REVIEW OF LITERATURE
The present review deals mainly with studies on various aspects of root nodule bacteria associated with wild legumes. However, whenevernecessary, relevant literature regarding studies on similar aspects of other cultivated legumes and their bacteria has also been incorporated. The review has been divided into the following parts
2.1 NODULATION STATUS
2.1.1 Black-coloured nodules.
2.2 STUDIES ON ROOT NODULE BACTERIA
2.2.1 Classification
2.2.2 Cultural and Morphological characteristics
2.2.3 Biochemical and Physiological characteristics.
2.3 PLANT INFECTIVITY AND CROSS-INOCULATION STUDIES
2.3.1 Plant infection test
2.3.2 Cross-inoculation studies
2.3.3 Nodulation test on Siratro
2A CROP RESPONSE TO INOCULATION
2.4.1 Competition studies
2.if.2 Application of rhizobia isolated from wild legumes on cultivated legumes.
2A.3 Evaluation of symbiotic nitrogen fixation efficiency.
2.5 NITROGEN FIXATION BY WILD LEGUMES. 2.1 NODULATION STATUS
The ecological uniqueness of the Leguminosae derives from the tubercles or nodules of their root systenns. A wide variation is seen in the nodulation ability of leguminous plants. Allen and Allen (1981) who have compiled extensive nodulation survey data of Leguminosae, claim that ^8% of leguminous genera have been examined for nodulation status. Out of this percentage, 84% were found to be nodulated. Of the genera examined in Mimosoideae and Papilionoideae,
83.87 % and 94.17 % respectively included nodulated species, whereas in
Caesalpinoideae only 40% of the genera examined showed nodulation ability.
Although tropical flora is rich in leguminous plants, there is very limited knowledge about the nodulation of tropical legumes (Banadoz and Fernandez, 1954;
Bowen, 1956; Lange. 1959; Desouza, 1966).
According to Allen and Allen (1947), Williams (1967) and Lim and Burton
(1982), the limited knowledge about the nodulation status of Leguminosae is due to the following major difficulties
(i) Inaccessibility of specimens, many of which are found in areas not easily
reached by collectors.
(ii) Confinement of some genera and species to certain limited parts of
the world.
(ill) Difficulty of examining woody tree genera for presence or absence of
nodules.
(iv) Non-availability or short life of seeds of tropical legume species which
are required for confirming nodulating ability.
(v) The fact that, if not all, most of the promising tropical legumes are 7
poor seed producers, or that the seeds they produce are difficult to
harvest for reasons of pod dehiscence, intermediate flowering habit,
or buried seed bearing pods in masses of foliage.
Beadle (196^) found that out of 80 legumes surveyed in arid or semi-arid regions of eastern Australia, 68 were nodulated. Norris (1969) surveyed rain forest legumes in Amazonia and Guyana and recorded nodules on 30 out of 53 tree species examined. Corby (1971) described the characteristics of wild legumes indigenous to Rhodesia, according to their tribal classification. Gallardo (1970) examined nodulating species in Argentina. Dubey e^_aj_ (1972) surveyed the
Island of Puerto Rico for indigenous nodulated legumes.
Lim and Ng (1977) examined the nodulation of legumes in Singapore.
•They reported for the first time the occurrence of nodules on Adenanthera pavonina.
Yanasugondha e t ^ (1977) studied the nodulation of 52 species of native legumes from various parts of Thailand.
The nodulating ability of legumes in sub-tropical Pakistan was studied by Athar and Mahmood (1979) who reported 52 species of Papilionoideae as having nodules, five of these being new records.
The work done in India on the nodulation of wild legumes spreads over
the last three decades - apart from isolated reports like that by Joshi (1920) who worked on root nodule bacteria in Crotalaria juncea. Nodulation on Crotalaria retusa and Clitorea ternatea was shown by Rangaswami and Oblisami (1962). Satyanarayan and Gaur ( 1965) observed that Atylosia scaraboides, Rhyncosia minima and
Tephrosia purpurea plants have very deep root systems and they are either devoid
of nodules or contain very few, limited to their lateral roots. The nodulation 8
and nitrogen fixation ability of Sesbania cannabina has been studied by Singh
(1971). Nodulation in 2^* wild legunne species growing in Hooghley and Burdwan districts of West Bengal was reported by Sinha e ^ ^ (1971).
According to Subba Rao (1972) hardly 10% - 12% of leguminous species have been examined for nodulation; and of these, 65% Caesalpinoideae, 10%
Mimosoideae and 6% Papilionoideae plants do not bear nodules.
Out of the 25 species of tree legumes belonging to the genera Acacia,
Albizia, Bauhinia, Colophospermum, Dichrostachys, Leucaena, Peltophorum,
Pithecellobium and Prosopis nodulation was observed by Basak and Goyal (1980a) in only 19 species.
Nodulation on Crotalaria angulata and Rhyncosia velutina was reported for the first time by Subramanian and Manjula (1986) who also reported that
Cassia auriculata and occidentalis lack nodulation although previously reported as nodulating species.
Few reports are available on the nodulation of wild legumes in Maharashtra
State. Bhelke (1972) reported nodulation for the first time on some species of Alysicarpus monj liferg^ A. tetragonolobus, Clitorj a biflora, Crotalaria filipes,
C. nana, Dalbergia sympathetica, Geissaspis cristata, Smithia capitata, S^. purpurea and pycnantha.
Nimbalkar . (1986) reported nodulation on 19 legume species during the survey of wild legumes in Western Maharashtra. 2.1.1 Black-coloured nodules.
