Tải bản đầy đủ - 0trang
Chapter 5. Nitrification Inhibitors for Agriculture, Health, and the Environment
RAJENDRA PRASAD AND J. F. POWER
Nitrification inhibitors (NIs) emerged as a group of agrichemicals with the development of N-Serve [2-chloro-6(trichloromethyl)pyridine](Dow Elanco trade
name for nitrapyrin) by Goring ( 1962ab), although inhibition of nitrification by a
number of herbicides, insecticides, nematicides, and fungicides were known long
before. Except for a few field experiments, research on nitrification inhibitors during the 1960s was mostly restricted to laboratory studies (Prasad el al., 1971).
Intensive field investigations were carried out in the late 1960s and 197Os, and the
American Society of Agronomy, the Crop Science Society of America, and the
Soil Science Society of America jointly sponsored a symposium on December 6,
1978, at Chicago, Illinois, the proceedings of which were published in 1980
(Meisinger et al., 1980). Three years later a technical workshop on the nitrification
inhibitor dicyandiamide (DCD) was held on December 4-5, 1981, in Muscle
Shoals, Alabama; this workshop was jointly sponsored by the National Fertilizer
Development Center, the Tennessee Valley Authority at Muscle Shoals, Alabama;
the International Fertilizer Development Center, Muscle Shoals, Alabama; and
SKW Trostberg AG, West Germany (Hauck and Behnke, 1981). A second workshop on DCD was held on December 4-5, 1987, at Atlanta, Georgia, and the
proceedings were published as a special issue in Communications in Soil Science
and Plant Analysis (Vol. 20, Nos 18 and 19, 1989) (Hauck et al., 1989).
In addition to specific chemicals such as nitrapyrin or DCD, natural products
like those from neem (Azadirachta indica Juss) are reported to have nitrificationinhibiting properties (Reddy and Prasad, 1975; Sahrawat and Parmar, 1975) and
have been widely evaluated in India. Prasad et al. (1993) addressed the N use
efficiency aspects of urea coated with neem cake and other neem products at the
Neem World Conference held at Bangalore, India (February 24-28, 1993).
An ideal nitrification inhibitor should be mobile, persistent, and, above all, economic in use (Hauck, 1972). It should also be nontoxic to other soil organisms,
animals, and humans and should move with the fertilizer or nutrient solution.
Compounds with high vapor pressure may move fast and compounds easily absorbed may not be very effective. An ideal NI should stay effective in soil for an
adequate time period; at least for the growth period of a crop. Above all, the real
testing ground is in the economics of use; most studies indicate that about a 0.3 to
0.5 mg ha - ' yield increase will pay for the cost. This one factor alone has stopped
many nitrification inhibitors from reaching the farm level. The major goal in using
a NI is to increase the efficiency of fertilizer N applied to agricultural/horticultural
crops by reducing nitrate leaching losses as well as nitrification losses as N,O or
N,. Thus ideal situations where NIs are likely to be the most effective are those
where such losses predominate, such as rice paddies, areas receiving heavy precipitation, irrigated areas (especially furrow) because of leaching, and crops receiving high rates of N fertilization or manures.
During the 1980s there was considerable effort by ecologists, environmentalists, and some agriculturists to reduce fertilizer N use on the farm, mainly due
to its likely role in increasing nitrate concentrations in groundwater and because
N fertilizers are manufactured from a nonrenewable natural resource (natural gas).
However, on a global scale this will neither be possible nor desirable if we are
to feed the increasing world population. The available estimates indicate that
2422 Tg of cereals will be required in 2000 AD (Prasad, 1986) compared to the
1991 estimated production of 1884 Tg of cereals (FAO, 1991). Thus an additional
28.5% of cereals will have to be produced in the next decade, most of it in the
While this increase in cereal production can be achieved in most African and
South American countries by bringing more land under cultivation, Asia has done
it by increasing productivity per unit land per unit time. This calls for a sizable
increase in the consumption of fertilizer, especially nitrogen. It is estimated by
2000 AD that 145.4 Tg of fertilizer N will be consumed annually (UNIDO, 1978),
which is nearly double the 1990-1991 consumption of 77 Tg of fertilizer N
(FAO, 1991). Furthermore, a large number of the developing countries, especially
those in south and southeast Asia, grow rice as a principal crop, the crop for which
fertilizer N losses are greatest (Prasad and De Datta, 1979; Fillery and Vlek, 1986;
Reddy and Patrick, 1986; De Datta, 1986). In addition to the large amounts of
fertilizer N needed in the developing countries, high costs involved in their production or purchase also need to be considered. Also the sustainability of synthetic
fertilizer production from natural gas at some time in the future is a concern. Thus,
efficient use of fertilizer N is necessary, suggesting that nitrification inhibitors
have a role to play. This chapter provides an overview of the literature available
on the use of nitrification inhibitors in relation to production and quality of agricultural and horticultural crops, human and animal health, and the environment.
