Plant Nutrient Management in Hawaii’s Soils

From: Plant Nutrient Management in Hawaii’s Soils, Approaches for Tropical and Subtropical Agriculture
J. A. Silva and R. Uchida, eds. College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, ©2000

Chapter 13

Biological Nitrogen Fixation

Nature’s Partnership for Sustainable Agricultural Production

NifTAL Center for BNF Technologies

M any agriculturally important plants in the legume

family can use nitrogen (N) from the atmosphere
for growth through biological nitrogen fixation (BNF).
Nature has an alternative method of providing N
to plants and enriching soil N resources—biological
nitrogen fixation. Many members of the legume plant
Legume BNF involves a symbiosis between legume family, such as beans, peas, alfalfa, and leucaena, have
plants and the rhizobia that live in nodules on their roots. the special ability to use BNF to meet their N needs.
There are economic, environmental, and agronomic Legume BNF involves a remarkable symbiosis, or
benefits from using BNF in cropping systems. mutually beneficial relationship, between the plant and
All living things require nitrogen. It is a key ele- N-fixing soil bacteria called rhizobia. The rhizobia in-
ment of amino acids, the building blocks of proteins. vade the host plant’s roots and cause the formation of
There is a huge reservoir of N in the atmosphere. Ap- structures called nodules. Within these nodules, the
proximately 80% of the air consists of nitrogen gas (N2), rhizobia use enzymes to biologically convert (“fix”)
but plants cannot use atmospheric N directly to make N2 gas from the air into a form of N that can be used by
protein. The gaseous N must first be converted, or its plant host to make proteins. In turn, the plant pro-
“fixed,” into forms plants can use. vides the rhizobia with products of photosynthesis:
Unless fertilizer N is applied, most plants obtain sugars and carbohydrates that fuel the bacteria and the
their nitrogen from natural sources in the soil. Natural BNF process (see Figure 13-1).
reserves of soil N are normally low, so N fertilizers
must be added to increase plant growth. “Fixing” the Benefits of using BNF
N from the air to manufacture fertilizers like urea re- Economics: BNF reduces costs of production. Field
quires extremely high temperatures and pressures, and trials have shown that the N captured by crops due to
the use of fossil fuels. Because the energy requirements the use of rhizobia inoculants costing $3.00/ha is equal
for production are high, and N is needed in large to fertilizer N costing $87.00.
amounts, N fertilizers are an expensive input for crop Environment: The use of inoculants as alternatives
production. to N fertilizer avoids problems of contamination of

121
College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa

Figure 13-1. The legume-rhizobia symbiosis.

1 Carbon dioxide, water, and solar energy

fuel plant photosynthesis

2 4
Products of photosynthesis Fixed N in the form of
from the plant fuel N2 fixation ammonia produced by
by rhizobia in root nodules rhizobia is used by the
plant

3 N2 gas in the air is “fixed” by rhizobia

water resources from leaching and runoff of excess fer- peas, beans, and peanuts; forage crops like alfalfa and
tilizer. Utilizing BNF is part of responsible natural re- clover; and useful trees like leucaena and acacias. By
source management. inoculating legume seeds with appropriate rhizobia,
Efficiency: Legume inoculants do not require high farmers can ensure that they take advantage of the ben-
levels of energy for their production or distribution. efits of BNF listed above. Estimates of the amounts of
Application on the seed is simple compared to spread- N fixed by various legumes are listed in Table 13-1.
ing fertilizer on the field. Long-term leguminous tree
crops are self-sustaining through BNF. Inoculating legumes with rhizobia
Better yields: Inoculants increase legume crop to maximize BNF
yields in many areas. BNF often improves the quality
of dietary protein of legume seed even when yield in- • Many soils do not have sufficient numbers of appro-
priate rhizobia for maximum BNF.
creases are not detected.
Increased soil fertility: Through practices such as
• Rhizobial inoculants and legume crops must be prop-
erly matched.
green manuring, crop rotations, and alley cropping, N-
fixing legumes can increase soil fertility, permeability, • There are several methods of inoculating legumes.
and organic matter to benefit nonlegume crops. • Inoculants require some special care to maintain their
viability.
Sustainability: Using BNF is part of the wise man-
agement of agricultural systems. The economic, envi- Although BNF is a natural process, many soils do
ronmental, and agronomic advantages of BNF make it not have sufficient numbers of appropriate rhizobia for
a cornerstone of sustainable agricultural systems. effective symbioses. Inoculating legume crops with
Legumes comprise one of the most important plant compatible rhizobia ensures maximal BNF. Inocula-
families in agriculture. Nitrogen-fixing members of this tion is especially important when introducing new
family include important food grains like soybeans, legumes to an area.