The internal pigmentation in the root nodules of legumes is indicative of their nitrogen-fixing effectiveness. Actively nitrogen-fixing nodules range from pink to red-brown in colour due to the presence of leghaemoglobin. Non nitrogen-fixing nodules, on the other hand, are normally white or green in colour.
Occasionally black nodules are formed. While screening rhizobial isolates from west Africa for effectiveness in the case of cowpea, Eaglesham et al (1982) detected black-coloured nodules in cowpea.
Instances of unusually black-coloured nodules have been reported for several tropical legumes; Vigna mariana (Allen and Allen, 1936), Doiichos lablab
(Cloonan, 1963), Mimosa spp. and Leucaena glauca (Campelo and Campelo, 1970),
Centrosema pubescens and Phaseolus atropurpureus (Vincent, 1970), Mucuna pruriens
(Nimbalkar, 1986). According to Cloonan (1963), the black pigment may be due to melanin formed by the enzyme phenolase in the nodules.
Eaglesham e^_a^ (1982) attempted to correlate nodule phenotype with colony morphology and found that only certain dry-colony type strains and not
the wet colony-type, caused black nodulation on cowpea.
Studies by Raffiquddin (198^) on competition between inoculated
and native rhizobia for nodulation of cowpea, Vigna unguiculata Walp. showed
the usefulness of black-nodule strain in evaluating nodulating competitiveness
of cowpea rhizobia in soils where black-nodule strains are not indigenous. 10
2.2 STUDIES ON ROOT NODULE BACTERIA
2.2.1 Classification :
In the 9th edition of Bergey's Manual of Systematic Bacteriology, nodule- forming bacteria have been classified into two genera ; Rhizobium and Bradyrhizobium
(Jordan, 198^). Differentiation of these genera is supported to a great extent by cross-inoculation groups. Fast-growing, nodule-forming bacteria having a generation time of less than six hours have been included in the genus Rhizobium whereas the nodule bacteria having a generation time of more than six hours form the genus Bradyrhizobium (Jordan, 1982).
In the new system of c la s s ific a tio n , Rhizobium phaseoli and
■Rhizobium trifolii have been fused with Rhizobium leguminosarum which contains
three biovars, viz. viceae, trifolii and phaseoli. Rhizobium meliloti (Dangeard)
and Rhizobium loti have been designated as separate species (Jarvis 1982).
A new species Rhizobium fredii is erected for fast-growing rhizobia that nodulate
soybean (Scholia and Elkan, 198^).
The genus Bradyrhizobium represents a heterogeneous group of nodule
bacteria of which the taxonomic relationships are not well understood. This
genus has only one designated species, viz. Bradyrhizobium japonicum. For the
present, it is suggested that, members of the genus Bradyrhizobium, other than
japonicum, be referred to as Bradyrhizobium sp. with the name of the appro
priate plant in parenthesis immediately following; e.g. Bradyrhizobium sp. (Vigna).
In this classification, no species epithet is given to the members of the cowpea
miscellany group. 11
At present not more than approximately 8% of the 1^,000 or so known
species of legunninous plants have been examined for nodulation, and less than
0.5% have been studied with respect to their symbiotic relationships with nodule
bacteria (Jordan, 1982; Tauro, 1986). Most of the plants examined have been
of agricultural importance, and the huge reservoir of wild tropical leguminous
plants is only beginning to be investigated. It thus becomes clear that the present
classification can not be accepted as final and it is quite likely that it will
be gradually modified after a larger number of Rhizobium strains from a wide
variety of leguminous plants will be studied.
2.2.2 Cultural and morphological characteristics :
The rhizobial cells are aerobic, Gram negative, motile rods measuring
G.5 - 3.5 )jm X 0.7 - 3 ym, non-spore forming, capsulated or non-capsulated with polar or peritrichous flagella. The typical rhizobia produce circular, convex,
white glistening colonies, with entire margins on Congo red yeast extract mannitol
agar medium. The fast-and slow-growing organisms produce in 7 days colonies
of k mm and 1 mm diameter respectively (Graham and Parker, 1964). Crow et al
(1981) designated strains with growth rate of less than five hours as fast-growing
and those with more than five hours as slow-growing.
Generally the rhizobia associated with wild tropical legumes are slow
growers (Allen and Allen, 1936; Norris, 1956 and Lange, 1961) but fast-growing
rhizobia have also been isolated from some species, e.g. Samanea, Andura, Alibizia
and Cytisus (Allen and Allen, 1936); Psoralea (Norris, 1965); Leucaena leucocephala.
Acacia farnesiana, Mimosa invisa and M. pudica (Trinick, 1980); Sesbania grandiflora,
Acacia pennatula and Gliricidia sepium (Roskoski and Wood, 1984). Lim (1976)
have isolated fast-growing rhizobia from tropical legumes of Singapore belonging 12
to subfamilies Caesalpinoideae and Mimosoideae.
Both fast- and slow-growing rhizobia of wild and cultivated legumes have been isolated from species surveyed in Western Maharashtra (Basnyat,
1979). Gangawane and Salve (1986) isolated fast-growing rhizobia from
Alysicarpus vaginalis, A. monolifera and tetragonolobus. Nimbalkar (1986) isolated 5 fast and 14 slow-growing rhizobia from wild arboreal and herbaceous legumes growing in Maharashtra.