11. NITRIFICATION INHIBITORS
A fairly large number of chemicals have been reported as nitrification inhibitors: Nitrapyrin (abbreviated as NP in this chapter) or N-Serve [2-chloro-6-(trichloromethyl)pyridine] (Goring 1962ab); AM (2-amino-4-chloro-6-methylpyrimidine) (Toyo Koatsu Industries, 1965); DCD (Amberger and Guster, 1978);
terrazole or Dwell or etridiazole (5-ethoxy-3-trichloromethyl- 1,2,bthiadiazole)
(Olin Corp., I976ab); DCS [N-(2,5-dichlorophenyl)succinamicacid] (Namioka
and Komaki, 1975ab); KN? (potassium azide) (Hughes and Welch, 1970); ATC
(4-amino- 1,2,4-triazole) (Guthrie and Bomke, 1980); TU (thiourea); MBT (2mercaptobenzothiazole); 2-ethynyl pyridine (McCarty and Bremner, 1986); MPC
(3-methyl-pyrazole-I-carboxamide) (McCarty and Bremner, 1990); ST (2-sulfanil-amido thiazole) (Mitsui Toatsu, 1968); CS, (Ashworth et ul., 1977); 2-mer-
RAJENDRA PRASAD AND J. F. POWER
capto- 1,2,4-triazole, sodium diethylthiocarbamate; 2,5-dichloroaniline; 4-amino1,2,4-triazole(Bundy and Bremner, 1973);C2H,(acetylene) (Hynes and Knowles,
1981; Berg et al., 1982); gaseous hydrocarbons such as C,H, (ethane), C,H,
(ethylene), and CH, (methane) (McCarty and Bremner, 1991); ammonium thiosulfate (Goos, 1985); and thiophosphoryl triamide (Radel er al., 1992). Of these,
only eight (NP, AM, DCD, ST, TU, Dwell, MBT, and C2H,) have been widely
In addition to specific chemicals, allelochemicals also have nitrification-inhibiting properties. For example, Rice ( 1984) postulated that because inhibition of
nitrification results in conservation of both energy and nitrogen, vegetation in late
succession or climax ecosystems contains plants that release allelochemicals that
inhibit nitrification in soil. However, a critical appraisal of the available information does not lend support to such a hypothesis (Bremner and McCarty, 1993). As
an example, terpenoids thought to be released by a ponderosa pine (Pinusponderosu Dougl.) and supposed to inhibit nitrification in soil had no such effects
(Bremner and McCarty, 1988). However, some natural products are reported as
nitrification inhibitors. These include “neem” (A. indica Juss.) cake or an acetone/alcohol extract of seed (Reddy and Prasad, 1975; Sahrawat and Parmar,
1975) and “karanj” (Pongarnia glabra Vent.) seed, bark, and leaves (Sahrawat
et a!., 1974).
OF M S
Rajale and Prasad (1970) found AM as effective as NP, while Bundy and Bremner (1973, 1974) found that AM was less effective than NP and DCD. Sommer
(1970) compared a number of NIs and ranked them in the following order:
Terrazole > NP > DCS > guanylthiourea > AM > MAST (2-amino-4-methyl6-trichloromethyltraizine) > ST. McCarty and Bremner (1989) compared 12
compounds and found 6 of them to be effective NIs: 2-ethynylpyridine > Dwell >
NP > MPC > ATC > DCD (Table I).
