15

CHAPTER 2

REVIEW OF LITERATURE

Pea (Pisum sativum L.), belongs to the Legumes (subfamily: Faboideae, tribe:

Fabeae and family: Fabaceae), which has an important ecological advantage because it

contributes to the development of low-input farming systems by fixing atmospheric

nitrogen and it serves as a break crop which further minimizes the need for external

inputs. Legumes constitute the third largest family of flowering plants, comprising more

than 650 genera and 18,000 species. Economically, legumes represent the second most

important family of crop plants after Poaceae (grass family), accounting for

approximately 27% of the world's crop production. Dry pea currently ranks second only

to common bean as the most widely grown grain legume in the world with primary

production in temperate regions and global production of 10.4 M tonnes in 2009. Pea

seeds are rich in protein (23–25%), slowly digestible starch (50%), soluble sugars (5%),

fiber, minerals and vitamins. On a worldwide basis, legumes contribute about one-third

of humankind's direct protein intake, while also serving as an important source of fodder

and forage for animals and of edible and industrial oils. One of the most important

attributes of legumes is their capacity for symbiotic nitrogen fixation, underscoring their

importance as a source of nitrogen in both natural and agricultural ecosystems. Legumes

also accumulate natural products (secondary metabolites) such as isoflavonoids that are

considered beneficial to human health through anticancer and other health-promoting

16

activities. Pea has also been a model system in plant biology since the work of Gregor

Mendel.

The fundamental discoveries of Mendel and Darwin established the scientific basis

of modern plant breeding in the beginning of the 20th century. Similarly, current progress

in molecular biology, genetic and biotechnology has revolutionized plant breeding,

allowing a shift toward molecular plant breeding and adding to its interdisciplinary

nature. However, although the methods have been available for over a decade, there is

still a large gap between plant biologists engaged in basic research and plant breeders. In

this review we summarize the current status of pea genetics, genomics and molecular

biology in a format relevant for application to pea breeding.

Origin of Pea:

Pea (Pisum sativum L.) is one of the world’s oldest domesticated crops. Its area of

origin and initial domestication lies in the Mediterranean, primarily in the Middle East.

Prior to cultivation, pea together with vetches, vetchlings and chickpeas was part of the

everyday diet of hunter-gatherers at the end of the last Ice Age in the Middle East and

Europe. Remains of these legumes occur at high frequencies in sites dating from the 10th

and 9th millennia BC suggesting that domestication of grain legumes could even predate

that of cereals. Thus, grain legumes were fundamental crops at the start of the

‘agricultural revolution’ which facilitated the establishment of permanent settlements.

Subsequently, during centuries of selection and breeding thousands of pea varieties were

developed and these are maintained in numerous germplasm collections worldwide. The

range of wild representatives of P. sativum extends from Iran and Turkmenistan through

17

Anterior Asia, northern Africa and southern Europe. However, due to the early

cultivation of pea it is difficult to identify the precise location of the center of its

diversity, especially considering that large parts of the Mediterranean region and Middle

East have been substantially modified by human activities and changing climatic

conditions. Moreover, reliable and thorough passport data are often missing or

incomplete for the early accessions that were collected. The genus Pisum contains the

wild species P. fulvum found in Jordan, Syria, Lebanon and Israel; the cultivated species

P. abyssinicum from Yemen and Ethiopia, which was likely domesticated independently

of P. sativum; and a large and loose aggregate of both wild (P. sativum subsp. elatius)

and cultivated forms that comprise the species P. sativum in a broad sense.

Importance of the genus Rhizobium:

Plant growth is often limited by the amount of available nitrogen when other soil

nutrient deficiencies have been corrected by amendments or fertilizations. The rhizobia

are able to supply available nitrogen to the soil by fixing the atmospheric nitrogen gas

into organic compounds. The members of the genus, Rhizobium are non-spore forming

Gram negative rods, usually containing poly-hydroybutyrate granules observable under

phase contrast microscopy. These organisms occur as free-living microorganisms in soil

or as micro-symbionts in root nodules of leguminous plants.

Rhizobia in root nodules are estimated to carry out between 50 to 70% of the

world’s biological nitrogen fixation, and the estimated annual biological fixation of

18

atmospheric nitrogen varies between 100 x 106 and 180 x 106 metric tons per year

(Burris and Roberts, 1993). .

Biological nitrogen fixation has of particular importance in agriculture.

Leguminous plants that fix nitrogen well may grow on soils that are poor in available

nitrogen, reducing the amendments with expensive nitrogen fertilizers (Burris and

Roberts, 1993). Leguminous plants are also of crucial importance as animal feed.

Alfalfa and clovers are grown over extensive areas as forage crops for grazing or as dry

hay, and they furnish not only high quality protein but also a variety of biologically active

molecules such as vitamins, minerals and other nutrients (Burris and Roberts, 1993).

Despite the fact that this process is free, self-sustaining and non-polluting, it does

not necessarily operate with optimum efficiency. Nitrogen fixation requires a significant

amount of energy by the cell and by the whole plant. It is possible to enhance the

nitrogen fixation in the Rhizobium-legume symbiosis by selecting host plant phenotypes

as well as effective rhizobial strains.

Although some soils contain high numbers of indigenous clover rhizobia the

introduction of superior nitrogen fixing strains is still considered an important

management practice (McInees et al., 2004). However, the inoculant strains may be

susceptible to loss of symbiotic traits such as infectiveness and effectiveness due to

environmental factors, and may not be competitive with the indigenous strains already

present in the soil (Amarger, 1981).

19

Need of legume crop for human being:

Legumes, broadly defined by their unusual flower structure, podded fruit, and the

ability of 88% of the species examined to date to form nodules with rhizobia (de Faria et

al., 1989), are second only to the Graminiae in their importance to humans. The 670 to

750 genera and 18,000 to 19,000 species of legumes (Polhill et al., 1981) include

important grain, pasture, and agroforestry species. Cohen (1977) reported domestication

of lentils (Lens esculenta) at a site in Iran dating to 9,500 to 8,000 B.P. Roosevelt et al.

(1996) noted the use of Hymenaea as a food source in Amazonian prehistory. Bean

(Phaseolus vulgaris) and soybean (Glycine max), the staple crops in the Americas and

Asia respectively, were each domesticated more than 3,000 years ago (Kaplan and

Lynch, 1999). Use of legumes in pastures and for soil improvement dates back to the

Romans, with Varro (Fred et al., 1932) noting “Legumes should be planted in light soils,

not so much for their own crops as for the good they do to subsequent crops.” This paper

briefly overviews the legumes and their importance in different agricultural and natural

environments.

Grain and pasture production:

Grain and forage legumes are grown on some 180 million Ha, or 12% to 15% of

the Earth's arable surface. They account for 27% of the world's primary crop production,

with grain legumes alone contributing 33% of the dietary protein nitrogen (N) needs of

humans (Vance and Hartley, 2000). Under subsistence conditions, the percentage of

legume protein N in the diet can reach twice this figure. In rank order, bean, pea (Pisum

20

sativum), chickpea (Cicer arietinum), broad bean (Vicia faba), pigeon pea (Cajanus

cajan), cowpea (Vigna unguiculata), and lentil constitute the primary dietary legumes

(National Academy of Science, 1994). Legumes predominantly soybean and peanut

(Arachis hypogeaea) provide more than 35% of the world's processed vegetable oil and

soybean and peanut are also rich sources of dietary protein for the chicken and pork

industries. The potential of legume crops is evident in the huge increase in soybean

production in Brazil, with national mean yields increased from 1,166 kg ha−1 in 1968 to

1969 to 2,567 kg ha−1 in 2001 to 2002 (Hungria and Bohrer, 2000; Hungria et al.,

2003). This followed selection for later maturity, aluminum tolerance, and calcium-use

efficiency (Spehar, 1995). In the same crop, the controversy over molecular

engineering, with some countries refusing to grow transgenic soybean illustrates the need

for balance in future breeding activities.

Unfortunately, improvement in legume crop yields has not kept pace with those of

cereals. Jeuffroy and Ney (1997) note that wheat (Triticum aestivum) yields in France

increased 120 kg ha/year between 1981 and 1996; those for pea increased only 75 kg

ha−1 year−1 over the same period. The situation is worse in the developing countries

where Oram and Agcaoili (1994) noted that pea yields are only 45%, and faba bean and

chickpea are only 75%, of those achieved in developed countries. In part, this difference

is due to the unfavorable environmental conditions under which many legume species are

grown. Legumes are often grown after corn or rice and are seeded toward the end of the

growing season. They may have short growing seasons and may be subject to

21

intermittent or terminal drought. Progressive soil chemical and physical degradation and

acid soil conditions may also limit their productivity.

Drought problems for legumes are likely to worsen with the projected rapid

expansion of water-stressed areas of the world from 28 to 30 countries today to 50

countries encompassing 3 billion people by 2030 (Postel, 2000). There is a crucial need

to increase drought tolerance in legumes; increasing salinity tolerance is a parallel

requirement in many areas. The more drought-tolerant legumes, such as cowpea, are

deeply rooted and may have reduced leaf size with thickened cuticles to reduce water

loss. Less tolerant legumes such as beans can be selected for early maturity, efficiency

in the partitioning of nutrients toward reproductive structures, and phenotypic plasticity

(Beaver et al., 2003). Pinto Villa, now grown over 90% of the pinto bean area in Mexico,

has these characteristics.

Nutrient depletion of soil is a particular problem for small landholders in

developing countries, where much grain-legume production occurs, and many farmers

cannot afford to use fertilizers. Sanchez (2002) suggests average annual nutrient

depletion rates across 37 African countries of 22 kg N ha−1, 2.5 kg P ha−1, and 15 kg K

ha−1. Soil acidity affects more than 1.5 billion ha worldwide, with acid soil constraints to

legume production likely to increase as the result of acid rain, long-term N fertilization,

and natural weathering (Graham and Vance, 2000). H ion concentration per se, Al and

Mn toxicity, and P, Mo, or Ca deficiency all contribute to the problem (Graham, 1992).

22

Diseases and pests are also major constraints to legume production, especially in

the tropics and subtropics. In common bean, for example, important pathogens include

several viruses, fungi-causing root rots, anthracnose, angular leaf spot, bean rust, white

mold and web blight, and the bacteria responsible for common bacterial blight and halo

blight (Coyne et al., 2003). In Minnesota alone, losses due to root rot are estimated at $4

million annually. A number of these pathogens are seed transmitted; others can be

carried by insects. Limiting crop losses requires an integrated approach that may include

certified seed programs, fallow periods to reduce vector populations, plowing to bury

infected plant tissue, biological control of root disease, chemical application, and

resistance breeding (Beaver et al., 2003; Coyne et al., 2003). Molecular markers have

permitted rapid progress in disease resistance breeding in beans (Kelly et al., 2003), but

many of the measures suggested above are beyond the resources of the subsistence

farmer, which is another reason why legume yields in third-world countries are low.