2.2.3 Biochemical cind Physiological characters ;
There are many reports (Fred 1932; Allen and Allen, 1950; Graham and Parker, 1964; Kleczkowska et 1968; Clark, 1969; Lange, 1961; Gaur ^ ah,
1973; Skinner, 1977; Gaur and Sen, 1981) on the usefulness of certain biochemical and physiological characteristics in the presumptive identification of Rhizobium.
2.2.3.1 Production of 3 ketolactose :
Most of the strains of Agrobacterium tumefaciens and _A. radiobacter produce 3 ketolactose on lactose-containing medium detectable as a yellow ring of precipitate of cuprous oxide around the growth when the plates are flooded with Benedict's reagent (Bernaerts and De Ley, 1963). Neither Rhizobium nor
Bradyrhizobium are able to produce 3 ketolactose (Gaur and Sen, 1981).
2.2.3.2 Growth on lactose manganous salt medium :
Clark (1969) examined many strains of different bacteria for manganese
toxicity based on the finding of Mustafa (1966) that manganous sulphate is toxic 13
to certain Gram negative bacteria. He found that agrobacteria tolerated 20 m.e.
Mn"^"^ per litre of medium but neither plant pathogenic bacteria nor rhizobia
could do so. The test thus becomes important for differentiation between the
two genera. Gaur and Sen (1981) confirmed Clark's finding with Cicer rhizobia.
2.2.3.3 Growth on glucose peptone agar :
In this medium all strains of rhizobia, except a few of medic rhizobia,
show little or no growth within 48 hours, whereas agrobacteria grow well with
a change in the reaction of the medium; hence this forms one of the important
preliminary differentiation tests (Kleczkowska ^ 1968). Gaur and Sen (1981)
while studying Cicer rhizobia showed that most of the strains exhibited no growth,
and that few which showed a little growth did not change the reaction of the
■medium in hours. Similar observations were made with rhizobia from wild
arboreal and herbaceous legumes by Nimbalkar (1986).
2 . 2 . 3 A Growth in Hofer's alkaline medium :
Hofer (1935) observed that none of the rhizobia except a few strains
from medic could grow in a moderately alkaline medium of pH 10, while in
a strongly alkaline medium of pH 11 agrobacteria, but not medic rhizobia, could
grow. This characteristic was considered suitable in negative selection of rhizobia
from agrobacteria (Allen and Allen, 1950; Vyas and Prasad, 1959). However,
many contradictory reports have been published (Graham and Parker, 1964;
Basak and Goyal, 1980b) which assign only a limited value to the test. 14
2.2.3.5 Nile blue reduction test ;
Hamidi (1969) has described a test to distinguish rhizobia from agrobacteria based on the greater ability of the latter to reduce Nile blue. Skinner (1977) modified Hamidi's Nile blue test and observed that 99% of rhizobia failed to reduce the dye whereas all strains of agrobacteria reduced it completely to a colourless state. None of the 19 rhizobia from wild arboreal and herbaceous legumes reduce the dye, as reported by Nimbalkar (1986).
2.2.3.6 Growth and reaction in Litmus milk :
Reactions in litmus milk after 1-6 weeks of incubation have been used to record changes in the acid or alkaline direction, and in the development of the digested serum zone, which is a feature characteristic of some strains of rhizobia. On the basis of changes in reaction and serum zone formation,
Stevens (1923) divided organisms into three groups :
(1) Serum zone positive and reaction acidic;
(2) Serum zone positive and reaction alkaline;
(3) Serum zone negative and reaction alkaline.
According to Fred et ^ (1932), the acid producing Rhizobium meliioti reduced litmus milk, the alkali-producing rhizobia of Glycine max and Phaseolus lunatus did not reduce litmus milk, while Rhizobium leguminosarum and trifolii were quite variable in thisproperty. Variation in the litmus milk test among the rhizobia of the same host species were also observed by others (Raju, 1936;
Allen and Allen, 1950; Vyas and Prasad, 1959; Graham and Parker 196^; Gaur and Sen, 1981). 15
2.2.3.7 Acid and alkali production :
The growth of rhizobia in a medium containing any carbon source is
accompanied by change in the reaction (acidity or alkalinity) of the medium
depending upon the kind of bacterium and carbohydrate used. This was first
demonstrated by Bialsuknia and Klott (1923). Subsequently, it was confirmed
by many others (Fred et 1932; Raju, 1936; 1938; Johnson and Allen, 1952;
Vyas and Prasad, 1939; Jones and Burrows, 1969). Acid and alkali production
with yeast-extract mannitol (YEM) as the culture medium has been employed
as a general taxonomic character for rhizobia (Norris, 1965). With a mannitol
medium, barring a few exceptions, rhizobia of soybean, lupin, and cowpea groups
produced alkali; whereas those of medic, clover, pea and bean groups produced
acid.
Norris (1965) advanced the hypothesis that the slow-growing non-acid
producing types of rhizobia, commonly associated with tropical legumes, represent
the ancient forms of symbiont, which have persisted unchanged because acid production during growth in the rhizosphere in acid soils would react unfavourably
against survival. The acid-producing type of rhizobia were considered to have
evolved at a later stage, as the host legume adapted to non-acid soils. Acid
and alkali production was considered the means for survival against adverse
soil conditions. If a group of legumes is found to be characteristically associated
with Rhizobium - that is strongly acid producing - it indicates that the host
group is an advanced cne that has become strongly adapted to non-acid soils.