In a number of U.S. studies NP and DCD were found to be equally effective. In
their studies in Illinois, Malzer et al. (1989) at Urbana, Monmouth (Typic Haplaquolls), and Dekalb (Aquic Arguidoll), showed that the disappearance of ammonium was similar between DCD ( 5 % DCD-N) and NP (0.5 kg ha ’). At Brownston (Mollic Albaqualf), however, ammonium disappearance was slower with
DCD than with NP. Bronson et al. (1989) from Alabama reported that DCD in
Norfolk loamy sand (Typic Paleudult) was equal to NP for up to 42 days, but was
less effective than NP in Decatur silt loam (Rhodic Paleudult).
Etridiazole and NP are equally effective in reducing nitrification of ammonium
N in soils up to 160 days after application on silty loam soils (Typic Ochraqualfs
and Aquic Hapludalfs) in Illinois (Shyilon et al., 1984).
Blending of urea with neem cake inhibited nitrification by 70, 40, and 5% at
Effects of 5 mg kg-' Soil with Different Compounds on Nitrification of
Ammonium in Soils"
3-Methylpyrazole- I -carboxamide
4-Amino- I ,2,4-triazole
"Samples of soil (20 g) were incubated at 25°C for 25 days after treatment
with 6 ml water containing 4 mg N as ammonium sulfate and 0 or 100 y g of
the compound specified. Adapted from McCarty and Bremner (1989).
the end of 1,2, and 3 weeks of incubation, respectively; the corresponding figures
for NP at 1% of N were 85,93, and 90% (Reddy and Prasad, 1975). Thomas and
Prasad (1982) evaluated neem cake-coated urea on a number of soils (Entisols,
Vertisols, Ultisols) and found it to be 50% as effective as NP. The active compounds in neem responsible for retardation of nitrification are thought to be meliacins (epinimbin, nimbin, desacetyl nimbin, salanin, desacetylsalanin, and azadirachtin) (Devkumar, 1986). Nitrification retardation after 2 weeks was 73.6,44.6,
and 12.5% for NP, epinimbin, and desacetylnimbin, respectively (Devkumar and
Neem cake and DCD were evaluated for their efficiency in inhibiting nitrification of prilled urea-derived NH,+-N in a wheat field (Joseph and Prasad,
1993a,b). Prilled urea was blended with 10 and 20% DCD-N or with 10 and 20%
neem cake and incorporated into the soil just before the wheat was sown. Both
DCD and neem cake partially inhibited the nitrification of prilled urea-derived
NH,; DCD was better than neem cake. The nitrification-inhibiting effects of DCD
lasted for 45 days, while that of neem cake lasted for only 30 days.
Most NIs inhibit nitrification by retarding the oxidation of NH,+-N to
NO,--N by Nitrosomonas sp. Research with different strains of Nirrosomonas
RAJENDRA PRASAD AND J. F. POWER
D A Y S
Figure 1. Effect of dicyandiamide (DCD),nitrapyrin (NP), and thiourea (TU)on the activity of
from Zacheri and Ainberger ( 1990).
Nitrosomonas eurupoeo in pure culture. Adapted
sp. showed remarkable differences in sensitivity to nitrapyrin (Belser and
Schmidt, 1981), and it was concluded that NP does not retard the activity of the
entire population of Nitrosomonas sp. Results of a study done by Zacheri and
Amberger (1990) on the effect of three NIs are shown in Fig. 1. Growth of a pure
culture N . europaea was completely suppressed by 10 ppm NP or 0.5 ppm TU;
inhibition by 300 ppm DCD was 83%. Ammonium oxidation and respiration of
Nitrosomonas cell suspensions were reduced by 93% with 10 ppm NP, 95% with
0.5 ppm TU, and 73% with 300 ppm DCD. When used at 1000 ppm, DCD had
bacteriostatic effects. Enzymatic investigations revealed that hydroxylamine oxidoreductase was not affected by high concentrations of inhibitors (200 ppm DCD,
100 ppm TU). Cytochrome oxidase activity was increased 10% with 200 ppm
DCD, was not affected by 100 ppm TU, and was inhibited by 52% with 100 ppm
NP. These results suggest that different NIs probably have different modes of
OF N I S
A number of studies have investigated the effect of different soil factors on the
effectiveness of NIs, and this subject has been well reviewed by Slangen and Kerkhoff (1984). The main findings are summarized below.
1. Organic Matter
Hendrickson and Keeney ( 1979b) found complete inhibition of nitrification
with NP at 0.5 mg kg - I in a soil with 1% organic matter and none in the same
soil when organic matter was raised to 5% by adding active carbon. Similar results
were obtained by McClung and Wolf (1980) with NP and terrazole when they
added compost to the soil. The influence of organic matter is probably due to its
effect on sorption and rate of decomposition of the chemical.