122 Biological Nitrogen Fixation

 Plant Nutrient Management in Hawaii’s Soils

the seed. Commercial stickers are recommended. If they

Table 13-1. Estimates of the amount of nitrogen fixed by
various legumes (FAO 1984).
are not available, a 10% sugar-water solution or veg-
etable oil can be used. Farmers should follow the in-
Nitrogen fixed oculation rates and procedures described by the inocu-
Plant Scientific name (kg N/ha/yr) lant producers.
Sources of commercial inoculants are listed at the
Horse bean Vicia faba 45 –552
Pigeon pea Cajanus cajan 168 –280 end of this section on BNF. Like fertilizers, inoculants
Cowpea Vigna unguiculata 73 –354 are inputs that can increase plant growth, but they are
Mung bean Vigna mungo 63 –342 very different than chemical fertilizers in several re-
Soybean Glycine max 60 –168 spects. Some important points to consider when using
Chickpea Cicer arietinum 103
Lentil Lens esculenta 88 –114
inoculants:
Peanut Arachis hypogaea 72–124 • Rhizobia are living organisms. Use inoculants be-
Pea Pisum sativum 55 –77 fore their expiration dates, and protect them from
Bean Phaseolus vulgaris 40 –70 sun and heat.
Leucaena Leucaena leucocephala 74–584
Alfalfa Medicago sativa 229 –290
• Never expose inoculants to temperatures above 35°C
Clover Trifolium spp. 128 –207 or below freezing. Inoculants should be stored in
sealed bags at 4–25°C.
• Inoculate seeds just before planting and protect the
coated seeds also from heat. Cover furrows and, if
possible, irrigate soon after planting.

Rhizobia and legumes have specific requirements,

and they must be properly matched. Legumes that are
effectively nodulated by the same rhizobia belong to
the same “cross-inoculation” group. Commercial in-
Inoculation is simply the process of applying suit- oculants contain tested, selected rhizobia that are ef-
able live rhizobia to the soil where they can infect the fective on legumes in specific groups listed on the la-
roots and form effective nodules. Inoculant producers bel. Always check the label to be sure you have the
grow pure cultures of selected, highly effective strains proper inoculant: different legumes require different
of rhizobia. The cultures are usually mixed with a car- inoculants.
rier material like powdered peat. Each gram of high-
quality peat inoculant can contain a billion rhizobia. Common methods of seed inoculation
There are several inoculation methods. Granular (The inoculant producer usually provides specific di-
inoculants can be applied directly to the field. Pow- rections for the use of the product.)
dered inoculants can be mixed with water for irrigat- The two-step method: Seeds are first coated with
ing the soil. This liquid application is suitable for in- a sticker solution at a rate of about 15 milliliters/kg
oculating trees in a seedling nursery. By far, the most seed, depending on the size of the seed. Inoculant is
common method of inoculating crops is to coat the seed added to the sticky seeds at a rate of 5–20 g/kg seed.
with the powdered inoculant. The rhizobia are then The slurry method: Powdered inoculants are
“planted” in the soil with the seed, and can infect the mixed with water or sticker solution to form a slurry.
emerging root as the seed germinates. Some dealers Seeds are mixed with the slurry until they are coated.
sell preinoculated seed, but these are not recommended Seed pelleting: Seeds are first inoculated by the
because the viability of the rhizobia is often low by the slurry or two-step method. Before the seeds dry, they
time they are planted. are mixed with finely ground, sifted lime or phosphate
There are several general variations of the seed in- to form a protective pellet.
oculation process. While the simplest is to dust the seeds Seed dusting: Powdered inoculants are simply
with the powdered inoculant, the use of a liquid shaken over the seeds. This method is the least satis-
“sticker” is recommended so the inoculant adheres to factory because relatively few rhizobia stick to the seed.