Norris, himself has pointed out the limitation of his hypothesis i.e. biased culture
collection (mainly herbaceous members of the Papilionoideae), from temperate
regions, and has suggested the need for further work especially with rhizobia
from tropical legumes in order to check the hypothesis. 16
Working with rhizobia isolated from the legumes growing in India, Gaur and Sen (1981) and Wange (1986) found that acid or alkali production is independent of the property of soil from which rhizobia have been isolated, and thus did not support Norris's hypothesis. This was further confirmed by Basak and Goya!
(1980b) and Nimbalkar (1986) while working with rhizobia from wild legumes in India.
2.2.3.S Sensitivity to Crystal violet :
The relation between the growth of root nodule bacteria and Crystal violet sensitivity has been studied by Stevens (1923), Johnson and Allen (1952),
Shete (1965), Konde (1975) and Nimbalkar (1986). Johnson and Allen (1952) and
Nimbalkar (1986) stated that the fast-growing rhizobia were more tolerant to
Crystal violet than the slow-growing strains. The former further stated that a relationship exists between tolerance of rhizobia! strains to Crystal violet and their ability to produce gum. Shete (1965) and Konde (1975) observed that ineffective strains could resist higher concentrations of Crystal violet, whereas effective strains could grow only at lower concentrations. Gaur and Sen (1981) while studying Cicer rhizobia, found that the rhizobial strains do not absorb
Crystal violet dye.
2.2.3.9 Starch hydrolysis :
Beijerinck (1888) stated that root nodule bacteria are unable to degrade starch. Except a few reports (Mukerjee and Johari 1967; Dawkhar, 1962; Muthusamy et 1973) the rhizobia have not been found to hydrolyse starch (Fred et al.,
1932; Conklin, 1936; Vyas and Prasad, 1959; Oblisami 1974). 17
2.2.3.10 Gelatin liquefaction :
Many workers reported inability of nodule bacteria to liquefy gelatin
(Fred et al_., 1932; Zipfel, 1911; Conklin, 1936; Gupta and Sen, 1962; Obiisami,
197^f; Nimbalkar, 1986). Burril and Hansen (1917) and Stapp (192^) demonstrated
slow liquefaction of gelatin, which was later confirmed by Allen and Allen (1950)
and Muthusamy ^ al_ (1973).
2.2.3.11 Reduction of nitrate :
Reduction of nitrate was investigated by Zipfel (1911), Allen and Allen
(1930), Jordan and San Clemente (1953), Graham and Parker (196^) and they
found that rhizobial strains under study reduced nitrate to nitrite. Muthusamy
- ^ •^(1973) found that the nitrate reducing rhizobia from groundnut were highly
efficient in nodulation and nitrogen fixation. Manhart and Wong (1973), however,
demonstrated that there was no correlation between high nitrogen fixation activit
ies and nitrate reductase activity. As per 3ain and Rewari (1983) nitrate reduction
was more in temperature - tolerant strains than in temperature-sensitive Rhizobium
strains of black gram, cowpea and pigeonpea.
2.2.3.12 Reduction of Methyl red :
Graham and Parker (196^) while characterizing strains of rhizobia from
various legume species, failed to record reduction of Methyl red, whereas Konde
and Moniz (1967) and Moniz et ^ (1968) have reported reduction of Methyl
red by rhizobia of medic, clover and lablab bean. 18
2.2.3.13 Production of hydrogen sulphide :
The claim that the rhizobiaJ strains do not produce H^S was confirmed by Vyas and Prasad (1959), Graham and Parker (196^+), Konde and Moniz (1967).
However, Adkoli (1952) reported H^S production in certain Cicer rhizobial strains.
2.2.3.1^ Production of indole :
Thiamann (1936) put forward the hypothesis that rhizobia convert tryptophan into heteroauxin which leads to nodule formation. It was suggested that tryptophan exuded by plants is converted into indole acetic acid by Rhizobium
(Kefford ^_aL, 1960). Some workers support Kefford's finding (Fred e^ 1932;
Muthusamy e ^ ^ ., 1973) but others do not (Graham and Parker, 1964; Konde and Moniz 1967; Moniz £t al_., 1968).
2.2.3.15 Production of enzymes :
Graham and Parker (1964) reported production of catalase and urease in strains of Rhizobium meliloti, _R. trifolii, leguminosarum, phaseoli,
lupini, japonicum, and Rhizobium spp. (Cowpea rhizobia). Francis and
Alexander (1972) observed significant differences in the catalase content in
nodules and bacteroids of varying degree of effectiveness, and suggested a possible
implication of catalase in the rhizobial effectiveness.
Graham and Parker (1964), showed that 37 out of 48 fast growing strains
and 25 out of 31 slow growers showed urease activity. Sadowsky et ai (1983),
found that all fast-and slow-growing isolates from soybean showed catalase,
urease and oxidase activity. 19
2.2.3.16 Carbohydrate utilization :
In many studies the utilization of different carbon sources by rhizobia have been examined (Table 2). It appears that all the rhizobia, irrespective of their host group, utilize almost all the different carbon sources. If there is any difference it may be quantitative depending upon the strain but not upon the group of rhizobia.
2.2.3.17 Glucose consumption :
Attempts have been made from time to time to work out some method for determination of efficiency of Rhizobium spp. from their easily assessible characteristics. Since sugar is used as energy material for fixation of nitrogen by, nitrogen fixing organisms, it was suspected that high sugar requirements might be related to their capacity for nitrogen fixation (Gupta and Sen, 1965).