Use of legumes in the human diet can also be problematic. Legume seeds

generally contain 20% to 30% protein and are Lys rich, complementing the nutritional

profiles of cereals and tubers in the diet (Duranti and Gius, 1997). However, legumes are

limited in sulfur amino acids, contain antinutritional factors including lectins and

flatulence factors, and are commonly hard to cook. Preference for particular grain types

or seed color also affects marketability.

Forage legumes have been the foundation for dairy and meat production for

centuries (Russelle, 2001). When properly managed, they are rich sources of protein,

23

fiber, and energy. Even in intensive animal and milk production, where grain crops are

major feed sources, forage legumes are required to maintain animal health (Wattiaux and

Howard, 2001). Meat and dairy production in developing countries is almost solely

dependent upon forage legumes and grasses. Alfalfa (Medicago sativa) is the prevalent

forage legume in temperate climates (Russelle, 2001), with more than 72 million Mg of

alfalfa worth $7 billion produced annually in the U.S. alone. Alfalfa is the third or fourth

most valuable crop in the U.S. Other important temperate pasture species include clovers

(Trifolium spp.), trefoil (Lotus corniculatus), sweetclovers (Melilotus spp.), and vetches

(Vicia spp.).

Inclusion of legumes is critical for sustainable meat and dairy production on the

infertile savannah soils of the tropics and subtropics (Consultative Group on International

Agricultural Research). Incorporation of improved legumes into these ecosystems has

lagged due to lack of information, seed costs, and poor infrastructure. Species from the

genera Aeschynomene, Arachis, Centrosema, Desmodium, Macroptilium, and

Stylosanthes offer promise for improved tropical pasture systems (Giller, 2001). Of

these, Stylosanthes spp. with some 30 species distributed throughout the tropics (de

Leeuw et al., 1994), has been most widely adopted, with Stylosanthes guyanensis and

Stylosanthes hamata now grown as improved pasture in Australia, China, Latin America,

and West Africa.

Presently under utilized crop and pasture legumes could still emerge. Ladizinsky

and Smartt (2000) address opportunities for improved adaptation via further

24

domestication. More exotic examples include marama bean (Tylosema esculentum;

Dakora et al., 1999), sword beans (Canavalia gladiate) (Ekanayake et al., 2000), and

Desmanthus illinoensis among grain crops, and annual medics and Biserrula pelecinus

among pasture species (Howieson et al., 1995; 2000). Again, germplasm collection and

evaluation must continue to be a research emphasis.

Ecological and economic importance of biological nitrogen fixation:

Nitrogen is an element of all proteins and is an essential component in both plant

and animal metabolism. Although elemental nitrogen makes up about 80 percent of the

atmosphere, it is not directly available to living organisms; nitrogen that can be

metabolized by living organisms must be in the form of nitrates or ammonia compounds.

Through a mutual benefit arrangement (symbiosis) between legumes and Rhizobium

bacteria, nitrogen gas (N2) is fixed into a compound and then becomes available to the

biotic world. The legume plant furnishes a home and subsistence for the bacteria in root

nodules. In a complex biosynthetic interaction between the host plant and the bacterium,

nitrogen compounds are formed that are used by the host plant. These compounds are

also available to other plants after decayed roots (and other plant parts) of the host plant

have allowed these nitrogen products to be released into the soil. Animals obtain

compound nitrogen by eating plants or other animals.

Consequently, the vegetation of the forests, prairies, and deserts of most of the

world is primarily dependent on the legume component of their vegetation and could not

exist without it. Only in a few ecosystems-—those that include few legume species—

25

have alternative biological nitrogen-fixing arrangements evolved. These include

symbiotic relationships between miscellaneous woody species other than legumes, and

certain actinomycetes or bacteria and are limited mostly to boreal evergreen forests,

certain coastal areas, and acid bogs. Nitrogen fixation by free-living cyanobacteria seems

to be important in aquatic ecosystems. On a worldwide scale, however, these alternate

arrangements of nitrogen fixation are relatively minor compared with those supported by

legumes.

Legume nitrogen fixation is of prime importance in agriculture. Before the use of

synthetic fertilizers in the industrial countries, the cultivation of crop plants, with the

exception of rice, was dependent on legumes and plant and animal wastes (as manure) for

nitrogen fertilization. A common procedure was the use of crop rotation, usually the

alternation of a cash grain crop such as corn (maize) with a legume, often alfalfa

(Medicago sativa), in the temperate world. Apart from the nitrogen contribution, the

legume in this case furnishes animal forage (hay or silage). Pastures or other grazing

areas must have legume components, such as a clover (Trifolium), as well as a grass

component.

The 20th-century substitution of petroleum-derived synthetic nitrogen fertilizers is

partly a consequence of economics in that a cash grain, such as corn or wheat, planted

every year provides a higher fiscal return than alternating it with a legume crop. In

addition, legume-Rhizobium nitrogen fixation is inhibited when the level of nitrogen in

the soil is high and is not sufficient for maximum yields of a grass crop. Therefore, in

26

developed countries chemical fertilizers have largely replaced biological fixation in row-

crop culture. On a worldwide basis, however, dependence on legumes is still preeminent.

Even in the United States, when rangeland and pasture agriculture are included, it has

been estimated that nitrogen production by biological fertilizers still exceeds chemical

application.

Other benefits accrue from the use of legumes to maintain soil nitrogen. Weed

control is facilitated by a crop sequence that alternately changes the growing

environment. Such legumes as alfalfa may be harvested for forage (hay or silage) or

grazed by livestock. As cover crops, legumes prevent or reduce soil erosion and may be

plowed under as “green manure.” Even though starch-producing grasses such as corn are

more efficient under favourable conditions in producing energy foods, grain legumes are

commonly grown in the tropics because they are more successful in depleted, nitrogen-

deficient soils.

Legume seeds constitute a part of the diet of nearly all humans. Their most vital

role is that of supplying most of the protein in regions of high population density and in

balancing the deficiencies of cereal protein (Poaceae). Except for the soybean and

peanut, the order is not noted for the oil content of the seeds since most seeds have only

about 10 percent oil content by weight. The legume seeds generally are highest in

carbohydrate compounds, followed by protein and fat. Legumes are thus considered to

be energy foods. Most legumes that are used for foods are multipurpose plants, serving

for animal forage and soil improvement as well. Some, notably the soybean, are also

27

important industrial crops. Fabaceae contains the more important crop plants, such as

soybeans, beans, cowpeas (Vigna), pigeon peas (Cajanus cajan), chick-peas (Cicer

arietinum), lentils (Lens culinaris), peas (Pisum sativum) and peanuts (Arachis

hypogeaea). Forage legumes (which concentrate their vitamins and proteins in their

young growing parts) also are grown as animal feed. Their role as such is especially

common in countries that can afford the luxury of meat (luxury because livestock

typically yield fewer calories than the plants they are fed). Some major forage legumes

of the temperate world include clovers, alfalfa, bird’s-foot trefoil (Lotus corniculatus),

and vetches. In the tropics or arid regions, some of the important elements of the habitat

are species of Glycine (soybean), Stylosanthes, and Desmodium.

Apart from the legume plants of worldwide importance, the following are

examples of locally significant legume species that are cultivated or gathered from the

wild. Some would plainly have substantial potential were they subject to genetic

evaluation and development through modern breeding techniques. They are still in the

same stage as teosinte (the ancestor of corn) or einkorn and emmer (the ancestors of the

modern varieties of cultivated wheats) in yield and utilization potential.

Notable among the locally useful plants of the legume family is Vigna subterranea

(Bambara groundnut), a leguminous plant that develops underground fruits in the arid

lands of Africa. Important too are the seeds of Bauhinia esculenta; they are gathered for

the high-protein tubers and seeds. Vigna aconitifolia (moth bean) and V. umbellata (rice

bean) are much used in the tropics for forage and soil improvement, and their seeds are

28

palatable and rich in protein. Psophocarpus tetragonolobus (winged bean) is collected in

Southeast Asia for the edible fruits and protein-rich tubers. Pachyrhizus (yam bean) is a

high-yield root crop of Central America.

Various forms of Leucaena (such as Leucaena leucocephala) have been developed

for animal forage, firewood, and construction, as well as for the high production of

nitrogen that enriches impoverished soils, especially in the Asiatic tropics. Other

important plants are acacia, used for animal food (both pods and leaf forage), for soil

improvement and revegetation, and as a source of tannin and pulpwood; Cordeauxia

edulis (yeheb), an uncultivated desert shrub of North Africa that has been so extensively

exploited for food (seeds) that it is in danger of extinction; Ceratonia siliqua (carob), a

Mediterranean plant whose fruits are used as animal and human food and in the

manufacture of industrial gums; and Tamarindus indica (tamarind) of Africa, now

primarily grown in India, which has food and medicinal uses and is also used as an

industrial gum.

The soybean is a bushy annual whose seeds are an important source of oil and

protein. An edible oil pressed from the seeds is used to make margarine and as a

stabilizing agent in the processing of food and the manufacture of cosmetics and

pharmaceuticals. The oil is employed in such industrial products as paint, varnish,

printing ink, soaps, insecticides, and disinfectants. Oil cakes pressed from the seeds are

used as protein concentrate in the mixed-feeds industry. The soybean is a good source of

29

vitamin B and is dried to produce soy milk, which is used in infant formulas. Fermented

pods are used in making soy sauce, flavouring common in Asian cooking.

The peanut, a native of South America, is high in vitamin B complex, proteins,

and minerals. The peanut is eaten raw or roasted or is processed into peanut butter. An

edible oil is pressed from the seed and is used as a cooking oil and in processing

margarine, soap, and lubricants. The oil also is employed by the pharmaceutical industry

in making medications. Pressed oil cake is fed to livestock. Peanuts are commercially

grown in the United States, Asia, Africa, and Central and South America.

Legumes in general are used to revitalize nutrient-depleted soils, especially

abandoned or abused agricultural and grazing lands. A more stringent revegetational

challenge is that following strip-mining. Generally speaking, native legumes are common

in these habitats because they are better able to thrive in nitrogen-poor soils than other

plants.