123
College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa

For example, Figure 13-2 shows that there was little dif-
Figure 13-2. Response of a soybean crop in Hawaii to
inoculation with rhizobia, with various levels of phos-
ference between yields of inoculated and uninocu-lated
phorus fertilizer added. soybeans in a P-deficient soil in Hawaii when no phos-
phorus was added, yet when 50 or more kg/ha of P was
applied, inoculation resulted in dramatic yield increases.
This example demonstrates an important point:
Seed yield (1000 kg / ha)

2 BNF is dependent on the plant’s total growth potential.

This type of study can help to identify situations in
which farmers can get the maximum benefit from their
inputs and from BNF. Phosphorus is the most limiting
1 nutrient in the soil in which the experiment shown in
Figure 13-2 was conducted. When the lack of P limits
plant growth, as shown in Figure 13-2 when zero P
was applied, inoculation has little effect on yields. When
0 P is added to the soil, N becomes the most limiting
P0 P50 P100 P200
nutrient, and inoculation results in large yield increases;
Phosphorus application (kg / ha)
however, the added P does not significantly improve
Inoculated Uninoculated the yields of the uninoculated plants. As the plant’s
growth potential improves with added P from P50 to
P100, the benefits of BNF also increase. Notice that when
P applications are increased from 100 to 200 kg/ha, the
benefits due to inoculation do not increase. At this point
Factors that affect a crop’s response to some other factor, possibly weather or the genetic yield
inoculation potential of the variety, limits further yield increases.
This “limiting factor” principle applies to other
• Inoculation failure may be caused by loss of viabil- factors that affect plant growth. Anything that limits
ity of the inoculant rhizobia. the yield of the legume will reduce the amount the
• Environmental and management factors that affect farmer will benefit from BNF. If the legume crop yield
the growth potential determine a plant’s BNF require- is limited by the availability of an essential nutrient
ment. like P, as in the previous example, or any other factor
• When soil N sources are high enough to meet the like soil pH, water, or pests, the crop will not require
crop’s N requirements, legumes will not use BNF. much N. Legumes will not form many nodules or fix
• Soils may have many native rhizobia that can infect much N when N requirements are low, and the addi-
the plant and fix N for the crop. tion of rhizobia through inoculants will not increase
yield.
Viability of inoculants In summary, farmers will get the most benefit from
Farmers in many parts of the world have received dra- inoculation when they combine it with good crop man-
matic increases in yields due to inoculating their le- agement. It is important to use inoculants with other
gumes, but positive results do not always occur. Inocu- agricultural inputs that increase plant health and yield.
lation failure is sometimes due to the loss of viability
of rhizobia in the inoculant due to exposure to heat or Available soil N
prolonged storage. Other causes may be improper han- Legumes can get their N from BNF and from available
dling, application, or planting methods that cause the N sources in the soil. Often they use both N sources —
rhizobia to die on the seed. fixation and the soil—to meet their N requirements.
Nitrogen in the soil comes from mineralized organic
Plant growth potential matter, manure, or residual fertilizer N from the previ-
If growth is limited by other factors, good inoculant ous crop. Legumes use these mineral sources of N be-
quality and proper application will not increase yields. cause it requires less energy for the plant to take up N