Most reports suggest that nitrification inhibitors are more effective at relatively
low temperatures, i.e., below 20°C (Goring, 1962a; Bundy and Bremner, 1973).
This is mainly due to the effect of temperature on degradation of a NI and the
consequent persistence. Herlihy and Quirke (1975) found that the half-life of NP
was 43 to 77 days at 10"C and 9 to 16 days at 20°C. Touchton et al. (1979) found
the half-life of NP to be 22 days at 4" C and less than 13 days at 2 1" C for a loamy
soil with pH 6.8 and an organic matter content of 2%. In a soil with pH 5.5 and
an organic matter content of 5%,the half-life of NP was 92,44, and 22 days at 4,
13, and 2 I " C, respectively. Touchton ef a/. ( 1979) reported that the half-life of
NP in a Cisne silt loam was 7 days at 2 1"C and 22 days at 8" C.
DCD is highly sensitive to temperature. Vilsmeier (1980) reported that after
60 days, 0.67 mg DCD-N 100 g I soil degraded to 0.60 mg at 8" C, 0.4 at 14" C,
and 0.1 mg at 20" C in a sandy silt loam soil of Germany with a pH of 6.2. Bronson
et a/. ( 1989) found that the half-life of DCD decreased from 52.2 days at 8" C to
22 days at 22°C in Norfolk loamy sand and from 25.8 days at 8°C to 7.4 days at
22" C in Decatur soils. Data of McCarty and Bremner (1989) for Iowa soils
showed that in 28 days inhibition of nitrification decreased from 72% at 15"C to
19% at 30°C in Harps silty clay soil when DCD was added at 10 mg kg - I soil
(Table 11). At 30°C the inhibitory effect at 10 mg kg - ' of soil with etridiazole
exceeded that at 100 mg kg soil with DCD and the inhibitory effect of 10 mg
Influence of Soil Temperature on Effectiveness of Dicyandiamide (DCD) for
Inhibition of Nitrification of Ammonium in Soils"
(mg kg ' soil)
Soil temperature ("C)
% inhibition of nitrification
"Samples of soil (20 g) were incubated at 15, 20, or 30°C for 28 days after treatment
with 6 ml water containing 4 mg N as ammonium sulfate and 0.0.2, or I .O mg of DCD.
Adapted from McCarty and Bremner (1989).
RAJENDRA PRASAD AND J. F. POWER
k g - ' soil with NP exceeded that at 50 mg kg - I soil with DCD (McCarty and
Bremner, 1989). In Illinois, DCD and NP were equally effective on Drummer silty
clay loam at 7.2"C. but DCD was more effective than NP at 15.5"C (Sawyer,
The influence of soil pH on the persistence of NP is reported to be minimal
(Hendrickson and Keeney, 1979a). This can be expected since a number of genes
of nitrifying organisms are involved in nitrification, each with different pH optima
(Bhuija and Walker, 1977).
4. Soil Water
Hydrolysis of NP is enhanced in water-saturated soils (Hendrickson and Keeney, 1979a) as compared to aerobic conditions in soils at field capacity (0.01 to
0.033 M Pa). Volatilization of NP is more pronounced in wet than in dry soils
(McCall and Swann, 1978).
In addition to these factors, method and time of fertilizer application and source
of N used can affect the effectiveness of NIs under field conditions (Singh and
Prasad, 1985; Sudhakara and Prasad, 1986a; Thomas and Prasad, 1987).
S AND NITROGENLOSSES
1. Urea Hydrolysis
Most of the NIs, such as NP, AM, ST, ATC, KN3, CS,, and DCD, have little
effect on urea hydrolysis. However, TU, ammonium, and potassium ethylxanthate
and thiosulfate retard urea hydrolysis (Mahli and Nyborg, 1979; Goos, 1985; Ashworth ef al., 1980).