As mentioned above, the legumes produce secondary compounds of an irritating

or poisonous nature that provide protection against predators. Some of these secondary

compounds are being studied for their pharmacological potential. They are found in the

leaves and fruiting parts and includes flavonoids, alkaloids, terpenoids, nonprotein amino

acids, and others. Some of these - for example, the amino acid canavanine - may

comprise up to 5 percent of the dry weight of seeds. The chemical compound rotenone,

which is toxic to a number of organisms, is sufficiently abundant in the roots and stems

of certain species belonging to the Papilionoideae that primitive peoples often used these

30

plants to poison fish. More recently it has been shown that serious bone and neural

diseases afflicting humans (e.g., lathyrism) and livestock may be caused by the ingestion

of unusually large amounts of certain free amino acids. In sheep, ingestion of large

quantities of the amino acid mimosine, found in Leucaena glauca and some other species

of the Mimosoideae, apparently halts the growth of hair or wool, and in certain cases the

fleece itself has been observed to shed. A wide variety of alkaloids are found in the

order, most of them restricted to Fabaceae, however. Some alkaloids occur in sufficient

concentration in range plants to be poisonous to livestock, especially in species belonging

to the large genus Astragalus. Species of Astragalus are commonly referred to as

locoweed in North America because, following excessive consumption of these plants,

cattle seem to become unmanageable and “go crazy” or “loco.” Astragalus is poisonous

in any of three ways: by promoting selenium accumulation, through locoine, and through

several nitrogen-containing toxins. In the early 20th century, several African species of

Crotalaria were brought to the United States for use as soil-improvement plants. Their

poisonous qualities were discovered in connection with animal stock loss, and

development was then halted, but several persist as common noxious weeds.

An interesting biochemical component of the legume seed is phytohemagglutinin,

a large protein molecule that is specific in its capacity to agglutinate certain human blood

types. Approximately 60 percent of the several thousand seeds belonging to this order

tested to date contain the compound. Phytohemagglutinin is particularly abundant in the

common bean and has been extracted in a relatively pure state on a commercial scale

31

from species belonging to this genus. In addition to its agglutination properties, the

compound has been of interest because of its other biological effects. It is toxic to rats,

inactivates some human tumor cells, and has beneficial effects in the treatment of aplastic

anemia, the shortage of blood cells in humans due to the destruction of blood-forming

tissues.

The subfamilies Caesalpinioideae and Mimosoideae do not contain many food

crops and are perhaps best known for their shade and ornamental species, such as Cercis

siliquastrum (the Judas tree, or redbud), Bauhinia bartlettii (orchid tree), and Acacia

farnesiana (sweet acacia), although some of the more rapid-growing weedy species—for

example, Leucaena leucocephala (white popinac) and Albizia species—are widely

employed as green manure and fodder crops. Acacia species are used extensively in the

production of gum exudates and wood, especially in South Africa and Australia, where

the species are known as wattle trees.

Natural ecosystems:

Nitrogen is the primary nutrient limiting plant production in most natural

ecosystems (Seastedt and Knapp, 1993; Vitousek et al., 1997). Legumes, through their

symbiotic abilities, can play an important role in colonizing disturbed ecosystems,

including those that are fire prone (Arianoutsou and Thanos, 1996). Rates of N2 fixation

in such environments are often low, but can still satisfy much of the legume's nitrogen

needs. Spehn et al. (2002) examined plant species and functional groupings among

grassland communities in seven countries in Europe. Two years after sowing, the

32

presence of legumes affected N pool size in five of the seven sites. Such a build-up in

soil N is probably not open ended, because Pearson and Vitousek (2001) noted a 10- to

20-fold decline in nodule mass and N fixation between 6- and 20-year-old regenerating

stands of Acacia koa. A model developed by Vitousek and Field (1999) associated

reduction in N2 fixation with shade, P limitation, and grazing. Atmospheric CO2

enrichment and N deposition, each a major ecological concern is likely to have opposing

effects in natural ecosystems. Hardy and Havelka (1976) showed N2 fixation enhanced

under CO2 enrichment, and both legume biomass and frequency were enhanced in free-

air CO2 enrichment studies (Reich et al., 2002). Total nitrogen in Lespedeza capitata and

Lupinus perrenis increased 58.3% and 32.0%, respectively at 560 µmol mol−1CO2

(Reich et al., 2002). In contrast, C3 and C4 grasses were responsive to nitrogen

deposition, whereas legumes showing little response. Influence of nitrogen on

legume/grass balance in pastures is well documented. In a submodel developed by

Thornley (2001), the legume fraction in pasture declined from 18% to 1% as nitrogen

supply was increased.

Industrial and medicinal use of legumes:

In addition to traditional food and forage uses, legumes can be milled into flour,

used to make bread, doughnuts, tortillas, chips, spreads, and extruded snacks or used in

liquid form to produce milks, yogurt, and infant formula (Garcia et al., 1998). Pop beans

(Popenoe et al., 1989), licorice and soybean candy (Genta et al., 2002) provide novel

uses for specific legumes. Legumes have been used industrially to prepare biodegradable

33

plastics (Paetau et al., 1994), oils, gums, dyes, and inks (Morris, 1997). Galactomannan

gums derived from Cyamopsis spp. and Sesbania spp. are used in sizing textiles and

paper, as a thickener, and in pill formulation.

Many legumes have been used in folk medicine (Duke, 1992). Isoflavones from

soybeans and other legumes have more recently been suggested both to reduce the risks

of cancer and to lower serum cholesterol (Kennedy, 1995; Molteni et al., 1995). Soybean

and soyfood phytoestrogens have been suggested as possible alternatives to hormone

replacement therapy for postmenopausal women.

Biological N fixation:

A hallmark trait of legumes is their ability to develop root nodules and to fix N2 in

symbiosis with compatible rhizobia. This is often a critical factor in their suitability for

the uses outlined above.

Formation of symbiotically effective root nodules involves signaling between host

and microsymbiont. Flavonoids and/or isoflavonoids released from the root of the

legume host induce transcription of nodulation genes in compatible rhizobia, leading to

the formation of lipochitooligosaccharide molecules that, in turn, signal the host plant to

begin nodule formation (Long, 1996). Numerous changes occur in host and bacterial

gene expression during infection, nodule development, and function (Vance, 2001), with

approximately 100 host legume and rhizobial genes involved.

34

Some 40 to 60 million metric tons (mt) of N2 are fixed by agriculturally important

legumes annually, with another 3 to 5 million Mt fixed by legumes in natural ecosystems

(Smil, 1999). This is amazing efficiency given the miniscule quantities of nitrogenase

involved (Delwiche, 1970).

In addition to its role as a source of protein nitrogen in the diet, nitrogen from

legume fixation is essentially “free” N for use by the host plant or by associated or

subsequent crops. Replacing it with fertilizer N would cost $7 to 10 billion annually,

whereas even modest use of alfalfa in rotation with corn could save farmers in the U.S.

$200 to 300 million (Peterson and Russelle, 1991). Furthermore, fertilizer nitrogen is

frequently unavailable to subsistence farmers, leaving them dependent on N2 fixation by

legumes or other N2 fixing organisms.

One of the driving forces behind agricultural sustainability is effective

management of N in the environment (Graham and Vance, 2003). Application of

fertilizer N increased approximately 10-fold to 90 million mt between 1950 and 1995

(Frink et al., 1999) with significant energy consumption for nitrogen fertilizer synthesis

and application. Further increases in N needs for agriculture are projected for the period

to 2030 and these needs will contribute to environmental pollution. To the extent that

farming practices can make use of the more economically viable and environmentally

prudent N2 fixation (Peoples et al., 1995; Vance, 2001), agriculture and the environment

will benefit. The ability of legumes to sequester C has also been seen as a means to

offset increases in atmospheric CO2 levels while enhancing soil quality and tilth. Resh et

35

al. (2002) found that soils under N2-fixing trees sequestered 0.11 ± 0.07 kg m2 year−1 of

soil organic carbon, whereas there was no change under Eucalyptus spp. Carbon

sequestration under Prosopis spp. has also been reported.

Giller (2001) suggests that rates of N2 fixation of 1 to 2 kg N ha−1 growing season

day−1should be possible in all legumes. Rates reported by Unkovich and Pate (2000) are

clearly less than this, with the latter authors reporting a decline in average N2 fixation rate

for both soybean and beans over the period since 1985. Constraints to N2 fixation include

drought (Sinclair et al., 1987), soil acidity, N fertilization, and nutrient limitations. Many

cultivars also show only limited ability to fix N2 in symbiosis.

Management of soil acidity for temperate and tropical regions has often differed

but increasingly depends on acid-tolerant legume cultivars and rhizobia (Howieson et al.,

2000), with soil liming only to a pH at which Al and Mn are no longer toxic. Acid soil

management was of critical importance in opening the Brasilian Cerrado to agriculture,

but it was serendipitous that the acid-tolerant Rhizobium tropici could replace other less

tolerant bean rhizobia (Hungria et al., 1997). Identification of additional acid-tolerant

host and rhizobial germplasm and the deployment of acid tolerance genes such as occur

in R. tropici CIAT899 (Graham et al., 1982) are priority areas.

Maximum benefits from N2 fixation depend on soil P availability (Kennedy and

Cocking, 1997), with 33% of the world's arable land limited in P (Sanchez and Euhara,

1980). Acid-weathered soils of the tropics and subtropics are particularly prone to P

deficiency. Even where P fertilization is adequate, <15% of that P may be taken up by

36

plants in the first year (Holford, 1998). Perhaps of greater concern, reserves of rock

phosphate could be depleted in only 60 to 90 years (Abelson, 1999). Plants dependent

on symbiotic N2 fixation have ATP requirements for nodule development and function

(Ribet and Drevon, 1996) and need additional P for signal transduction and membrane

biosynthesis. Phosphorus concentrations in the nodule are often significantly higher than

those in shoot or root tissue (Israel, 1987). Al-Niemi et al. (1997) suggest that bacteroids

can be P limited even when plants have received otherwise adequate P levels. Given this

requirement for symbiosis, approaches leading to improved P acquisition and use in

legumes (rhizosphere acidification, acid phosphatase secretion, root architectural changes

at low P, enhanced P transport and use-efficiency, and functional differences in

mycorrhizal symbioses) all need further study. White lupine (Lupinus albus) and

common bean are excellent model legumes for such studies. Each undergoes change in

root architecture and rhizosphere chemistry at low P (Johnson et al., 1996; Nielsen et al.,

1998; Miller et al., 2001), improving soil exploration and phosphate scavenging.

Transgenic alfalfa has proved extremely useful in understanding the genetic and

molecular basis of low soil P, acid and aluminum stress responses. Results in this study

highlight the need for more effective transformation and regeneration protocols in the

more recalcitrant legumes, including bean and cowpea. Progress in the study of

nodulation and N2 fixation under drought or salinity stress has been minimal, largely

because the legume and the process of nodulation are more susceptible to these

constraints than is the microorganism.

37

Legumes play a critical role in natural ecosystems, agriculture, and agroforestry,

where their ability to fix N in symbiosis makes them excellent colonizers of low-N

environments, and economic and environmentally friendly crop, pasture, and tree species.

Legume yields unfortunately continue to lag behind those of cereals. A research

orientation that better balances the needs of third-world or sustainability-oriented

agriculture with the breakthrough technologies of genomics and bioinformatics is needed.