124 Biological Nitrogen Fixation

 Plant Nutrient Management in Hawaii’s Soils

directly from the soil than to fix N2 through BNF. If Effective nodules usually are large and clustered
there is enough N available in the soil to meet plant on the primary and upper lateral roots. When they are
requirements, there will be no added benefit from in- sliced open, they have a deep pink or red color inside.
oculation. If through good management the plant’s In contrast, ineffective nodules often are small, numer-
growth potential increases, the resulting increased N ous, and distributed throughout the root system. They
requirement for N may be met by BNF. have white or green interiors. A single plant may be
Although legumes use available soil N first, a nodulated by both effective and ineffective rhizobia.
farmer who can establish effective BNF with his le- Figure 13-3 shows commonly observed conditions
gumes will profit more than the farmer who adds fer- and how to diagnose them. Pot and field experiments
tilizer N to the crop. Legumes can have low fertilizer can provide further assessment of the need for inocula-
use efficiency, and the farmer would have to apply at tion and other management inputs that can improve
least two to three times as much N in fertilizer as can BNF. These types of experiments should be conducted
be fixed by the legume. For example, the farmer would with care to ensure that there are proper controls. For
have to add 200 to 300 kg/ha N in fertilizer to get the further information on conducting tests to assess BNF,
same yield as a crop that gets 100 kg/ha of N from contact the NifTAL Center for BNF Technologies, 1000
BNF. Holomua Rd., Paia, Hawaii, 96779, USA.

Native rhizobia in the soil Sources of inoculants

Some soils have high populations of native rhizobia High-quality legume inoculants can be obtained from
that are compatible with the legume crop. If the crop’s garden shops and other retailers of agricultural supplies,
requirement for BNF can be met by these native rhizo- or directly from the following producers:
bia, inoculation may not increase yield. Large native
rhizobial populations often occur when legume crops LiphaTech, 3101 W. Custer Ave., Milwaukee, Wiscon-
are grown in the same field for many crop cycles or sin 53209, USA. Phone (414) 462-7600, fax (414)
when crops have been previously inoculated and the 462-7186
rhizobia persist. Despite this occurrence, many farm- MicroBio RhizoGen, 3835 Thatcher Ave., Saskatoon,
ers continue to inoculate their legumes each season Saskatchewan S7R 1A3, Canada. Phone (306) 373-
because this practice is considered “cheap insurance.” 3060, fax 306 374 8510, e-mail <mbrsask@sk.
Native rhizobial strains may not be as effective as the sympatico.ca>.
tested strains in inoculants, and inoculation assures the Urbana Laboratories, P.O. Box 1393, St. Joseph, Mis-
farmer that there will be sufficient numbers of superior souri, 64502, USA. Phone (816) 233-3446, (800)
rhizobia for the crop. 892-2013, fax (816) 233-8295, Web <www.
seedsolutions.com>.
Assessing BNF
Legume BNF can be assessed by nodule color, obser- Acknowledgments
vations of plant growth, and field and pot trials. NifTAL is supported by the U.S. Agency for Interna-
Nodules should be visible on inoculated legumes tional Development to promote BNF for sustainable
within 21 to 28 days after planting. They should not be agricultural development. This publication is based on
confused with nematode galls. The nodules can be de- the following references:
tached from the roots, unlike the galls, which are actu- Burton, J. 1984. Legume inoculants and their use. Food
ally root swellings. Root nodules have characteristic and Agriculture Organization, Rome.
shapes, depending on the legume species. For example, Singleton, P., P. Somasegaran, P. Nakao, H. Keyser, H.
soybeans, cowpeas, and peanuts have spherical nod- Hoben, and P. Ferguson. 1990. Applied BNF tech-
ules; clovers and leucaenas have elongated, finger-like nology: a practical guide for extension specialists.
nodules; and chickpea and alfalfa nodules are fan or NifTAL, Paia, Hawaii.
coral shaped. The presence of many effective nodules
on or near the tap root generally indicates that a quality
inoculant has been properly applied to the crop.

125
College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa

Figure 13-3. Legume growth conditions commonly observed in farmers’ fields, and the explanations for them.

126 Biological Nitrogen Fixation