2. Ammonia Volatilization
Since NIs retard nitrification, ammonium-N can accumulate and result in a
higher soil pH (Bundy and Bremner, 1974), which is conducive to NH3 volatilization. Enhanced NH3 volatilization losses due to application of NI have been reported (Bundy and Bremner, 1974; Smith and Chalk, 1978; Prakasa Rao and Puttanna, 1987). While Bundy and Bremner (1974) reported a 28-34% N loss from
volatilization of added urea N with a NI (NP, ATC, CL- 1850) and 9% without,
Smith and Chalk (1978) found NH3-N losses of 86 and 92 mg kg I soil without
and with NP. High NH3-N losses reported by Bundy and Bremner (1974) could
be due to high rates of N applied (400 mg N kg - I soil). Volatilization losses of
NH, with or without NI can be reduced by incorporation of the fertilizer N. Clay
et al. (1990) reported that NH, volatilization from bare soil was lower with urea
and DCD than with untreated urea. However, when the soil surface was covered
with residue, NH, volatilization was similar with or without DCD. Sudhakara and
Prasad (1 986a) reported that when 120 kg N ha ' was applied 20 days after sowing rice, the NH, volatilization loss was 8.37% of the applied N from urea compared to 3.89% with neem cake-coated urea. Thus at rates of N generally applied
in field crops, an increase in NH, volatilization due to NIs can be considerably
reduced by the incorporation of fertilizer N and NIs in soil. Another possibility
for reducing NH, volatilization is the use of dual purpose (NIIurease inhibitor)
compounds such as thiophosphoryl triamide (Radel et ai., 1992).
By retarding nitrification, NIs slow down and reduce the potential for N loss by
denitrification as N,O or NZ;this, however, should not be confused with the reduction of denitrification per se. For example, some workers reported that NIs
(NP, DCD, NaN,, Dwell, KN3, ST, PM, ATC) directly retard denitrification
(Mitsui et ul., 1964; Henninger and Bollag, 1976; McElhannon and Mills, 1981),
especially when added at the rate of 50 or 100 mg kg ~I soil (Bremner and Yeomans, 1986). Such rates are too high for general applications to field crops. Bremner and Yeomans (1986) evaluated the effect of 28 NIs and found that only KN,
and 2,4-diamino-6-trichloromethyl-Striazine, when added at the rate of 50 mg
kg ~I soil, inhibited denitrification. The other NIs had no appreciable effect on
4. Nitrogen Losses from Plants
Plants also lose some amount of N from the foliage (Wetselaar and Farquhar,
1980; Patron et al.. 1988; Francis et al., 1993). A high loss of N was observed by
Daiger e t a / . (1976) from winter wheat at different locations in western Nebraska,
following different rates of N application. In general, dry matter and N content of
tops and roots reached a maximum at anthesis. Thereafter, dry matter declined by
about lo%, while losses of N from the tops plus roots ranged from about 20 to
over 60% depending on the fertilizer N rate. Tanaka and Navasero (1964) reported
a loss of 47 kg ha I in the N content of rice tops in the 3 weeks before flowering
and maturity at high N rates. Patron et ai. (1988) reported a NH, loss of 60120 ng N m - > sec - I from spring wheat plants during the presenescence time
period (before milk stage) and 200 to 300 ng m - ? sec ~I during final plant senescence. They found that NH, loss rates on a leaf area basis were similar for the low
and high N plants despite significantly higher N concentrations in high N plants.
RAJENDRA PRASAD AND J. F. POWER
Twice the leaf area was attained by the high N plants, resulting in similar NH,
volatilization rates per plant which translates into nearly twice as high on a plant
N basis for the low N plants. Farquhar et al. (1979) reported an evolution of
0.6 nmol m -,sec - I (36 g N ha I day I at LA1 5) from senescing leaves of corn.
In a study at Lincoln, Nebraska, postanthesis fertilizer N losses as NH, from the
aboveground biomass of corn plants ranged from 10 to 25% of the fertilizer applied (Francis et al., 1993); the apparent total N losses from the aboveground plant
material ranged from 49 to 81 kg N ha-'. Francis er al. (1993) observed that
postanthesis N losses from aboveground plant biomass in corn accounted for 52
to 73% of the total unaccounted for fertilizer N and suggested that failure to include such losses can lead to overestimation of N losses from soil by denitrification and leaching.
Mosier er al. (1990a,b) reported a N, + N,O gas flux of 270 g N ha - I
day ~I 15 days after transplanting rice where plants were included in the measuring chamber as compared to only 240 g N ha - I day - I when the plants were not
included in the chamber. They concluded that young rice plants facilitated the
efflux of N, and N,O from the soil to the atmosphere. Effects of NI on such losses
of N from the plants have so far not been reported.