The relationship between glucose consumption by Rhizobium japonicum and its nitrogen fixation efficiency has been studied by Sen (1965) who observed a significant positive relationship between the two parameters. Gupta et ^
(1971) reported wide variation in glucose consumption by strains of Rhizobium belonging to the same species. Jain and Rewari (1983) found more glucose consump tion in temperature-tolerant than in temperature-sensitive Rhizobium strains of black gram, cowpea and pigeonpea.
2.2.3.18 Tolerance to sodium azide :
Studies on azide resistance were first carried out with a wild
R. leguminosarum strain and its mutant L4 (Ram et aL, 1978) and a positive 20
ON O s Os CD lA kA lA ON On ON kC ON r-^ O CJ lA kT\ T- LA T-
c 'I
D O IG u u
CDc 00 co GO I CO d) J3 uT. Ct) CM lA LA CO CO CO lA l/> a (/) CSJ lA «— CM lA lA lA r- 8 w (D i 0) a ■o a CJ !£ Mm E u 0 3 1 2P
c c S (0 lA rs a CO = 5 ? : lA lA ■i « I >> 06I ja • • 0) (M > o lA OJ U CN 1 lA lA kA u '■5 0) a) to (D V5 0) o O O W _o c (/) c c S O O o o u o CD !5 E c £ c 03 CD o iS CJ O c (D 5 0) >v CD 3 D CD ( j CL cs: X O C3 2 21 OJCO ON Ql $ VO IK o ON CTs o (J ON irC ltT ltT D O c 0>CD Xi CD O W) 0)(U a o o!E CDc Q) CD lA 0}CO a eg 03 (D ;d ;d *b>CD £ -CCO U u CDo .fc o ^ ^ -g ■fiCO 5o CO o 0) r ^ o o V> a : CL Q £ < cn 22 correlation was discovered between azide resistance and nitrogen fixation efficiency on Pisum sativum. There is evidence that azide (N^ ) is one of the several substances available in Azotobacter vinelandii and Clostridium pasteurianum for reduction by nitrogenase (Hardy and Knight, 1967). Since the subunits of a rhizobial nitrogenase are complementary to those of _A. vinelandii, it seems possible that may be a substrate for nitrogenase coded by rhizobia (Burris, 1971). The lethal action of in bacteria is mainly due to impairment of the respiratory electron transport chain. it can be argued that if the nitrogenase - catalysed reduction of should occur quantitatively vitro, then N^~ will not be available in sufficient amount for inhibitory action on an energy generating system. Thus, there can exist resistant strains in which relief from N^"- caused inhibition will be due to dilution of by its intracellular reduction. 2.2.3.19 Studies on pH, temperature and salt tolerance : Highly active nitrogen fixing strains of Rhizobium often fail to survive during storage of the inocula in different carrier materials or following their application to seed. This is primarily due to various stresses experienced by the organisms; hence it becomes important to find out the survival ability of the selected rhizobial cultures under the influence of these stresses. 2.2.3.19.1 pH tolerance : Although neutral pH appears to be most favourable for the growth of rhizobia (Allen and Allen, 1930), tolerance to a wide range of pH has been shown (Graham and Parker, 196^*; Habish and Khairi, 1970; Yadav and Vyas, 1973; Scholia and Elkan, 198«t). Yadav and Vyas (1973) have found that some strains of cowpea rhizobia could grow at pH values as low as 3.5. They found however, 23 that the rhizobia did not nodulate at this pH. While the literature shows a wide variability in nninimum pH tolerance of rhizobia the indications are that the cowpea group probably tolerate lower pH values than others. 2.2.3.19.2 Salt tolerance : Yadav and Vyas (1971) and Graham and Parker (1964) observed that at 2 % NaCl concentration in the medium, Agrobacterium radiobacter, _A. tumefaciens and a few _R. meliloti strains grew while none of the rhizobial strains belonging to the other six Rhizobium cross-inoculation groups could grow. At 3% NaCl concentration, except for Agrobacterium, none of the rhizobial strains could grow. These authors therefore claimed this test to be of diagnostic importance. This finding was further confirmed with rhizobia from clover (Pilai -and Sen, 1969), soybean (Wilson and Norris, 1970) and lotus (Jarvis et ^ ., 1982; Scholia and Elkan, 198^+). Steinborne and Roughley (1975) showed that salt tolerances of the strains were not related to their ecological origin. They further stated that many rhizobial strains can not only withstand the stress but may even grow at salt concentrations in excess of those tolerated by most agriculturally important legumes. 2.2.3.19.3 Temperature tolerance : It has been suggested that growth and survival of rhizobia in soils and their symbiotic association with leguminous plants are adversely affected by high soil temperatures. Hence the ability to tolerate higher temperatures is a desirable property of any Rhizobium strain to be used in the tropics. The survival of rhizobia in inoculant materials has also been found to be severely 24 affected by high storage temperatures among species and strains of Rhizobium has been recognized, and selection of strains for temperature tolerance has been suggested as a means of overcoming temperature stress. Allen and Allen (1950) stated that the optimum temperature for most rhizobia occurs between 2 5 ° C and 30°C, with 35°C preferred by meliloti isolates. Graham and Parker (1964) obtained growth at 39°C with only eight strains, all belonging to Medicago group; other rhizobial types failed to survive. The rhizobial isolates from wild arboreal legumes could grow well at 35-40°C but the growth of all the isolates was adversely affected at 45-50°C (Basak and Goyal, 1980b). ,2.2.3.20 Antibiotic resistance : Most rhizobia are sensitive to a wide spectrum of antibiotics, and many workers (Davis, 1962; Graham, 1963; Cole and Elkan, 1979; Nimbalkar, 1986) have tested a large number of fast and slow-growing rhizobia against them. The strain to strain variation within a rhizobial group is large enough to make any generalization. Graham (1963) observed that slow-growing rhizobia were less susceptible to antibiotics than the fast-growing ones. He further suggested that pasture legumes requiring slow-growing rhizobia would establish more easily. Sinclair and Eaglesham (1984) studied the intrinsic antibiotic resistance of 128 strains and observed multiple resistance against rifampicin, gentamycin and penicillin by most of the strains having wet colonies (transluscent, copious watery slime, confluent with raised elevation) whereas the strains having dry colonies (opaque, paste like, granular, never confluent, punctiform with raised 25 elevation) showed resistance against only one or two antibiotics. Intrinsic antibiotic resistance to a range of antibiotics may contribute to the saprophytic competence of a strain introduced into a soil as an inoculant. The intrinsic antibiotic resistance pattern is generally used for strain identification in ecological studies (Gupta 1983). 