It requires stronger and more adventurous breeding programs, better use of marker-

assisted technologies, and emphasis on disease resistance, enhanced N fixation, and

tolerance to edaphic soil constraints. It also requires extension of existing low-cost

technologies, such as rhizobial inoculation, to the small farmer. To paraphrase a

comment by Catroux et al. (2001) “we enter the era of biotechnology knowing more and

more about the growth of legumes at the gene level, but except for some producers in

developed countries, unable to effectively translate these into major gains in

productivity.”

Crop production in India and the world:

Legumes have been used in agricultural production since the earliest of

civilizations. They have served as the primary source of nitrogen for many cropping

systems, as well as providing food for humans and domestic animals. In many

developing agricultural regions of the world, legumes are still used extensively for these

purposes.

38

In the least several decades, the widespread availability of synthetic nitrogen

fertilizer in many nations has resulted in a major decrease in the cultivation of legumes.

An exception to this trend has occurred for soybean (Glycine max), a widely grown grain

legume that has increased within the least five decades in the United States from almost

nothing to tens of millions of hectares. Although soybean grown in rotation with corn

(Zea mays) enhances corn production (Voss and Shrader, 1984; Hesterman et al., 1986),

studies with N-isotopes indicate that after harvest of the soybean seed, soybeans

contribute little if anything to total soil N (Heichel, 1987).

The exclusion of other legumes from cropping systems these last several decades

resulted not only from the availability of fertilizer nitrogen but was also accelerated by

the conversion to fossil energy as a source of power for American agriculture. Because

of this change, farmers were no longer compelled to use as much as 25 percent of their

land for the production of forage and grain for draft animals. Many farmers eliminated

all livestock from their operations, moving to cash grain enterprises and making them

entirely dependent upon nitrogen fertilizers as their source of added N.

The energy crisis of the 1970's, with attendant increases in cost and reduced

availability of N fertilizers, caused many producers to evaluate the stability of continued

dependence on synthetic N fertilizers as their only N input. Even though the initial

energy crisis has abated somewhat in recent years, economic stress in agriculture,

coupled with knowledge that N fertilizer prices probably will again greatly increase at

39

some time in the near future, has resulted in many farmers again considering the use of

legumes in their enterprises.

Benefits from legumes:

The ability of legumes to fix atmospheric N2 and thereby add external N to the

crop-soil ecosystem is a distinct benefit of legume culture. When fertilizer-N is

expensive or unavailable, crop production systems depend on the N fixed by legumes to

maintain the N cycle at a sustained productive lever. Such limitations of fertilizer-N

availability and cost are not uncommon in many developing countries.

The quantity of N biologically fixed each year by legumes varies greatly from zero

to several hundred kg N per ha (Table 1). Many grain legumes are efficient at N

fixation. Variables affecting quantity of nitrogen fixed include not only legume species

and cultivar, but also such factors as soil type and texture, pH, soil nitrate-N level,

temperature and water regimes, availability of other nutrients, and crop (especially

harvest) management. The latter factor is extremely important. For instance, alfalfa

(Medicago saliva) may add up to several hundred kg N/ha to the soil if a final cutting of

hay is not removed, compared to less than 150 kg N if only the roots and stubble remain

(Heichel, 1987).

40

Table 1. Area, production and other characteristics of important legume crops, India

Legume crop

Planted area (1000

ha)

Production

(1000 mt)

Average yield (Kg/ha)

Experimental yield (Kg/ ha)

Growing season

Maturity (Days)

Pigeon

pea

2660 1911 663 1500-3500 Rainy 120-270

Chickpea 8221 5450 718 2000-4500 Post-rainy 100-150

Mungbean 2525 854 338 1000-2400 Rainly, Post-rainy, summer

65-80

Urdbean 2260 698 309 1000-2000 Rainy, post rainy, summer

80-110

Horse

gram

2053 710 345 500-1000 Rainy, post rainy

100-120

Lentil 987 432 438 1000-1500 Post rainy 90-100

Khesari 1564 609 389 400-700 Post rainy 100-120

Pea 582 348 600 2000-3000 Post rainy 100-140

Groundnut 7548 6387 846 2000-3000 Rainy, Post rainy, summer

90-130

Soybean 303 292 964 1500-3500 Rainy, post rainy, summer

90-140

Source: Directorate of Economics and Statistics, Ministry of Agriculture and Irrigation, Government of India, 1979.

41

The economic value of the nitrogen fixed by legumes also varies widely. One must

consider the cost of production of the legumes, the amount of fixed nitrogen returned to

the soil, and the availability of this nnitrogen for future crops. Often, these costs are

compared directly against the cost of purchasing and applying an equivalent amount of

nitrogen fertilizer plus the net income lost by producing a legume instead of a grain crop

(if the legume is grown in rotation). In the past several decades, the cost of production

and price of nitrogen fertilizers have been such that this type of calculation would

generally favor the use of nitrogen fertilizer. This fact is largely responsible for the

decreased use of legumes in our crop production systems these past 40 years.

There are other benefits from using a legume on a cropping system that should be

figured into any comparison with fertilizer-N, but unfortunately, they are often omitted

because of difficulty in quantifying them. Usually, yields of a grain crop grown in

rotation are at least 10 to 20 percent greater than those for continuous grain, regardless of

the amount of fertilizer applied to the continuous grain. This response is often referred to

as the rotation effect. Because additional N will not entirely eliminate this yield

difference, much of the response must be due to factors other than N availability. Cook

(1984) and others have shown that rotation of crops reduces population and activity of

some pathogenic soil organisms. Likewise, rotations break the weed and insect cycles

that often predominate with contiguous cropping. In addition, there is the possibility

42

(although often difficult to prove) that rotations may enhance soil structure and improve

air-water relations in the soil.

Legumes may have long-term benefit on some soils that again are difficult to

convert into monetary value. Usually legume rotations, compared to continuous grain

cropping, result in enhanced soil organic master content and mineralizable N. This

provides not only better control of N availability, but also improved soil structure, less

energy for cultivation, and less erosion (Hoyt and Hargrove, 1986). Reduction in erosion

rate, over a period of decades, can have a major influence on the properties and

productivity of some soils (Mielke and Schepers, 1986). The enhanced mineralizable N

levers in soils with legume rotations compared to those for continuously cropped soils

may aid greatly in control of "round water quality. With legumes, not only is less

fertilizer-N required, but the lever of nitrate N in the soil at any one time is usually less,

so there are fewer nitrates to leach below the root zone.

Food legume crops represent an important component of agricultural food crops

consumed in developing countries and are considered a vital crop for achieving food and

nutritional security for both poor producers and consumers. As a matter of fact, in

dietary terms, food legumes complement cereal crops as a source of protein and minerals

while agronomically they serve as rotation crop with cereals, reducing soil pathogens and

supplying nitrogen to the cereal crop. Food legumes also serve as a feed crop in many

farming systems and fetch higher prices compared to cereals and are increasingly grown

to supplement farmers’ incomes. The important and diverse role played by food legumes

43

in the farming systems and in diets of poor people, makes them ideal crops for achieving

the CGIAR’s (Consultative Group on International Agricultural Research)

developmental goals of “reducing poverty and hunger, improving human health and

nutrition, and enhancing ecosystem resilience.”

Traditional cropping systems of legume grown in India and the status of the

agronomic research for legume improvement:

The low yields of legume crops and the shortage of pulses as indicated by

increasing prices have emphasized a greater need for research and as a result the "All-

India Coordinated Projects" for the improvement of grain legumes and oilseeds

(including groundnut) were established in the late 1960s. The setting up of the

International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) in India has

given further impetus to legume research. Improved genotypes of legume crops suitable

for intercropping and multiple cropping systems have recently been developed.

As rainfed crops, legumes are mostly mixed or intercropped with cereals or long-

season and widely-spaced crops. Cropping systems research has identified - compatible

crops for intercropping and the optimum proportions and spatial arrangements that yield

maximum intercropping advantage. Responses of legumes to inoculation and nitrogen

have so far been inconsistent but considering the low cost involved in the Rhizobium

culture, the use of inoculum seems to ensure proper nitrogen nutrition. The response of

legume crops to phosphorus is significant when applied to phosphorus-deficient soils and

rates between 30-40 kg P2O5/ha are worth recommending. Pests and diseases restrict

44

legume production and even as chemical methods of control are known, the development

of cultivars resistant to pests and diseases is emphasized. There is considerable scope for

increasing pulse production by popularizing the improved methods of intercropping,

growing pulses even in non-traditional areas, cultivating them in off-season, or growing

them together with another crop in a traditionally mono-cropped area.

Pulses are the chief source of dietary proteins in India. They are grown on 23.5

million ha with an annual production of 12.2 million mt, Even as this quantity represents

27.8 per cent of world production and puts India as one of the leading grain legume

producing nations, pulses are still in short supply in the country. The net per capita

availability of pulses has decreased from 75 g/day in 1958 to 40 g/day in 1974 which is

far below the daily nutritional requirement. This shortfall in domestic supply may be

attributed to a shift from pulse production to the production of cereals which, in turn, is a

result of expanded irrigated areas and marked improvement in the yield performance of

some cereal crops. The steady increase of population and the rising costs of animal

proteins, however, will continue to influence greater demand for pulses.

The statistics of important grain legumes in India are shown in Table 1. Chickpea

or gram (Cicer arietinum L.) and pigeonpea or "tur" (Cajanus cajan) which together

account for 61 per cent of the total pulse production, are the principal grain legumes;

chickpeas are grown in the post-rainy season (October - March) and pigeonpeas are

planted in the rainy season (June - October). Other legumes grown exclusively in the

post-rainy season are lentil (Lens culinaris Medic.), "khesari" or grasspea (Lathyrus

45

sativus L.) and peas (Pisum sativum L.). Mungbean or green gram (Vigna radiata) "urd"

or black gram (Vigna mungo) and cowpea (Vigna sinensis) are grown in both seasons but

the post-rainy season crop is possible only in the warmer parts of the country. Where

irrigation is available these can also be grown in the summer. Other grain legumes grown

in the rainy season with limited regional importance are field beans (Lablab purpureus

L.) Sweet mothbean (Vigna acantifolia), cluster beans (Cyamopsis tetragonolobus) and

soybean (Glycine max). Groundnut (Arachis hypogaea) is the major oil seed legume

grown in both seasons but in the post-rainy season the crop is mostly confined to the

irrigated areas of peninsular India.

Breeding for Better Nitrogen Fixation in Grain Legumes:

All of the elements needed to significantly enhance N2 fixation in grain legumes

by plant breeding are currently available, but attention to this problem has been limited.

This paper considers genetic variation in traits associated with nodulation and N2 fixation

and how they might be utilized. It also considers the role of rhizobia in an effective

grain-legume breeding program.

Elements of an Effective Applied Breeding Program:

An effective applied breeding program must be based on certain elements. These include:

• traits whose improvement will enhance crop production and/or profitability;

• genetic variation in the trait to be advanced;

46

• identification of parents differing in that trait;

• understanding of trait genetics;

• formulation of appropriate breeding strategy; and

• development of a field-based, simple, and inexpensive method of progeny selection.

