol mint. (Mentha arvensis)
Treatment Mineral-N
s (mgkg-
soil)
Week Week Week Week Week Total
t II tII IV V
NH, NH, NH, NH, NH, NH,
NO~ NO~ NO~ N03 NO~ N03
Control 6.5 6.4 - 4.8- 6.8 - 9.3 2.8 3.5 9.3 30.8
(no
fertilizer
and no
coating
material)
Urea alone 20.0109 25.51208.0 45 - 34 10.218.963.8 324
(no
coating
maerials)
Urea+DCD 45.039.050.025.012.014.010.520.06.5 12.512.4 110
Urea+Artemisia25.049.020.535.06.5 17.5- 22.85.9 11.258.0 135
LSD (P=0.05)3.8 5.2 2.9 5.42.5 5.0 - 6.5 1.9 2.8 6.9 13.5
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Table 3. Influence of different coating materials on urease activity (in terms
of ~g NH4-N
liberated/g soiUhour)
Treatments* Urease activity
Control (No fertilizer and 24.5
no coating materials)
Urea alone (No coating materials)66.5
Urea+DCD (0.50) 56.7
(1.00) 45.1
Urea + Artemisia oil
(0.50) 45.2
( 1.00) 43.5
LSD(P=0.05) 8.23
* Urea applied @ 100mg kg's soil; coating materials applied @ 0.50 and 1.00%
(w/w
basis)
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Table 4: Influence of coating materials on Nitrosomomus Nitrobacter, total
actinomycetes
and bacterial population in soil.
Treatment* Nitrosomonus Nitrobacter Actinomycetes Total
X103 g-~ soil x 103 g-~ soil x 104 g-lsoil bacteria
x 105 g-~ soil
Control (no fertilizer and 1.10 2.50 6.50 2.00
no coating materials)
Urea alone (no coating 3.30 14.00 5.60 4.00
materials)
Urea+ DCD(0.50) 3.00 4.30 1.00 1.00
(1.00) 2.85 1.80 1.00 1.00
Urea + Artemisia oil
(0.50) 2.10 4.90 6.00 3.50
( 1.00) 1.80 3.20 6.00 2.50
LSD (P=0.05) 0.25 0.50 0.45 0.45
*Urea applied @ 100 mg kg ~ soil; coating materials @ 0.50 and 1.00 % (w/w
basis)
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Table S: Influence of different coating materials on herb yield, total N
accumulation and
apparent N recovery in menthol mint.
Treatment Herb yield N accumulation Apparent
N
(g pot' (mg pot 1) recovery
)
Level of Coating (%)
urea-
N (mg kg't)materials
( 1.00%)
0 (Control)Nil 32.5 170 -
100 Nil 44.5 467 37.12
DCD 59.2 603 54.12
Artemisia 58.9 612 55.25
LSD (P=0.05) 5.20 11.8
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Advantages of the inventions
1. This product is as effective as dicyandiamide (DCD) as nitrification
inhibitor.
2. This product has higher urease inhibitory properties.
3. This product doesn't allow high accumulation of NH.~-N following hydrolysis
of urea
Higher accumulation of NH4-N helps in increasing soil pH and thereby causing
volatilization of NH3.
4. This product being natural and having low persistence, is expected to leave
no adverse
effect in soil, on beneficial microflora and related transformation processes
and
environment.
5. This product is better than other essential oils and derivatives.
6. This product is cheaper than many synthetic inhibitors.