2.3 PLANT INFECTIVITY AND CROSS-INOCULATION STUDIES 2.3.1 Plant infection test : The tests mentioned for distinguishing Rhizobium from Agrobacterium and other non-rhizobial contaminants such as ketolactose test, growth in Hofer's alkaline medium, Nile blue reduction test, etc. are of limited value and offer only presumptive evidence that root nodule bacteria under study are either Rhizobium or Agrobacterium species. The only technique by which this identifi cation can be confirmed is through their nodulation ability. When specific seeds are not available, cross testing with another species in the same genus or plant species with closely related genera in the same tribe has to be carried out (Allen and Allen, 1981). Such a plant infectivity test supplies a positive proof that the reported results are obtained with bona fide Rhizobium or Bradyrhizobium species and that the results are reproducible. 2-3.2 Cross-inoculation studies : The ability of rhizobia to infect leguminous plants is highly specific and this host specificity is primarily used for the classification of these organisms. Fred et al (1932) for the first time put forward the concept of cross-inoculation 26 groups. This classification assumes that species of Rhizobium will nodulate only those plants which fall within a particular "infective" or "cross-inoculation" group, and that within such groups, rhizobia from one plant will nodulate all other plants and vice versa. Under this system, seven cross-inoculation groups are recognized, viz.. Pea, Bean, Alfalfa, Clover, Soybean, Lupin and Cowpea miscellany group. Graham (1976) has pointed out the following major limitations of the cross-inoculation concept. (A) Cross-infection i.e. nodulation of plants from one affinity group by rhizobia from another group. (B) Insufficient nodulation data i.e. out of l^f,000 known species of legumes only 8% - 9% have been examined for nodulation and only 0.3% - 0 . k % have been studied for the root nodule bacteria. (C) Lack of supporting biochemical data :- Many of the biochemical studies have been carried out on only a few strains and hence it is difficult to generalize regarding the taxonomic significance of the results. According to Elkan (1981) the assumption that each species of Rhizobium nodulates only a plant within a specified cross-inoculation group has lost crec(ib)lity. However, Somasegaran and Hoben (1985) advocate that cross-inoculation grouping is the best practical system currently available in spite of criticisms levied against it. There are a number of reports on cross-inoculation groupings of rhizobia from wild legumes, and the results showed that most of them belong to the 27 cowpea cross-inoculation group (Table 3). Table 3 shows that there are no reports on cross-inoculation grouping of the genera Butea, Gliricidia and Taverniera. The rhizobia from these genera are being examined in the present investigation for the cross-inoculation grouping. 2.3.3 Nodulation tests on siratro : Most of the tree legume rhizobia belong to the cowpea miscellany cross inoculation group, for which siratro (Macroptilium atropurpureum (DC.) Urb.) is used as a test host for authentication. While studying rhizobia from Acacia dealbata and _A. melanoxylon, Rangarajan and Balaji (1985) found that the strains could nodulate Vigna unguiculata Walp. within 21 days, but that they failed to nodulate Macroptilium atropurpureum even after ^5 days. 2 A CROP RESPONSE TO INOCULATION Inoculation of legume seeds with a culture of nodule bacteria (Rhizobium spp.) was originally introduced as a means of ensuring the establishment of seedlings in nitrogen-deficient soils, which lack adequate population of suitable rhizobia (Fred et^^-, 1932). Rhizobium inoculation is a cheaper and widely accepted technique. To achieve the highest yield potential of different legume crops, however, it is essential to ensure the presence of the correct type and number of rhizobia either on the seed or in the soil to meet the nitrogen require ment of these legumes (Khurana and Shartna, 1986). Simple Rhizobium inoculation increased yields of certain leguminous crops in certain soils upto a maximum of 71% over a corresponding uninoculated 2 8 Table 3 ; Reports on cross-inoculation studies of the twenty genera under study. Sr. Genus Reports No, Acacia Walker, 1928; Allen and Allen, 1936; 1939; MacKnight, 1949; Lange, 1961; Habish and Khairi, 1968; 1970. 2 Albizia Allen and Allen, 1936. 3 Alysicarpus Allen and Allen, 1936; 1939; Wilson, 1945; Sandmann, 1970. 4 Butea No Report. 5 Cassia Allen and Allen, 1936. 6 Crotalaria Allen and Allen, 1936; 1939; Conklin, 1936; Burton, 1952. 7 Dlbergifit Narayan and Monga, 1979. 8 Desmodium______Carroll, 1934; Allen and Allen, 1939; Bhide, 1956; Bowen, 1956. 9 Erythrina Allen and Allen, 1939; Erdman, 1948. 10 Geissaspis Shinde, 1981. 11 Gliricidia No Report. 12 Goniogyna Shinde, 1981. 13 Indigofera Allen and Allen, 1936; 1939; 1940; Raju, 1936; Wilson, 1939. 14 Ponqamia Allen and Allen, 1939. 15 Pterocarpus Allen and Allen, 1936. 16 Sesbania Rangaswami and Oblisami, 1962. 17 Smithia Shinde, 1981. 