In the case of breeding for enhanced nitrogen (N2) fixation in grain legumes, all of these

elements have been demonstrated, yet progress in all but a few cases has been limited.

Symbiotic Nitrogen Fixation in Legumes:

Essentially all agriculturally important legume species have the ability to symbiose

with a group of bacteria collectively known as rhizobia. In this symbiosis the bacteria

derive energy from the host for growth and N2 fixation, and are protected from external

stresses; the host accesses a form of nitrogen it could not otherwise utilize. Worldwide

some 44 to 66 million tons of N2 are fixed annually, providing nearly half of all the

nitrogen used in agriculture. The quantity of nitrogen needed for agriculture is projected

to increase in the period to 2030 and in the USA could lead to greater environmental

pollution. Reduced dependence on fertilizer nitrogen and attention to farming practices

that favor the more economically viable and environmentally prudent N2 fixation will

benefit both agriculture and the environment (Vance, 2001)

Currently, the contribution of N2 fixation to the production of grain legumes in the

USA is consistently less than what could be achieved. Only 15% of farmers use

47

inoculants, while the percentage N derived from fixation in soybean and common bean is

thought to have declined from 65% to only 54%, and from 44% to 37%, respectively

since 1985 (Van and Hartley, 2000). A recent report even suggests that corn-soybean

production in the American Midwest is not sustainable, and depends on government

subsidy (Randall, 2001). Factors that have contributed to a reduced dependence on N2

fixation include the availability and low cost of N fertilizers and manures, the use of plant

varieties limited in their ability to fix N2 in symbiosis, and edaphic constraints that

include soil acidification, drought, and shortage of specific nutrients (Graham and Vance,

2003)

Rates of N2 fixation vary with plant species and environment, but with grain

legumes potential rates of as much as 0.9 to 1.8 lb/acre per day have been suggested

(Giller, 2001) -- a rate that would satisfy essentially all of the plant’s nitrogen needs.

Unfortunately, this level of N2 fixation is rarely achieved, though benefits from a

program aimed at enhanced N2 fixation in grain legumes can be dramatic. This is evident

in the case of soybeans in Brazil where yields have increased more than 4-fold since

1968, and where average yields in the Cerrado now outstrip those achieved in the USA

(Hungria et al., 2003). In this region more than 90% of farmers use inoculants and over

60% repeat inoculate. Percent N derived from fixation in studies conducted across Brazil

varies from 69% to 94%, with an economic return of a dependence on symbiotic N2

fixation in soybean of $1.95 billion (US) annually (Hungria et al., 2003b). Similar

responses to inoculation of soybean have been obtained recently in parts of the American

48

Midwest and are beginning to stir greater interest in the inoculation of this and other

crops.

Cultivar Variation in Traits Associated with N2 Fixation:

Cultivar variation in traits associated with N2 fixation has been demonstrated in

essentially every legume studied to date. Selected important examples include clover

(Nutman, 1967), soybean (Hardy et al., 1973; Pazdernik et al., 1996), common bean

(Graham and Rosas, 1977; Rennie and Kemp, 1983) and alfalfa (Degenhart et al., 1992;

Jessen et al., 1988). Further, these differences have been shown using a range of

different traits each associated with N2 fixation. They include cultivar differences in

nodule number and mass, speed of nodulation, lateral root nodulation post flowering,

nitrogen accumulation, acetylene reduction activity, allantoic acid production, and nodule

enzyme production and function.

Not all of these cultivar differences would be of value in a breeding program.

Commonly, for example, N2 fixation levels are correlated with time to flowering, but in

the northern USA at least, the use of cultivars with a longer pre-flowering period is only

useful if overall time to maturity can be held constant. Similarly, nodule number and

mass are usually inversely related, with an ineffectively nodulated cultivar having many

small nodules (Nutman, 1967). Nodule mass per plant is a more useful indicator of

symbiotic potential.

49

Nodulation and N2 fixation in legumes are generally thought to be quantitatively

inherited traits (Bliss, 1993). Because of this the emphasis has been given on the

identification of parents that differ in the contributions each might bring to the symbiosis.

The criteria used for this are nodule mass, activity (carbohydrate supply and form,

specific nodule activity, or nodule enzyme function), and duration (senescence, lateral

root nodulation) functions.

Fedorova et al. (2002) reported more than 300 tentative consensus sequences in

nodule libraries of Medicago truncatula that are not found in other libraries prepared

from this host. Additional gene products are undoubtedly needed for infection and

nodule initiation. While many of these are not vary with legume or cultivar, the

probability is that future studies leads to identify many additional traits useful in

characterizing host variability in nodulation and N2 fixation.

Breeding and Selection for Enhanced N2 Fixation in Grain Legumes:

A number of different breeding approaches have been used to improve N2 fixation

levels in legumes. They include backcross-inbred methods for population development

(Bliss, 1993); recurrent selection for enhanced nitrogen fixation (Elisondo et al., 1999);

bidirectional selection for specific nodule enzymes (Degenhart et al., 1992); and simple,

double and three-way crosses (Hungria et al., 1997). All have succeeded in raising levels

of N2 fixation. In fact, even selection for crop yield without particular attention to N2

fixation can enhance total N and N2 fixed (Coale et al., 1985). While a number of

different traits should be considered in careful parental selection, initial field screening of

50

the progeny can be simply and inexpensively undertaken. Common bean varieties were

selected for superior N2 fixation using low nitrogen soil conditions and only seed yield

and plant biomass nitrogen as selection criteria. The latter trait is included to distinguish

between superior N2 fixation levels and high harvest N index. Both Elissondo (1999) and

Hungria and Bohrer (2000) pointed to losses in N2-fixing ability in breeding populations

evaluated on the basis of seed yield or seed nitrogen alone.

Heritability estimates in fixation studies vary with trait measured, but range from

0.22 to 0.76. A problem in many of the studies cited above is that few progeny perform

better than the superior N2-fixing parent. This is not surprising if N2 fixation is

quantitatively inherited, and if parental selection is based mainly on nodule mass or

overall N2 fixation. Transgressive segregation toward markedly superior N2 fixation will

require the pyramiding of genes contributing to nodule mass, activity, and duration, and

approaches that parallel those used in yield enhancement. At higher levels of crop

production, competition between seeds and nodules for nutrients and energy could mean

that one couldn’t be improved without decline in the other. Fortunately, that time does

not appear to be near. In the meantime a priority must be the development of markers

with which superior nodulation or N2 fixation can be dissected. To this point, there has

been a surprising dearth of studies on marker-assisted selection for traits associated with

nodulation and N2 fixation.

51

Where do the Rhizobia fit in?

Ability to nodulate and fix N2 must be a part of the breeding program for all

agronomically important legumes. At some stage, all breeding lines need to be tested in

low N soils, and those that are weak to average in N2 fixation discarded. Inoculant strains

and inoculation practices used in these trials need to be the best available, and any

variable likely to reduce the potential for nodulation and N2 fixation (seed treatment, soil

compaction, inappropriate N fertilization, and the limited availability of fixation-specific

nutrients (including P, Ca, Fe, and Mo) avoided or corrected.

In an age of rapidly changing, often bioengineered varieties, rhizobial strain

selection needs to be revisited. Hungria et al. (2003) reviewed changes in the inoculant

strains used for soybean in Brazil since 1960. These authors noted that soils in much of

Brazil were initially devoid of rhizobia, and were generally inoculated with strains from

the USA and Australia. More recently, variants of these initial inoculant strains have

been recovered from soil and found to be better adapted to local soil conditions, more

competitive, and higher yielding than the original inoculant strains. In one study, plants

inoculated with a variant of Bradyrhizobium japonicum CB1809 out yielded those

inoculated with the wild type strain by more than 600 lb/acre. Similar studies need to be

undertaken in the USA, where some inoculant strain recommendations have not been

changed in 20 years.

Three additional areas stand out as warranting particular research attention:

52

• Host-Rhizobium interaction, and accounting for it in breeding programs;

• Differences in root colonization and saprophytic competence;

• Stress tolerance and its impact on host-strain interaction.

Host-Rhizobium interaction:

Host-Rhizobium interaction, with some strains performing better with specific

cultivars than with others, has been evident in some, but not all germplasm evaluation

studies. Mytton et al. (1977) with Vicia faba found that host-Rhizobium interaction

explained 74% of the variation in symbiotic response. In contrast, Roskothen (1989)

found limited host-strain interaction in Vicia faba, attributing this difference in results to

the limited diversity of rhizobia used in the earlier study. Bernal and Graham (2001)

found a local common bean cultivar to recover different bean rhizobia from soil than an

introduced one, while Bernal (1993) noted that cultivars derived from different races of

bean in Latin America differed in speed of nodulation with rhizobia from the Andean

region. Similar results have been obtained with different introductions of Glycine max

into Africa (Mpepereki et al., 2000).

53

Root colonization and saprophytic competence:

Gibson et al. (1976) and others have reported instances in which inoculant strains,

applied under near-optimum conditions, did not persist in the soil, while McDermott and

Graham (1989) noted that soybean inoculant rhizobia were competitive in nodule

formation in the crown region of the plant near where they were placed, but failed to

colonize the root system. These studies suggest problems in rhizosphere colonization and

saprophytic competence, which might be overcome by a better understanding and

management of legume rhizodeposition. This would require both a detailed study of

cultivar variation in rhizodeposition and of rhizobial differences in root colonization.

Results obtained in these studies might also contribute to a better understanding of the

basis for preferential nodulation by specific cultivars (Rosas et al., 1998). Recent

advances in understanding microbial community structure in the rhizosphere should

facilitate such a study. Grayston et al. (2001) have already noted differences in

rhizosphere community structure associated with plant improvement, while Siciliano et

al. (1998) found that Pseudomonas inoculants used in phytoremediation had significant

impacts on root associated microbial communities.

Stress tolerance and its impact on host - strain interaction. Soil acidity, drought,

low soil P, or high soil N levels can all affect symbiosis between host and rhizobia,

influencing rhizobial survival in soil, the host, or the process of nodulation itself

(Graham, 1992). The clearest example of how this can affect plant breeding is with soil

pH. Differences in both host and rhizobial strain tolerance to soil pH exist in Medicago

54

(Howieson, 1988, 1989) and Phaseolus (Graham et al., 1994; Vargas and Graham, 1988)

and can interact to enhance plant establishment and growth. In the case of Medicago, this

has meant the introduction of annual species into more than 800,000 acres of Australia

where the soils were previously too acidic for the growth of many Medicago species. In

the acid soils of Brazil, Rhizobium tropici can account for 97% of the nodules associated

with beans. For both pH and low soil phosphorus, cultivar use can significantly affect

strain recovery from soil (Christiansen and Graham, 2002). Careful selection of

inoculant strain is essential for any legume breeding nursery likely to be grown under

stress.