Immobilization of fertilizer N by soil microorganisms is significantly enhanced
in the presence of a nitrification inhibitor (Osiname et al., 1983; Juma and Paul,
1983). This has been attributed to the NIs maintaining more of the applied fertilizer N as NH,+ for a longer period of time (Prasad et al., 1983; Shyilon et al.,
1984; Norman and Wells, 1989) and preferential utilization of NH,+-N by heterotrophic microorganisms (Broadbent and Tyler, 1962; Alexander, 1977). Bjarnason
(1987) reported that not only is NH,' preferentially immobilized, but its remineralization is at a slower rate.
Norman and Wells (1 989) found that immobilization of fertilizer N by soil microorganisms in a Crowley silt loam (Typic Albaquelf) in Arkansas was approximately the same in urea and urea DCD-amended soils during the 4-week period
when the soils were not flooded (Fig. 2 ) . Immobilization appeared to level off after
2 weeks and stayed relatively constant for the remaining 2 weeks. After flooding,
immobilization of fertilizer N was much greater in the urea + DCD-amended soil
than in the urea-amended soil, and by the end of 8 weeks soil with urea DCD
had nearly 1.5 times more NH,+ than that treated with urea only. Osiname et al.
(1983) reported more N immobilization with NP than with DCD. Preferential immobilization of NH,+-N rather than NO,- -N has been suggested by a number of
researchers (Wickramsingha et al., 1985; Rice and Tiedje, 1989). This would support higher immobilization of fertilizer N with NIs.
Budot and Chone (1985) suggested an interesting pathway of nitrite incorpo-
INCUBATION TIME ( w e e k s )
Figure 2. Immobilization of fertilizer N under nonflooded and flooded conditions. Adapted from
Urea; (A)urea + DCD.
Norman and Wells (1989). (0)
ration into the organic N fraction via nitrite self decomposition and fixation on
organic matter in a humic-rich acid forest soil (pH 4.5; organic matter 46%).
Azhar et al. (1986a,b,c) also supported this pathway. NP not only reduced the loss
of nitrite via chemodinitrification, but Nelson ( 1982) also discussed the incorporation of nitrite into the organic N fraction.
Thus it appears that NIs increase immobilization of N by increasing the persistence of ammonium-N. Also, NIs retard nitrite accumulation in soils and thus
reduce fixation of nitrites into organic matter.
III. NIs, NH,+/NO,- RATIOS, AND PLANT GROWTH
Because NIs maintain a higher concentration of NH,' in the soil/solution for a
longer time by retarding nitrification, these chemicals have a role in determining
amounts of NH,+ and NO,- and their ratios to crop plants during different stages
of crop growth.
Ammonium can be more efficiently metabolized than NO, -N because it does
not need to be reduced when incorporated into amino acids or other organic materials. However, NH,', or rather NH;', is toxic to all plants at certain concentrations (Magalhaes and Wilcox, 1984) and this toxicity is related to the pH of the
growing media (Pill and Lambeth, 1977). Magalhaes and Huber (1989) reported
that NH,' toxicity was more severe at lower (3.5) than at higher pH (5.7). There
is also a difference between crops with respect to tolerance to NH,'. Prasad et al.
RAJENDRA PRASAD AND J. F. POWER
WEEKS AFTER FERTILIZER APPLICATION
Figure 3. Ammonium-N and nitrate-N concentration in maize and rice soils. 0,without nitranitrate-N. Adapted from Prasad ef al. (1983).
pyrin; 0,with nitrapyrin; -, ammonium-N; -.-,
(1983) suggested the term "ammoniphilic plants" for species growing better with
NH,'. They maintained high concentrations (40-60 mg kg I NH,+-N soil) using NP (Fig. 3) and found that while maize plants suffered in growth, rice plants
did not (Table 111). Rice absorbed more N with NH4+,while maize absorbed less
N in the presence of higher concentrations of NH,'. They identified rice as an
ammoniphilic plant. Other species of ammoniphilic plants are known (Gigon and
Plant Height and Dry Matter Accumulation in Rice and Maize Plants
Affected by N-Serve (NP) Treatment"
Plant height (cm)
(g per plant)
LSD ( P= 0.05)
"Adapted from Prasad e t a / . (1983).