18 Taverniera No Report. 19 Tephrosia Allen and Allen, 1939. 20 Vigna Shinde, 1981. 29 control. These included Cicer arietinum, Cajanus cajan, Glycine max, and Lens culunaris (Subba Rao, 1976). However, the artificial inoculation of Arachis hyposaea L. seeds with laboratory grown cultures has not increased the yield as shown by the results of field trials carried out in different parts of the country ( G a u r ^ ^ ., 197^1; Nambier, 1985). ^ " The studies on groundnut have shown that not only the microsymbiont (rhizobia) but also the macrosymbiont (groundnut host) possess "promiscuity". Cross-infectivity tests have shown that groundnut Rhizobium nodulates other cowpea hosts (Rajgopalan and Sadasivan, 196^) and becomes nodulated by rhizobia which also nodulate many species of tropical leguminous plants, and these rhizobia are classified as cowpea-miscellany (Allen and Allen, 1981). Most of the soil under cultivation in the tropics appears to have relatively large populations (>10^ cells per gram dry soil) of these rhizobia. These types of rhizobia which cause nodulation in groundnut are not usually efficient, and owing to their large population colonize early in the rhizosphere, cause early infection, which develops immunity to further infection (Dunham and Baldwin, 1931); the infection sites also become, covered with the nodules produced by them (Chen, 19^1). Thus the failure to obtain response to inoculation through efficient nitrogen fixing strains may be attributed to the competitiveness for infection by the inefficient native population of infecting rhizobia (Thornton, 195^), since an efficient strain may not be necessarily competitive in its character (Nicol and Thornton, 19^fl). Commenting on inoculation experiments conducted by various authors, Hegde (1982) noticed that, "in India the necessity to inoculate groundnut has neither been shown conclusively nor investigated thoroughly." Dadarwal ^ ^ ( 1 9 7 9 ) carried out a survey of native rhizobia that infect 3 0 green gram for nitrogen fixation efficiency. The results of their studies revealed that 5 0 % of the rhizobia were highly ineffective. They further demonstrated that the nodulation by these rhizobia creates a condition in which the plant spends energy for no return. Owing to the ubiquitous nature of cowpea rhizobia in tropical soils, positive inoculation response in cowpea hosts is very unlikely (Dobereiner, 1977). On the other hand Ahmad et _^(I981) put forth another view, that the question of inoculant response in cowpea hosts has not been approached on a significant scale. According to Sylvester (198^) the soil contains a number of effective rhizobial strains, and the selection of such strains by simple agronomic practice, though important in inoculation programmes, is relatively neglected due to the proliferation of advanced techniques such as a strain improvement programme through molecular biology. 2.^.1 Competition studies To understand the potential for improving nitrogen fixation where the indigenous population of rhizobia is abundant, knowledge of rhizobial characteristics IS required, particularly of the ranges of competitiveness and effectiveness. Competitiveness has been shown to be independent of effectiveness, and highly effective strains vary greatly in their degree of competitiveness (Vincent, 1970; Franco and Vincent, 1976). The competitiveness of an inoculated strain is a very important attribute (Ghai ^ a h , 1982; Materon and Hagedorn, 1982; Khurana and Sharma, 1986) for displacing the native rhizobia which lack the capability of causing effective nodulation. 31 The dynamics of the Rhlzobium population in a legume field is poorly understood. One reason for this is that there has been no adequate method of identifying indigenous rhizobial strains. Various established methods such as serological techniques (Ikram et 1978) or antibiotic resistance markers (Gupta et 1983) have been employed to understand the contribution of introduced Rhizobium to the native population with respect to nodulation. According to Khurana et ^ (1978) the rhizobial culture supplied with the seed can very well compete with native strains, and showed 60% competitive ability in the case of certain chickpea cultures. Studies on fast-growing gram rhizobia by Chahal e^ ^ (1978) showed the competitive ability of these rhizobia in the range of 16% to 85%. Competition studies of fast-and slow-growing soybean rhizobia by Dowdle and Bohlool (1985) showed that fast-growing rhizobia were highly competitive and formed 86% of the nodules. While studying the inoculation response of mungbean rhizobia, Gupta e^ ^ (1 9 8 3 ) encountered certain mungbean strains having a competitive ability as high as 50%. Dadarwal e^ ^ (1 9 7 9 ) showed that the competitive ability of the strain can be increased against native rhizobia upto 70% - 80% through proper seed bacterization. 2 A . 2 Application of rhizobia isolated from wild legumes on cultivated legumes : There are many reports in the literature to the effect that rhizobia from wild legumes cross-inoculate cultivated legume hosts (Rangaswami and Oblisami, 1962; Shinde, 1981; Gaur et 197^; Singh et 1976; Lim and Ng, 1977; Yanasugondha et 1977). However, very few studies have been attempted to evaluate the efficiency of these strains in increasing the nitrogen fixation capacity, and ultimately the grain yield, of cultivated pulses. Ramachandran et al (1980) found that a strain from wild sunnhemp gave significantly higher 32 dry weights of cowpea plants. Basak and Goyal (1980b) showed that six rhizobial strains from tree legumes, when tried on green gram and black gram, were found superior to the uninoculated control and performed better than homologous (specific) rhizobia. Nimbalkar (1986) showed that seventeen out of nineteen strains of rhizobia from wild legumes effectively nodulated cowpea (Vigna unguiculata Walp.) plants. He also observed that the fast-growing strains from Erythrina subrosea, Prosopis cystisoides, and the slow-growing strains from Mucuna pruriens and Tephrosia tinctorea, gave significantly higher dry weights of plant tops than the homologous strain of cowpea. This indicates that there is hardly any literature on the applicability of rhizobia from wild legumes to cultivated legumes. 