Crop Improvement:

A recent paper in Nature (Knight, 2003) laments the decline in public and

institutional plant breeding in the face of genetic engineering encroachments into this

field. Yet with few exceptions, molecular approaches achieve with N2 fixation that

traditional breeding method. Rengel (2002) reviews some molecular options, but

discusses mainly supernodulation, opine exudation, and flavonoid expression. More

likely to have success are studies such as those of Tesfaye et al. (2001) with over-

expression of malate dehydrogenase, a trait that could affect not only aluminum and low

P tolerance, but also levels of N2 fixation (Vance, 2001).

55

Biofertilizers:

For the better crop production biofertilizers are important which are of various

types. Blue-green algae cultured in specific media. Blue-green algae can be helpful in

agriculture as they have the capability to fix atmospheric nitrogen to soil. This nitrogen

is helpful to the crops.

A biofertilizer is a substance which contains living microorganisms which, when

applied to seed, plant surfaces, or soil, colonizes the rhizosphere or the interior of the

plant and promotes growth by increasing the supply or availability of primary nutrients to

the host plant (Vessey, 2003). Bio-fertilizers add nutrients through the natural processes

of nitrogen fixation, solubilizing phosphorus, and stimulating plant growth through the

synthesis of growth-promoting substances. Bio-fertilizers can be expected to reduce the

use of chemical fertilizers and pesticides. The microorganisms in bio-fertilizers restore

the soil's natural nutrient cycle and build soil organic matter. Through the use of bio-

fertilizers, healthy plants can be grown, while enhancing the sustainability and the health

of the soil. Since they play several roles, a preferred scientific term for such beneficial

bacteria is "plant-growth promoting rhizobacteria" (PGPR). Therefore, they are

extremely advantageous in enriching soil fertility and fulfilling plant nutrient

requirements by supplying the organic nutrients through microorganism and their

byproducts. Hence, bio-fertilizers do not contain any chemicals which are harmful to the

living soil.

56

Bio-fertilizers are eco friendly organic agro-input and more cost-effective than

chemical fertilizers. Bio-fertilizers such as Rhizobium, Azotobacter, Azospirillum and

blue green algae (BGA) have been in use a long time. Rhizobium inoculant is used for

leguminous crops. Azotobacter can be used with crops like wheat, maize, mustard,

cotton, potato and other vegetable crops. Azospirillum inoculations are recommended

mainly for sorghum, millets, maize, sugarcane and wheat. Blue green algae belonging to

a general cyanobacteria genus, Nostoc or Anabaena or Tolypothrix or Aulosira, fix

atmospheric nitrogen and are used as inoculations for paddy crop grown both under

upland and low-land conditions. Anabaena in association with water fern, Azolla

contributes nitrogen up to 60 kg/ha/season and also enriches soils with organic matter

(Peters and Meeks, 1989).

Other types of bacteria, so-called phosphate-solubilizing bacteria, such as Pantoea

agglomerans strain P5 or Pseudomonas putida strain P13 (Chen et al., 2006) are able to

solubilize the insoluble phosphate from organic and inorganic phosphate sources. In fact,

due to immobilization of phosphate by mineral ions such as Fe, Al and Ca or organic

acids, the rate of available phosphate (Pi) in soil is well below plant needs. In addition,

chemical Pi fertilizers are also immobilized in the soil, immediately, so that less than 20

percent of added fertilizer is absorbed by plants. Therefore, reduction in Pi resources, on

one hand, and environmental pollutions resulting from both production and applications

of chemical Pi fertilizer, on the other hand, have already demanded the use of new

57

generation of phosphate fertilizers globally known as phosphate-solubilizing bacteria or

phosphate bio-fertilizers.

Benefits of biofertilizers

A bio-fertilizer provides the following benefits:

1. Since a bio-fertilizer is technically living, it can symbiotically associate with plant

roots. Involved microorganisms could readily and safely convert complex organic

material in simple compounds, so that plants are easily taken up. Microorganism

function is in long duration, causing improvement of the soil fertility. It maintains the

natural habitat of the soil. It increases crop yield by 20-30%, replaces chemical nitrogen

and phosphorus by 25%, and stimulates plant growth. It can also provide protection

against drought and some soil-borne diseases.

2. Bio-fertilizers are cost-effective relative to chemical fertilizers. They have lower

manufacturing costs, especially regarding nitrogen and phosphorus use.

3. It is environmentally friendly in that it not only prevents damaging the natural source

but also helps to some extent cleanse the plant from precipitated chemical fertilizers.

Current status of biofertilizer in the world:

Global warming and climate change have resulted in unexpected drought, stormy

rainfalls, extremely high temperature, cold damage, hurricanes and tornadoes in many

places around the world where such disastrous tragedy had never occurred in the past

58

decades. Heavy casualties and agricultural losses in these areas therefore significantly

affected the lives and security of residents and consequently, the regional economy.

Establishing an environmental friendly co-existing mechanism on earth is of vital

importance. For agricultural practices, effective strategy must be conducted to reach the

goal of a friendly environment.

In recent years, chemical pesticides and fertilizers were extensively applied to

maintain high crop yield. Overusing agrochemicals led to several agricultural problems

and poor cropping systems. The excessive application of chemical nitrogen fertilizer not

only accelerated soil acidification but also risked contaminating groundwater and the

atmosphere, and weakened the roots of plants that made them easy prey on unwanted

diseases. Biofertilizers are microorganisms that assist plants to grow by increasing the

quantity of nutrients. The living microorganisms that co-exist with the plants will

promote the supply of important nutrients and, consequently are crucial for the overall

productivity of the soil. Using biofertilizers offers a better option in reducing

agrochemical inputs, and helps maintain soil fertility and strength.

An increasing number of farmers and agriculturists are choosing biofertilizers over

their chemical counterparts as they are found to be gentler on the soil. The value of

biofertilizers has further increased in an increasingly eco-conscious world. In addition,

soil quality is also improved through the uptake of these environmental friendly

fertilizers. Biofertilizers also contributes in reducing the negative impact of global

warming.

59

Rhizobial inoculants:

Nitrogen is one of the major plant nutrients, which are referred to as the master

key elements in crop production. It can be made available through chemical or biological

processes, though chemical nitrogen fertilizers are relatively expensive (Zilli et al.,

2004). Symbiotic nitrogen fixer and phosphate solubilizing microorganisms play an

important role in supplementing nitrogen and phosphorus to the plant, allowing a

sustainable use of nitrogen and phosphate fertilizers. The use of these microbes as

fertilizers in the field has been reported as beneficial to crop yield. This is especially

important in the developing countries where farming will continue to be in the hands of

small farmers (Rao, 1999).

Study on the selection of efficient rhizobial strains for inoculation in Taiwan

started in 1958. Collection, isolation and subsequent selection of effective rhizobial

strains have yielded fruitful results in agriculture. Nevertheless, marked variations were

observed among rhizobial strains (Young and Chao, 1983).

Fast and slow-growing soybean rhizobial strains were isolated and selected from

Taiwan soils for inoculation (Young et al. 1982; Young and Chao 1983) in 1980s.

Several effective isolates were deposited in the Culture Collection and Research Center

(CCRC) of the Food Industry Research and Development Institute in Taiwan (CCRC

1991).

60

A few field experiments were conducted to determine the effects of single and

mixed inoculations with Rhizobium and Arbuscular-Mycorrhiza (AM) in six different

tropical soils in Taiwan (Young et al., 1988b). The results indicated that inoculation with

rhizobial strains alone increased N2 fixation and soybean yield in three out of six fields.

Inoculations with rhizobial strain singly, or in combination with AM, without any N2

fertilizer applications, significantly increased soybean yield from 5% to 134% in the field

experiments. The results from other experimental sites also showed that a mixed

inoculum of Rhizobium and AM can be an efficient biological fertilizer that maximizes

soybean yields. The combined effect of the mixed inoculum was a striking achievement

in the field of bio-fertilization. AM might have provided the essential phosphorus for the

growth of soybean plants.

Improvement in biofertilizer by strain carrier interaction:

Environmental issues such as freshwater pollution, energy saving, and soil erosion

are forcing the farmers to introduce methods of cultivation that have a lower impact on

the environment. In this context, the reduced use of chemical fertilizers with increased

application of organic fertilizers is considered a compulsory route to alleviate the

pressure on the environment derived from agricultural practices.

Several organic fertilizers have been introduced in the recent years, which are also

acting as natural stimulators of plant growth and development (Gousterova et al., 2008).

A specific group of this kind of fertilizers includes products based on plant growth-

promoting microorganisms (PGPM). Three major groups of microorganisms are

61

considered beneficial to plant nutrition: arbuscular mycorrhizal fungi (AMF) (Jeffries et

al., 2003), plant growth-promoting rhizobacteria (PGPR) (Podile and Kishore, 2006), and

nitrogen-fixing rhizobia, which are usually not regarded as PGPR (Franche et al., 2009).

Microbial inoculants based on these microorganisms can be divided into different

categories depending on their use, even though exact definition of these categories is still

unclear. Nevertheless, the category of biofertilizer most commonly refers to products

containing soil microorganisms increasing the availability and uptake of mineral nutrients

for plants (like rhizobia and mycorrhizal fungi). According to the definition proposed by

Vessey (2003), biofertilizers are substances which contain living microorganisms which,

when applied to seed, plant surfaces, or soil, colonize the rhizosphere or the interior of

the plant, and promote growth by increasing the supply or availability of primary

nutrients to the host plant. Another category of PGPM-containing products is that of

phytostimulators which are generally containing auxin-producing bacteria, inducing root

elongation (Lugtenberg et al., 2002).

The interest in the application of these products is rising due to the enhancement in

nutrient uptake efficiency (Adesemoye et al., 2008; Malusà et al., 2007) and society

demands for more green technologies in production (Lempert et al., 2003), increasing

costs of agrochemicals. Furthermore, biofertilizers and phytostimulators possess

secondary beneficial effects that would increase their usefulness as bioinoculants. Indeed

microorganisms such as Rhizobium and Glomus spp. have been shown to also play a role

in reducing plant diseases.

62

The practice of inoculating plants with PGPM can be traced back to the early 20th

century, when a product containing Rhizobium sp. was patented (Nobbe and Hiltner,

1896). Mycorrhizal fungi, even though utilized as biofertilizers since few decades, were

reported to promote plant growth through P uptake since the late 1950s (Koide and

Mosse, 2004). Since then, research efforts in these fields have steadily increased,

resulting, in recent years, in the selection of numerous strains showing several beneficial

features (Podile and Kishore, 2006; Vessey, 2003; Koide and Mosse, 2004; Okon and

Labandera-Gonzalez, 1994).

PGPM inoculants can be defined as formulations containing one or more

beneficial microorganism strains (or species) prepared with an easy-to-use and

economical carrier material. The development of techniques for the production of large

quantities of pure inocula, with high infectivity potential, is the main issue to be tackled

in order to allow a wide use of biofertilizers. The key aspects in PGPM inoculation

technology are the use of a proper formulation of inocula preparations, the selection of an

adequate carrier, and the design of correct delivery methods.

Rhizobium Strain Selection

After rhizobial strains have been isolated from nodules, they must be evaluated for

their ability to form nodules and fix nitrogen with targeted legumes. The source of

rhizobial strains for a strain selection program can range from local isolates, to strains

already tested in other parts of the region or country, to cultures from various overseas

collections. Preliminary screening is performed in the greenhouse, where numerous

63

strains can be tested on several host varieties. If the inoculated plants form nodules and

produce healthy green leaves when grown in nitrogen-free media, it can be assumed that

an effective symbiosis has been established. Rhizobia selected in greenhouse trials, where

conditions are usually optimal, must then be evaluated in the field. Rhizobia which adapt

to the agronomic conditions under which the host legumes will be cultivated and which

enhance crop production through nitrogen fixation can then be selected for inoculant

production.

The legume-rhizobial symbiosis exhibits widely differing degrees of specificity.

In some instances, the symbiosis is highly specific in that a particular species or strain of

Rhizobium or Bradyrhizobium can form an effective symbiotic association with only one

particular legume species or variety, such as the temperate legumes, Trifolium, Cicer,

Phaseolus, Medicago and tropical species like Glycine max, Leucaena and Lotononis.

There are also intermediate cases which exhibit varying degrees of cross-inoculation

capability as in Centrosema, Phaseolus acutifolius, P. lunatus, some Desmodium spp. and

Acacia spp. At the opposite extreme are the promiscuous associations, in which diverse

legumes may be infected by one or more of several rhizobia.

This condition is more prevalent in the tropical legumes than in the temperate

species. Because the earlier studies of symbiotic nitrogen fixation were initiated in

temperate regions, the taxonomy of the genera Rhizobium and Bradyrhizobium were

based on a host-dependent classification system which emphasizes temperate

associations. A large number of tropical rhizobia which form symbiotic associations with

64

Vigna, Macroptilium, Arachis, Cajanus, Lablab, and other genera of legumes are simply

labelled as the "cowpea miscellany" or Bradyrhizobium spp. In some cases it is desirable

to select a strain for a wide range of hosts. An example would be the Bradyrhizobium sp.

(CB756; TAL 309) isolated from the nodule of Macrotyloma africanum. This strain

effectively nodulates approximately 40 of the promiscuous tropical legumes. This

`broad-spectrum strain' characteristic would be advantageous if this superior strain of

Bradyrhizobium sp. were to be introduced to locations where those diverse legumes are to

be grown. In a different situation, it might be advisable to work with a very specific

symbiosis to ensure infection by a particular inoculant strain that competes with native

soil rhizobia. Due to these and other considerations, characterizing rhizobial associations

is of utmost importance when a legume cultivar is being developed through breeding or

when a legume is being introduced into a new environment.

Field evaluation of effective rhizobia is critical because the symbiosis may be

affected by a number of environmental factors discussed earlier. The ability of an

inoculant strain to persist in a particular environment, while in some cases competing

against a resident soil population of rhizobia, is of critical importance. A combination of

the above factors should be anticipated in the selection process to ensure good

performance at different geographical locations. The task of introducing superior strains

into soils that are alreadyinhabited by effective rhizobia is difficult, and evaluation

methods are an important key to success.

65

Final evaluation of the symbiosis is based on several measurable parameters.

Short term trials with Leonard jars or sterile sand culture pots can provide an adequate

basis forgross comparison of strains. The shoot dry weight of plants harvested at floral

initiation or after significant plant biomass accumulation is the generally accepted

criterion for nitrogen-fixing effectiveness, but nodule dry weight may also be employed.

Nodule number is a less reliable indicator of strain effectiveness. The measurement of

activity in the nodules by the nitrogen-fixing enzyme, nitrogenase, may also be done.

This is accomplished by means of the acetylene reduction assay, which is a measure of

ethylene production and indicates nitrogenase activity. However, the results of this assay

should not be used to conclude on the actual amounts of nitrogen fixed. This assay

requires the availability of a gas chromatograph and other rather sophisticated equipment

and materials. Total nitrogen accumulation in the shoot can be measured by the Kjeldahl

method. Since total nitrogen content and nodule dry weight frequently correlate well

with shoot dry weight, the latter parameter provides an acceptable basis for strain

comparison. The final proof of inoculation response must come from the field when the

seed and nitrogen yields at harvest are determined for grain legumes or from the dry

matter production for forage legumes.

Identification and Characterization of Rhizobium:

Several phenotypic and genotypic methodologies are being used to classify

bacteria. Although, phenotypic methods play a key role in identification but genotypic

techniques are more authenticated, reliable and useful for identification and diversity

66

studies of bacterial isolates. Phenotypic methods rely on morphological tests,

biochemical tests, carbohydrate metabolism and enzyme production tests (Holt et al.,

1994), fatty acid analysis (Miller, 1982), intrinsic antibody resistance (Beynon and Josey,

1980), fluorescent antibody technique and polyacrylamide gel electrophoresis of total

proteins(Roberts et al., 1980; Dughri and Bottomly, 1983). Genotypic methods include:

PCR, PCR-RAPD (Polymerase Chain Reaction-Randomly Amplified Polymorphic DNA)

Rep-PCR, DNA-RNA, DNA-DNA hybridization, TP-PCR (Two primer-Polymerase

chain reaction) (Rivas et al., 2001), LMW-RNA profiles (Cruz-Sanchez et al., 1997) and

RFLP (restriction fragment length polymorphism).

A large numbers of molecular methods based on PCR reaction have been proposed

to characterise the R. leguminosarum strains and to provide a high degree of

differentiation of these strains (Laguerre et al., 1996; Moschettia et al., 2005).

2.3 Random Amplification of Polymorphic DNA:

The Polymerase Chain Reaction (PCR) has been utilized for the analysis of natural

microbial diversity (Giovannoni et al., 1990) and for identification of particular microbial

strain (Tsai and Olson, 1992). A modification of PCR referred to as RAPD analysis has

been developed (Welsh and McClelland, 1990). This method is based on amplification

of genomic DNA sequence by using a single oligonucleotide as primer. This

oligonucleotide may not be complimentary to specific DNA sequence in the genome but

under low stringency conditions a number of different sites of annealing are present on

genome depending on the length, the nucleotide sequence and the G+C content of primer

67

which allow amplification. The polymorphic fragments of DNA named RAPD

(Randomly Amplified Polymorphioc DNA) can be used as genetic makers (Hardy et al.,

1992). The RAPD technique is a potential tool for the identification of the genetics and

systematic of different populations. This technique use arbitrary primers to detect

changes in the DNA sequence at sites in the genome which anneal by the primer.

Phylogenetic relationships are constructed from RAPD fingerprints by statistical analysis.

The preliminary findings indicated that RAPD could provide us with a rapid and efficient

method for screening the differences between genomes. The RAPD markers were shown

to be highly useful in the construction of genetic maps, referred to as RAPD mapping.

In, conclusion, the RAPD technique is a potentially useful tool for the study of genetics

and systematic (Young and Cheng, 1998). The development of randomly amplified

polymorphic DNA (RAPD) markers provided a new tool for investigating genetic

polymorphisms in many different organisms, including bacteria (Cancilla et al., 1992;

Fani et al., 1993; Jayarao et al., 1992), and recently this method has been used for

Rhizobium identification and Bradyrhizobium genetic analyses (Kay et al., 1994).

Dragana et al. (2002) assessed phenotypic and genotypic characteristics of 42 indigenous

Rhizobium leguminosarum bv. trifolii by using different techniques. Genetic

polymorphism in Rhizobium strains can also be found out by newly developed techniques

based on proteins or nucleic acids (Young et al., 1991).

68

16S-rRNA gene Sequencing:

Currently by far the preferred phylogenetic marker used in microbial ecology

is16S rRNA sequence (Normand et al., 1996) 16S-rRNA is highly conserved molecule,

its sequence pattern is used to indicate phylogenetic relationship between two bacterial

species. As molecular chronometers, rRNA sequences have preserved their evolutionary

history (Woese, 1987). Highly conserved portions carry the information of early

evolutionary events and changes that are more recent occur with in less conserved

portions or streches. The degree of divergence of present day rRNA sequences gives an

estimation of their phylogenetic distances (Weisburg et al., 1991). The rRNA genes

(rDNAs) are similar in length throughout the bacterial kingdom and contain highly

conserved regions as well as regions that vary according to species and family (Woese,

1987). Consequently, sequence analysis of the 16S rRNA gene, which is the most

extensively studied, is a well established, standard method for Phylogenetic analysis

(Young et al., 1991). These 16S rDNA sequence analyses support the well established

subdivision of rhizobia into species and genera (Willems and Collins, 1993).

Direct detection of rhizobia:

Culture-independent methods involving polymerase chain reaction (PCR)-based

approaches have potential for specific detection of rhizobia in the environment (soils,

nodules, roots, inoculants) without the step of cultivation. Depending on the

discrimination level (species or strain detection), it is possible to identify specific DNA

69

(oligonucleotoides from total DNA, chromosomal, or symbiotic genes) which can be

used in PCR-based protocols or in hybridization methods.

The variations in rhizobial genome were initially studied to determine the diversity

and to type and identify rhizobia from culture collections. Genes coding for 16S rRNA

are used to identify rhizobia at the species and higher levels, while intergenic spacer (IS:

16S–23S rDNAIGS) genes allow the differentiation of strains within a same species.

DNA fingerprints obtained by using repetitive extragenic palindromic (REP) and

enterobacterial repetitive intergeneric consensus (ERIC) primers have been used to

identify and classify members of several rhizobial species (Laguerre et al. 1996).

Symbiotic genes are useful to type and classify rhizobia, as shown by the use of nod

probes (from nodulation genes) in hybridization protocols. Insertion sequences (IS) or

repeated DNA sequences (RS) are used for strain identification and for evaluating the

genetic structure of populations (Hartmann et al. 1992).

PCR-based protocols require firstly the extraction of microbial DNA from

environment samples. In soils, this step may be more difficult than anticipated, as soils

have complex composition. Compounds in the DNA extracts may inhibit subsequent

PCR amplification and different DNA extraction methods affect the abundance and

composition of bacterial community (Martin-Laurent et al., 2001). In nodules, DNA

extraction is not necessary, as PCR can been preformed directly from crushed nodules

(Tas et al., 1996). However, DNA extracted from nodules was used in a microarray

assay (Bontemps et al., 2005). Recent advances on isolation of DNA and detection of

70

DNA sequences in environmental samples have been recently published (Kowalchuk et

al., 2004).