2.^.3 Evaluation of symbiotic nitrogen fixation efficiency : Symbiotic nitrogen fixation by rhizobia with leguminous hosts is an important biological activity affecting crop yield and soil fertility, and thereby greatly increasing economic value in agriculture. The effectiveness of rhizobial strains is measured in terms of the amount of nitrogen fixed. In the early days of investigation, the number of nodules formed by a particular strain was taken as the index of effectiveness, but with the discovery of parasitic strains by Erdman (1948), the number of nodules has lost its significance and the amount of nitrogen fixed, as determined by quantitative analysis, is today taken as the only criterion lor determining the elfectiveness oi any pariiruiar siiam. There are a number of methods of evaluating symbiotic nitrogen fixation efficiency, such as determination of nodule nitrogen, nitrogenase activity of nodulated roots and grain yield. In experiments with pea, clover and medic, Schwinghamer et ^ (1970), obtained a significant positive correlation between the nitrogenase activity of 33 nodulated roots and the dry weight of shoots of nodulated plants. Gupta e ^ ^ (1983) studied the correlation coefficient between yield and yield attributing characters while evaluating the response of inoculation in mung. It was indicated that the yield is correlated with dry matter which in turn is dependent on nitrogenase activity at an early stage of crop growth and this would be the nnost reliable parameter for estimating the yield. 2.5 NITROGEN FIXATION BY WILD LEGUMES. The research on root nodule bacteria done so far primarily concerns the agriculturally important legumes occurring in temperate and tropical regions. As compared with tropical regions, temperate regions produce fewer legumes and their flora is less predominant in terms of genera and species. Only 12% of the legume genera are typical of temperate climates (Eli, 1977). The tropical legumes^ on the other handj form an important group with reference to their nitrogen - fixing ability because they can leave upto 30% of fixed nitrogen in the soil for the succeeding crops (Oke, 1966) as against 10% to 15% contributed by temperate legumes (Parker, 1977). Out of the 18,000 species of leguminous plants growing in the tropics, only 100-150 species are cultivated (Lim and Burton, 1982). The uncultivated wild legumes, which comprise a large portion of Leguminosae, are neglected. MacConnel and Bond (1957) have defined "wild legumes" as a legume of no agricul tural significance or one which, though used in agriculture, is growing in a natural community. Although the wild legumes contribute a large amount of nitrogen to the soil ecosystem, few systematic efforts have been made so far to study their actual contribution. These studies are mainly concentrated on nitrogen 34 fixation by herbaceous legumes and their contribution to adjacent crops (Henzell and Norris, 1962; Whitney 1967; Akinola et 1972). Under the pressure of increasing population, sharply diminishing supplies of food and fuel wood, and the alarming rate of loss of the world's natural forests, a new branch of science-Agroforestry has been developed, which plays an important role in preventing soil erosion, retaining more rain water, and improving soil productivity. Felker and Bandurski (1979) developed an ' 'idiotype' for the plants to be used in agricultural practices which include (i) growth conditions for minimal soil and nutrient loss; (ii) little or no need for irrigation; (iii) high yield; (iv) ability to fix nitrogen (v) production of large amounts of high quality protein. They found that legume trees have the characteristics of an 'idiotype'. Hegde (1986) added further that the tree species to be introduced in agroforestry must have the following properties; (1) capacity to grow without suppressing arable crops; (2) easy establishment; (3) fast growth; (^) capacity to withstand frequent pruning; (5) production of easily degradable litter and (6) high yield of food, fodder and other valuable products. In agroforestry, woody legumes are very useful. According to Allen and Allen (1961) the number of woody legumes forming nitrogen-fixing root nodule symbiosis with Rhizobium spp. is very high in tropical areas, amounting to some thousand species. Despite the high number of tree species possibly fixing nitrogen, there is yet no information available on the actual capacity for nitrogen fixation apart from scattered reports on the nitrogenase activity of a few tree legumes (Dreyfus and Dommergues, 1981; Roskoski 1981; Hogberg and Kvarnstrom, 1982). Leucaena leucocephala (subabhul) is one of the most commonly cultivated 35 species throughout the tropical areas in agroforestry. However, it is not free from demerit. In addition to this tree species, there are number of multipurpose trees useful for food, fuelwood, fodder, and herbal medicines which can also be accommodated in agroforestry. These include Albizia lebbeck, Erythrina subrosea, Gliricidia sepium, and Sesbania grandiflora which have almost all the characteristics of 'idiotype' (Felker and Bandurski, 1979) and additional properties reported by Hegde (1986). One more additional criterion is that except Gliricidia sp. the remaining three species are indigenous fast-growing tree legumes. It is indeed unfortunate that a study on nitrogen fixation by legume trees in a natural ecosystem or agricultural field setting has not yet been reported from India.