Although PCR is recognized as the most sensitive qualitative method for the

detection of specific DNA in environmental samples, its quantification has become

restricted to the clinical area (Jansson and Leser, 2004). Until now, there is no

standardized and robust screening tool for the direct detection and counting of rhizobia in

soils. Only few protocols have been developed to trace specific strains, such as in

competition studies for nodulation (Tas et al., 1996) or in root colonization (Tan et al.,

2001).

Symbiotic Nitrogen-Fixing Efficiency of Rhizobia:

An efficient Rhizobium is a strain that is able to compete in the field with other

indigenous rhizobia for the colonization of the rhizosphere of its homologous legume

partner, under various soil physical and chemical conditions. This efficient strain forms

many large nitrogen-fixing nodules on the roots of the plant host that supply, for most

legumes, from 70% to 90% of the nitrogen. Thus, the best way to estimate the symbiotic

efficiency of rhizobial isolates is to do plant inoculation trials in field plots. However, as

only about 10% of field-isolated strains are very efficient. Therefore the symbiotic

efficiency of a large number of isolates has to be tested. Because of that, a first screening

is performed under artificial axenic conditions in tubes, growth pouches, Leonard jars, or

pots filled with sand or vermiculite or a mixture of both. These laboratory methods allow

the identification of strains with high N2-fixing ability, but they do not reflect the

71

competitive ability of the strains. This can be alleviated by performing assays in potted

field soils (Somasegaran and Hoben, 1994). However, the real symbiotic efficiency of a

strain cannot be determined without field plot inoculation trials. All legume inoculation

experiments require prior elaboration of a proper experimental design. Usually

treatments in addition to selected rhizobial strains include uninoculated and nitrogen

fertilized controls (Vincent, 1970). As commercially available legume inoculants include

very efficient strains of rhizobia intensively tested under field conditions, if available for

the area such an inoculant can be considered as a proper control.

Although rhizobia have several plant growth promoting mechanisms of action

(Antoun and Pre´vost, 2005), symbiotic N2 fixation is the most important mechanism in

legumes. The different methods used for measuring symbiotic N2 fixation in legumes

were appraised by (Azam and Farooq, 2003).

Rhizobium, Root Nodules & Nitrogen Fixation:

Rhizobium

Rhizobium is the most well known species of a group of bacteria that acts as the

primary symbiotic fixer of nitrogen.These bacteria can infect the roots of leguminous

plants, leading to the formation of lumps or nodules where the nitrogen fixation takes

place. The bacterium’s enzyme system supplies a constant source of reduced nitrogen to

the host plant and the plant furnishes nutrients and energy for the activities of the

bacterium. About 90% of legumes can become nodulated. In the soil the bacteria are

72

free-living and motile, feeding on the remains of dead organisms. Free living rhizobia

cannot fix nitrogen and they have a different shape from the bacteria found in root

nodules. They are regular in structure, appearing as straight rods; in root nodules the

nitrogen-fixing form exists as irregular cells called bacteroids which are often club and

Y-shaped.

Root nodule formation:

Sets of genes in the bacteria control different aspects of the nodulation process.

One Rhizobium strain can infect certain species of legumes but not others e.g. the pea is

the host plant to Rhizobium leguminosarum biovar viciae, whereas clover acts as host to

R. leguminosarum biovar trifolii. Specificity genes determine which Rhizobium strain

infects which legume. Even if a strain is able to infect a legume, the nodules formed may

not be able to fix nitrogen. Such rhizobia are termed ineffective. Effective strains

induce nitrogen-fixing nodules. Effectiveness is governed by a different set of genes in

the bacteria from the specificity genes. Nod genes direct the various stages of nodulation.

The initial interaction between the host plant and free-living rhizobia is the release

of a variety of chemicals by the root cells into the soil. Some of these encourage the

growth of the bacterial population in the area around the roots (the rhizosphere).

Reactions between certain compounds in the bacterial cell wall and the root surface are

responsible for the rhizobia recognizing their correct host plant and attaching to the root

hairs. Flavonoids secreted by the root cells activate the nod genes in the bacteria which

73

then induce nodule formation. The whole nodulation process is regulated by highly

complex chemical communications between the plant and the bacteria.

Once bound to the root hair, the bacteria excrete nod factors. These stimulate the

hair to curl. Rhizobia then invade the root through the hair tip where they induce the

formation of an infection thread. This thread is constructed by the root cells and not the

bacteria and is formed only in response to infection. The infection thread grows through

the root hair cells and penetrates other root cells nearby often with branching of the

thread. The bacteria multiply within the expanding network of tubes, continuing to

produce nod factors which stimulate the root cells to proliferate, eventually forming a

root nodule. Within a week of infection small nodules are visible to the naked eye. Each

root nodule is packed with thousands of living Rhizobium bacteria, most of which are in

the misshapen form known as bacteroids. Portions of plant cell membrane surround the

bacteroids. These structures, known as symbiosomes, which may contain several

bacteroids or just one, are where the nitrogen fixation takes place.

Nitrogenase:

An enzyme called nitrogenase catalyzes the conversion of nitrogen gas to

ammonia in nitrogen-fixing organisms. In legumes it only occurs within the bacteroids.

The reaction requires hydrogen as well as energy from ATP. The nitrogenase complex is

sensitive to oxygen, becoming inactivated when exposed to it. This is not a problem with

free living anaerobic nitrogen-fixing bacteria such as Clostridium. Free living aerobic

bacteria have a variety of different mechanisms for protecting the nitrogenase complex,

74

including high rates of metabolism and physical barriers. Azotobacter overcomes this

problem by having the highest rate of respiration of any organism, thus maintaining a low

level of oxygen in its cells.

Rhizobium controls oxygen levels in the nodule with leghaemoglobin. This red,

iron-containing protein has a similar function to that of haemoglobin; binding to oxygen.

This provides sufficient oxygen for the metabolic functions of the bacteroids but prevents

the accumulation of free oxygen that would destroy the activity of nitrogenase. It is

believed that leghaemoglobin is formed through the interaction of the plant and the

rhizobia as neither can produce it alone.

Observation of a cut root nodule:

If a root nodule is cut open and the inside is pink/red the nodule is active and

fixing lots of nitrogen for the plant. The colour is due to the presence of plenty of

leghaemoglobin. The redder the nodule, the more active it is. When nodules are young

and not yet fixing nitrogen they are white or grey inside. Legume nodules that are no

longer fixing nitrogen turn green and may be discarded by the plant. This may be the

result of an inefficient Rhizobium strain or poor plant nutrition.

Selection of Improved Strains of Root Nodule Bacteria:

The abundant diversity clearly present in soil populations of root nodule bacteria

provides a large resource of natural germplasm to screen for desired characteristics

(Sadowsky and Graham, 1998; Dilworth et al., 2001). Useful variation in many of the

75

appropriate characteristics required in inoculums strains seem to exist in this natural pool

of soil root nodule bacteria.

What characteristics are needed?

An essential desired characteristic for inoculums strains of root nodule bacteria is

highly effective nitrogen-fixation with the intended host species, and in some instances

there is a requirement for the strain to effectively nodulate a wide range of host legume

species. Other beneficial characteristics include stress tolerance, competitive ability

against the indigenous strains, genetic stability and satisfactory growth and survival

during procedures for manufacture of inoculum (Howieson et al., 2000)

Screening of Rhizobia from root nodule bacteria:

The current techniques used at the Centre for Rhizobium Studies to select

appropriate strains of root nodule bacteria require a combination of glasshouse and field

work (Howieson et al., 2000). At present screening and selection of root nodule bacteria

in the laboratory is neither possible nor worthwhile to attempt. The criteria for successful

laboratory screening for the key characteristics, such as acid tolerance, are not known

(Dilworth et al., 2001). There is need of the host plant and soil environment in any

screening program. The strain selection program developed at the Centre for Rhizobium

Studies has four principle phases. Firstly, there is germplasm acquisition, isolation of

strains and their storage and maintenance in a stable secure environment. Often isolates

of root nodule bacteria are obtained from the centres of origin of the host legume and/or

76

stressful soils. At the Centre for Rhizobium Studies strains of root nodule bacteria are

stored as lyophilised cultures, and in deep frozen suspensions in 20% v/v glycerol.

The second phase involves glasshouse experiments to authenticate isolates as root

nodule bacteria and screen for effective nitrogen fixation. A pool of effective strains is

then taken to the field for assessment of their adaptation to the edaphic environment

where they are intended to be used. These field trials are conducted over two or three

years to determine the capacity for the strains to survive in, and colonize, the soil. There

is often considerable variation between effective strains in their performance in the field.

The final phase involves validation of strain performance using a smaller number of

strains in larger scale rotation trials in farming systems, often carried out independently

by researchers in different locations. Even at this stage, there may be significant

variation in strain performance.

High-Efficiency Transformation of Rhizobium leguminosarum:

The Rhizobium-legume symbiosis accounts for a significant proportion of nitrogen

available to leguminous plants. Thus, there is a need to manipulate rhizobia to increase

their symbiotic efficiency and host range. An important prerequisite for genetic

improvement of any bacterial species is the availability of a highly efficient gene transfer

system. Transformation systems developed for rhizobia (Raina and Modi, 1969) are far

less efficient than those for other bacteria. So, introduction of foreign DNA into rhizobia

has been possible exclusively via conjugal matings with Escherichia coli (Ditta et al.,

77

1980); such procedures are time-consuming, however, and limited to special plasmids

having the mob gene.

Electroporation involves the use of a high-intensity electric field of short duration

to induce reversible permeabilization in the cell membrane to facilitate the entrance of

macromolecules such as DNA (Chassy et al., 1988). Electroporation was applied

initially for transformation studies in mammalian cells (Neumann et al., 1982) and was

found to be effective with bacterial protoplasts as early as 1983 (Shivarova et al., 1983).

Shortly thereafter, this technique was applied successfully to transform intact cells of

both gram-positive and gram-negative bacterial species with plasmid DNA (Dower,

1987; Harlander, 1986). Now, electroporation is a novel approach for introduction of

foreign DNA into bacterial species poorly transformable or for which transformation

protocols have yet to be established. The electroporation conditions required for

maximum transformation efficiency vary from cell to cell.

A plasmid-free, chloramphenicol-sensitive rhizobial strain, R. leguminosarum T-

19 C (Department of Microbiology, CCS Haryana Agricultural University, Hisar, India),

was used in the study and was grown in yeast extract-mannitol (YEM) broth (Fred et al.,

1932). E. coli S-17-1 (Simon et al., 1983) harboring plasmid pMP154 was grown at

37°C in Luria-Bertani (LB) broth (Sambrook et al., 1989) containing 20 µg of

chloramphenicol per ml. Plasmid pMP154, used for electrotransformation study, was an

IncQ transcriptional fusion plasmid (15 kb) containing the nodA promoter of R.

leguminosarum Sym plasmid pRL1JI cloned as a 114-bp restriction fragment in front of

78

the E. coli lacZ gene and also carries a chloramphenicol resistance marker (Spanik et al.,

1987).